Figure 7 ( A ) Correlation of ZEB1 mRNA expression with the expression of YAP , FOSL1 and JUN across all breast cancer cell lines included in the cancer cell line encyclopedia ( CCLE ) . rs : Spearman ' s rank correlation coefficient . ( B ) Heatmap of RNA - seq expression data of all CCLE breast cancer cell lines .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O

Figure 7(A) Correlation of ZEB1 mRNA expression with the expression of YAP, FOSL1 and JUN across all breast cancer cell lines included in the cancer cell line encyclopedia (CCLE). rs: Spearman's rank correlation coefficient.(B) Heatmap of RNA-seq expression data of all CCLE breast cancer cell lines.


Figure 8 ( A ) Kaplan Meier plots from survival analyses of human ER - / PR - breast cancers showing distant metastasis free survival ( DMFS ) based on the expression of the indicated genes or gene combinations . HR : hazard ratio . p - value from log - rank test . ( B ) Hypergeometric testing showed a significant association of nuclear ZEB1 and FOSL1 expression on the protein level as determined by immunohistochemistry on 81 human breast cancer samples . The upper panel shows staining of a serial section of a negative and a positive case . Note that stromal cells ( asterisks ) are negative for FOSL1 , and partially positive for ZEB1 and YAP , and that expression of ZEB1 in tumour cells ( arrows ) of positive cases is weaker than in stromal cells . Odds ratio ( OR ) : measure of association between categorical variables with OR > 1 indicating a positive association . ( C ) Heatmap of mRNA expression data of ZEB1 , YAP , JUN and 9 selected activated ZEB1 / YAP / AP - 1 target genes in breast cancer patient samples ( GSE18229 ) . Genes were selected for known roles in tumour promoting properties like cell migration . Tumours in which ≥ 6 out of 9 genes of the selected ZEB1 / YAP / AP - 1 target gene set were expressed higher than the 60th percentile of expression were defined as " tumour promoting gene set high " ; when expression of ≥ 6 out of the 9 genes was below the 40th percentile , tumours were defined as " tumour promoting gene set low " . Tumours were annotated according to their molecular subtype . ( E ) Kaplan Meier plots from survival analyses of human breast cancers patient samples ( GSE18229 ) showing relapse free survival ( RFS ) based on the combined expression of the 9 selected ZEB1 / YAP / AP - 1 activated targets HR : hazard ratio . p - value from log - rank test . ( F ) Kaplan Meier plots from survival analyses of human ER - / PR - breast cancers showing distant metastasis free survival ( DMFS ) based on the combined expression of the 9 selected ZEB1 / YAP / AP - 1 activated targets HR : hazard ratio . p - value from log - rank test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Kaplan Meier plots from survival analyses of human ER-/PR- breast cancers showing distant metastasis free survival (DMFS) based on the expression of the indicated genes or gene combinations. HR: hazard ratio. p-value from log-rank test.(B) Hypergeometric testing showed a significant association of nuclear ZEB1 and FOSL1 expression on the protein level as determined by immunohistochemistry on 81 human breast cancer samples. The upper panel shows staining of a serial section of a negative and a positive case. Note that stromal cells (asterisks) are negative for FOSL1, and partially positive for ZEB1 and YAP, and that expression of ZEB1 in tumour cells (arrows) of positive cases is weaker than in stromal cells. Odds ratio (OR): measure of association between categorical variables with OR > 1 indicating a positive association.(C) Heatmap of mRNA expression data of ZEB1, YAP, JUN and 9 selected activated ZEB1/YAP/AP-1 target genes in breast cancer patient samples (GSE18229). Genes were selected for known roles in tumour promoting properties like cell migration. Tumours in which ≥ 6 out of 9 genes of the selected ZEB1/YAP/AP-1 target gene set were expressed higher than the 60th percentile of expression were defined as "tumour promoting gene set high"; when expression of ≥ 6 out of the 9 genes was below the 40th percentile, tumours were defined as "tumour promoting gene set low". Tumours were annotated according to their molecular subtype.(E) Kaplan Meier plots from survival analyses of human breast cancers patient samples (GSE18229) showing relapse free survival (RFS) based on the combined expression of the 9 selected ZEB1/YAP/AP-1 activated targets HR: hazard ratio. p-value from log-rank test. (F) Kaplan Meier plots from survival analyses of human ER-/PR- breast cancers showing distant metastasis free survival (DMFS) based on the combined expression of the 9 selected ZEB1/YAP/AP-1 activated targets HR: hazard ratio. p-value from log-rank test.


Figure 1Morphology of four - week - old soil - grown plants of Col - 0 , snc1 , and two independent transgenic lines of SNIPER1 OE into snc1 background . OE stands for overexpression of SNIPER1 . Scale bar = 1 cm . SNIPER1 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . Quantification of H . a . Noco2 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . m . ES4326 on four - week - old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0 . 001 . Error bars represent mean ± SD ( n = 4 for day 0 ; n = 5 for day 3 ) . Three independent experiments were carried out with similar results . Morphology of four - week - old soil - grown plants of two independent transgenic lines of SNIPER1 OE in Col - 0 background . Scale bar = 1 cm . No difference in growth and development was observed . SNIPER1 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . Quantification of H . a . Noco2 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . t . DC3000 on four - week - old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0 . 001 . Error bars represent mean ± SD ( n = 4 for day 0 ; n = 5 for day 3 ) . Three independent experiments were carried out with similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Morphology of four-week-old soil-grown plants of Col-0, snc1, and two independent transgenic lines of SNIPER1 OE into snc1 background. OE stands for overexpression of SNIPER1. Scale bar = 1 cm. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.Morphology of four-week-old soil-grown plants of two independent transgenic lines of SNIPER1 OE in Col-0 background. Scale bar = 1 cm. No difference in growth and development was observed. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.t. DC3000 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.


Figure 2Morphology of four - week - old soil - grown plants of Col - 0 , chs1 - 2 ( A ) , chs2 - 1 ( B ) and two independent transgenic lines of SNIPER1 OE into chs1 - 2 ( A ) and chs2 - 1 ( B ) backgrounds . Plants were grown at 16 ° C under long day conditions . Scale bar = 1 cm . Morphology of four - week - old soil - grown plants of Col - 0 , chs3 - 2D ( C ) , snc2 - 1D eds5 npr1 ( D ) and two independent transgenic lines of SNIPER1 OE into chs3 - 2D ( C ) and snc2 - 1D eds5 npr1 ( D ) backgrounds . Scale bar = 1 cm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Morphology of four-week-old soil-grown plants of Col-0, chs1-2 (A), chs2-1 (B) and two independent transgenic lines of SNIPER1 OE into chs1-2 (A) and chs2-1 (B) backgrounds. Plants were grown at 16 °C under long day conditions. Scale bar = 1 cm.Morphology of four-week-old soil-grown plants of Col-0, chs3-2D (C), snc2-1D eds5 npr1 (D) and two independent transgenic lines of SNIPER1 OE into chs3-2D (C) and snc2-1D eds5 npr1 (D) backgrounds. Scale bar = 1 cm.


Figure 3Morphology of four - week - old soil - grown plants of Col - 0 , mekk1 - 5 ndr1 , and two independent transgenic lines of SNIPER1 OE into mekk1 - 5 ndr1 background . Scale bar = 1 cm . The mekk1 ndr1 double mutant was used instead of the mekk1 - 5 single mutant is to overcome the sterility . SNIPER1 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . Quantification of H . a . Noco2 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . m . ES4326 on four - week - old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0 . 001 . mn stands for mekk1 - 5 ndr1 double mutant . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . In vitro ubiquitination assay using E . coli - expressed SNIPER1 - myc ( E3 ) , AtUBC8 ( E2 ) , AtUBA1 ( E1 ) and / or HIS - FLAG - Ubiquitin . The molecular mass markers are indicated on the left ( kDa ) . Asterisk indicates SNIPER1 - myc . Arrow points to His - FLAG - Ub and straight lines on the right indicate self - ubiquitinated SNIPER1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Morphology of four-week-old soil-grown plants of Col-0, mekk1-5 ndr1, and two independent transgenic lines of SNIPER1 OE into mekk1-5 ndr1 background. Scale bar = 1 cm. The mekk1 ndr1 double mutant was used instead of the mekk1-5 single mutant is to overcome the sterility. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. mn stands for mekk1-5 ndr1 double mutant. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.In vitro ubiquitination assay using E. coli-expressed SNIPER1-myc (E3), AtUBC8 (E2), AtUBA1 (E1) and/or HIS-FLAG-Ubiquitin. The molecular mass markers are indicated on the left (kDa). Asterisk indicates SNIPER1-myc. Arrow points to His-FLAG-Ub and straight lines on the right indicate self-ubiquitinated SNIPER1.


Figure 4Immunoblot analysis of SNC1 ( A - B ) , SUMM2 - 3HA ( C ) , RPS4HA ( D ) , RPP4myc ( E ) , RPS2HA ( F ) and RPM1myc ( G ) protein levels in the indicated genotypes . For all panels , equal loading is shown by Ponceau S staining of a non - specific band . The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD ( n = 3 ) . Molecular mass marker in kilo Daltons is indicated on the left . Three independent experiments were carried out with similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Immunoblot analysis of SNC1 (A-B), SUMM2-3HA (C), RPS4HA (D), RPP4myc (E), RPS2HA (F) and RPM1myc (G) protein levels in the indicated genotypes. For all panels, equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.


Figure 5Growth of P . s . t . avrRps4 ( A ) , avrRpt2 ( B ) and avrRpm1 ( C ) on four - week - old leaves of the indicated genotypes at 3 dpi with bacterial inoculum of OD600 = 0 . 0001 . Error bars represent mean ± SD ( n = 5 ) . Three independent experiments were carried out with similar results . Appendix Figure S4 shows the growth of P . s . t . avrRps4 , avrRpt2 and avrRpm1 at 0 dpi . Quantification of H . a . Emwa1 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Growth of P.s.t. avrRps4 (A), avrRpt2 (B) and avrRpm1 (C) on four-week-old leaves of the indicated genotypes at 3 dpi with bacterial inoculum of OD600 = 0.0001. Error bars represent mean ± SD (n=5). Three independent experiments were carried out with similar results. Appendix Figure S4 shows the growth of P.s.t. avrRps4, avrRpt2 and avrRpm1 at 0 dpi. Quantification of H.a. Emwa1 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.


Figure 6Morphology of four - week - old soil - grown plants of Col - 0 and two independent transgenic lines of 35S - SNIPER1H129Y . Scale bar = 1 cm . PR1 and PR2 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . Morphology of four - week - old soil - grown plants of Col - 0 , sniper1 - 1 , sniper2 - 1 , eds1 - 2 , SNIPER1H129Y , SNIPER1H129Y eds1 - 2 , sniper1 - 1 sniper2 - 1 eds1 - 2 and sniper1 - c1 sniper2 - 1 eds1 - 2 . sniper1 - 1 is an exonic T - DNA insertional mutant . sniper1 - c1 is a SNIPER1 knockout mutant containing 807bp deletion that was generated by CRISPR - Cas9 system . Scale bar = 1 cm . Quantification of H . a . Noco2 sporulation in the indicated plants 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Two independent experiments were carried out with similar results . Immunoblot of RPS4HA ( E ) , SNC1 ( F ) , RPS2HA ( G ) , and RPM1myc ( H ) in SNIPER1H129Y background . Equal loading is shown by Ponceau S staining of a non - specific band . The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD ( n = 3 ) . Molecular mass marker in kilo Daltons is indicated on the left . Three independent experiments were carried out with similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Morphology of four-week-old soil-grown plants of Col-0 and two independent transgenic lines of 35S-SNIPER1H129Y. Scale bar = 1 cm.PR1 and PR2 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results.Morphology of four-week-old soil-grown plants of Col-0, sniper1-1, sniper2-1, eds1-2, SNIPER1H129Y, SNIPER1H129Y eds1-2, sniper1-1 sniper2-1 eds1-2 and sniper1-c1 sniper2-1 eds1-2. sniper1-1 is an exonic T-DNA insertional mutant. sniper1-c1 is a SNIPER1 knockout mutant containing 807bp deletion that was generated by CRISPR-Cas9 system. Scale bar = 1 cm.Quantification of H.a. Noco2 sporulation in the indicated plants 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Two independent experiments were carried out with similar results.Immunoblot of RPS4HA (E), SNC1 (F), RPS2HA (G), and RPM1myc (H) in SNIPER1H129Y background. Equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.


Figure 7Immunoprecipitation and biotinylation of SUMM2NB - FLAG ( A ) , RPP4NB - FLAG ( B ) , or CHS1NB - FLAG ( C ) by SNIPER1 H129Y - HATurboID in N . benthamiana . Two biological repeats were carried out with similar results . Interaction of SNIPER1 H129Y - CLuc and SUMM2NB - NLuc ( D ) , RPP4NB - NLuc ( E ) or CHS1NB - NLuc ( F ) as tested by split - luciferase complementary assay in N . benthamiana . Three biological repeats with four technical repeats each were carried out with similar results .

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Figure 7Immunoprecipitation and biotinylation of SUMM2NB-FLAG (A), RPP4NB-FLAG (B), or CHS1NB-FLAG (C) by SNIPER1 H129Y-HATurboID in N. benthamiana. Two biological repeats were carried out with similar results.Interaction of SNIPER1 H129Y-CLuc and SUMM2NB-NLuc (D), RPP4NB-NLuc (E) or CHS1NB-NLuc (F) as tested by split-luciferase complementary assay in N. benthamiana. Three biological repeats with four technical repeats each were carried out with similar results.


Figure 8Ubiquitination of the NB domains from SUMM2 ( A ) , RPP4 ( B ) and CHS1 ( C ) . Immunoblot of bacterial lysates form E . coil strains expressing AtUBA1 - S , MBP - NB - HA , AtUBC8 - S , HIS - FLAG - UBQ10 and SNIPER1 - myc , or strains without one of these components . Two biological repeats were carried out with similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Ubiquitination of the NB domains from SUMM2 (A), RPP4 (B) and CHS1 (C). Immunoblot of bacterial lysates form E. coil strains expressing AtUBA1-S, MBP-NB-HA, AtUBC8-S, HIS-FLAG-UBQ10 and SNIPER1-myc, or strains without one of these components. Two biological repeats were carried out with similar results.


figf1 ( a , b ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP ( a ) . The high and low 20 % were sorted ( a ) , ( a , b ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP ( a ) . The high and low 20 % were sorted ( a ) , re - plated and treated with lysosomal protease inhibitors pepstatin and E - 64d for 1 h ; lysates were then immunoblotted for the indicated proteins ( b ) . ( c ) Densitometry of LC3 - II and p62 western blots ( normalized to actin and hour 0 , mean ± s . e . m . , n = 3 blots from 2 independent experiments , * P = 0 . 051 , * * P = 0 . 0091 ) . ( d ) ; autophagic LC3 puncta were assessed by quantitative microscopy ( d ) ; autophagic LC3 puncta were assessed by quantitative microscopy ( e ; punctate area per cell , mean ± s . e . m . , n = 50 fields , * P = 0 . 010 , * * P = 0 . 053 ) . f ) Electron micrographs of HeLa mCherry - GFP - LC3 cells sorted for autophagic flux as in a . Yellow arrows denote autophagosomes ; red arrows indicate autolysosomes . ( g ) Quantification of autophagosomes and autolysomes from electron micrographs ( mean ± s . e . m . , n = 50 fields ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a),(a,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a), re-plated and treated with lysosomal protease inhibitors pepstatin and E-64d for 1 h; lysates were then immunoblotted for the indicated proteins (b). (c) Densitometry of LC3-II and p62 western blots (normalized to actin and hour 0, mean ± s.e.m., n = 3 blots from 2 independent experiments, *P = 0.051, **P = 0.0091).(d); autophagic LC3 puncta were assessed by quantitative microscopy(d); autophagic LC3 puncta were assessed by quantitative microscopy (e; punctate area per cell, mean ± s.e.m., n = 50 fields, *P = 0.010, **P = 0.053).f) Electron micrographs of HeLa mCherry-GFP-LC3 cells sorted for autophagic flux as in a. Yellow arrows denote autophagosomes; red arrows indicate autolysosomes. (g) Quantification of autophagosomes and autolysomes from electron micrographs (mean ± s.e.m., n = 50 fields). Uncropped images of blots are shown in Supplementary Fig. 6.


figf2a - c ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP . Cells were then re - plated in growth medium and autophagic flux was again measured by flow cytometry at the indicated time points ( median ratio of mCherry / GFP fluorescence ± s . e . m . , n = 3 wells ) . Lysates from cells collected at the indicated time points were immunoblotted with the indicated antibodies ( b ) . Representative flow cytometry histograms for cells at 0 h and 24 h after sorting ( c ) . ( d ) BJAB mCherry - GFP - LC3 cells were sorted for autophagic flux as in a ( top and bottom 20 % ) . Following treatment with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) , cell viability was determined by MTS assay ( a tetrazolium reduction - based metabolic measurement ) at 24 h ( percentage of untreated control , mean ± s . e . m . , n = 3 wells , * P = 2 . 3 × 10 − 4 , * * P = 0 . 0036 ) . ( e ) Long - term growth of BJAB mCherry - GFP - LC3 cells sorted for autophagic flux followed by treatment with Fas ligand ( 4 ng ml − 1 ) or TRAIL ( 15 ng ml − 1 ) for 24 h . Cells were then re - plated and allowed to recover for 5 ( Fas ligand ) or 6 ( TRAIL ) days then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 012 ) . ( f ) BJAB mCherry - GFP - LC3 cells were sorted for autophagic flux as above and apoptosis was measured at 1 h by flow cytometry using AnnexinV and DAPI ( mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) . ( g ) Cells sorted as in a , were re - plated and , starting at the indicated times following sorting , treated with Fas ligand ( 4 ng ml − 1 ) for 24 h ; cell viability was then determined by MTS ( percentage of untreated control , mean ± s . e . m . , n = 3 wells ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2a-c) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP. Cells were then re-plated in growth medium and autophagic flux was again measured by flow cytometry at the indicated time points (median ratio of mCherry/GFP fluorescence ± s.e.m., n = 3 wells). Lysates from cells collected at the indicated time points were immunoblotted with the indicated antibodies (b). Representative flow cytometry histograms for cells at 0 h and 24 h after sorting (c).(d) BJAB mCherry-GFP-LC3 cells were sorted for autophagic flux as in a (top and bottom 20%). Following treatment with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1), cell viability was determined by MTS assay (a tetrazolium reduction-based metabolic measurement) at 24 h (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 2.3×10−4, **P = 0.0036).(e) Long-term growth of BJAB mCherry-GFP-LC3 cells sorted for autophagic flux followed by treatment with Fas ligand (4 ng ml−1) or TRAIL (15 ng ml−1) for 24 h. Cells were then re-plated and allowed to recover for 5 (Fas ligand) or 6 (TRAIL) days then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.012).(f) BJAB mCherry-GFP-LC3 cells were sorted for autophagic flux as above and apoptosis was measured at 1 h by flow cytometry using AnnexinV and DAPI (mean ± s.e.m., n = 3 wells, *P = 0.0046).(g) Cells sorted as in a, were re-plated and, starting at the indicated times following sorting, treated with Fas ligand (4 ng ml−1) for 24 h; cell viability was then determined by MTS (percentage of untreated control, mean ± s.e.m., n = 3 wells). Uncropped images of blots are shown in Supplementary Fig. 6.


figf3 ( a ) BJAB and Jurkat cells were treated with vehicle or chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by Fas ligand ( 1 . 5 ng ml − 1 ) . Cell viability was determined by MTS 24 h later ( percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 0 . 0058 ) . ( b ) Immunoblots of cells in a , c probed for the indicated antibodies . CQ , chloroquine ( c ) BJAB and Jurkat cells were treated with chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by treatment with Fas ligand ( 4 ng ml − 1 ) for 24 h ( same technical replicate as a ) . Cells were then re - plated at low density in growth media and allowed to recover for 5 days , and then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0024 ) . ( d ) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs , followed by 3 days of puromycin selection . Selected cells were plated and treated with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) for 24 h and viability was assessed by MTS ( percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 , * P = 1 . 7 × 10 − 4 , * * P = 0 . 024 ) . ( e ) Immunoblots for Atg5 , Atg7 and p62 confirm protein depletion and autophagy inhibition in d , f . ( f ) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand ( 15 ng ml − 1 ) or TRAIL ( 12 . 5 ng ml − 1 ) for 24 h ( same technical replicate as d ) . Cells were then re - plated at low density and allowed to recover for 5 - 6 days , and then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0089 ) . Atg7 knockdown data were not included for long - term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment . Uncropped images of blots are shown in Supplementary Fig . 6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) BJAB and Jurkat cells were treated with vehicle or chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (1.5 ng ml−1). Cell viability was determined by MTS 24 h later (percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 0.0058).(b) Immunoblots of cells in a,c probed for the indicated antibodies. CQ, chloroquine(c) BJAB and Jurkat cells were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by treatment with Fas ligand (4 ng ml−1) for 24 h (same technical replicate as a). Cells were then re-plated at low density in growth media and allowed to recover for 5 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0024).(d) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs, followed by 3 days of puromycin selection. Selected cells were plated and treated with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1) for 24 h and viability was assessed by MTS (percentage of control (no ligand), mean ± s.e.m., n = 3, *P = 1.7×10−4, **P = 0.024).(e) Immunoblots for Atg5, Atg7 and p62 confirm protein depletion and autophagy inhibition in d,f.(f) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand (15 ng ml−1) or TRAIL (12.5 ng ml−1) for 24 h (same technical replicate as d). Cells were then re-plated at low density and allowed to recover for 5-6 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0089). Atg7 knockdown data were not included for long-term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment. Uncropped images of blots are shown in Supplementary Fig. 6.


figf4 ( a , b ) BJAB and Jurkat cells stably expressing mCherry - GFP - LC3 were flow sorted for high and low autophagic flux , treated with Fas ligand ( 1 . 5 ng ml − 1 ) for 24 h and viability was assessed by MTS ( a ; percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) . ( b ) Immunoblots following sorting of the samples in a . ( c , d ) The indicated cell lines were treated with chloroquine ( BJAB , CEM 20 μM ; SKW6 . 4 , 25 μM ; Jurkat , 10 μM ) for 16 h , followed by Fas ligand ( BJAB , 12 . 5 ng ml − 1 ; SKW6 . 4 , 50 ng ml − 1 ; Jurkat , 0 . 4 ng ml − 1 ; CEM , 40 ng ml − 1 ) . Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies ( c ) . ( c , d ) The indicated cell lines were treated with chloroquine ( BJAB , CEM 20 μM ; SKW6 . 4 , 25 μM ; Jurkat , 10 μM ) for 16 h , followed by Fas ligand ( BJAB , 12 . 5 ng ml − 1 ; SKW6 . 4 , 50 ng ml − 1 ; Jurkat , 0 . 4 ng ml − 1 ; CEM , 40 ng ml − 1 ) . Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies ( c ) . Cell viability was assessed by MTS 24 h following Fas ligand treatment ( d ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 2 . 7 × 10 − 4 , * * P = 1 . 6 × 10 − 4 ) . ( e , f ) BJAB and Jurkat cells were transduced with control , Atg5 , Atg7 or Vps34 shRNA lentiviruses , followed by three days of puromycin selection . Cells were then treated with Fas ligand ( 1 . 25 ng ml − 1 ) or TRAIL ( 1 . 25 ng ml − 1 ) for 24 h and viability was assessed by MTS ( e ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 1 . 4 × 10 − 6 , * * P = 2 . 3 × 10 − 5 , * * * P = 3 . 9 × 10 − 5 , § P = 9 . 2 × 10 − 6 , § § P = 3 . 4 × 10 − 6 , § § § P = 1 . 7 × 10 − 5 ) . Immunoblots demonstrate Atg5 , Atg7 and Vps34 knockdown , autophagy inhibition and altered Fap - 1 levels . The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other ; the Fap - 1 blot is not separated to demonstrate the lack of Fap - 1 protein in Type II Jurkat cells ( f ) . Fap - 1 blots ( b , c , f ) were run on separate gels owing to the quantity of protein required for detection ( see Methods ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a,b) BJAB and Jurkat cells stably expressing mCherry-GFP-LC3 were flow sorted for high and low autophagic flux, treated with Fas ligand (1.5 ng ml−1) for 24 h and viability was assessed by MTS (a; percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0046). (b) Immunoblots following sorting of the samples in a.(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c).(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c). Cell viability was assessed by MTS 24 h following Fas ligand treatment (d; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 1.6×10−4).(e,f) BJAB and Jurkat cells were transduced with control, Atg5, Atg7 or Vps34 shRNA lentiviruses, followed by three days of puromycin selection. Cells were then treated with Fas ligand (1.25 ng ml−1) or TRAIL (1.25 ng ml−1) for 24 h and viability was assessed by MTS (e; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 1.4×10−6, **P = 2.3×10−5, ***P = 3.9×10−5, §P = 9.2×10−6, §§P = 3.4×10−6, §§§P = 1.7×10−5). Immunoblots demonstrate Atg5, Atg7 and Vps34 knockdown, autophagy inhibition and altered Fap-1 levels. The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other; the Fap-1 blot is not separated to demonstrate the lack of Fap-1 protein in Type II Jurkat cells (f). Fap-1 blots (b,c,f) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.


figf5 ( a , b ) BJAB and Jurkat cells expressing the indicated Fap - 1 wild - type ( WT ) and catalytically inactive ( ΔCD ) constructs were treated with chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by Fas ligand ( BJAB , 1 . 5 ng ml − 1 ; Jurkat 15 ng ml − 1 ) for 24 h . Cell viability was determined by MTS ( a ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 2 . 7 × 10 − 4 , * * P = 2 . 4 × 10 − 5 , * * * P = 0 . 0024 ) . Cell lysates were blotted and probed with the indicated antibodies ( b ) . c - e ) BJAB cells expressing control or Fap - 1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand ( 12 . 5 ng ml − 1 ) or TRAIL ( 25 ng ml − 1 ) for 24 h ; cell viability was then determined by MTS ( c ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 4 . 8 × 10 − 6 , * * P = 8 . 1 × 10 − 5 , * * * P = 8 . 0 × 10 − 4 , § P = 0 . 013 , NS P > 0 . 05 ) . ( d ) Immunoblots of lysates collected following chloroquine treatment . Long - term growth was assessed in cells from c by re - plating them at low density and allowing them to recover for 5 days , followed by viability assay ( percentage of untreated control , mean ± s . e . m . , n = 3 wells , * P = 0 . 043 ; e ) . Fap - 1 blots ( b , d ) were run on separate gels owing to the quantity of protein required for detection ( see Methods ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a,b) BJAB and Jurkat cells expressing the indicated Fap-1 wild-type (WT) and catalytically inactive (ΔCD) constructs were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (BJAB, 1.5 ng ml−1; Jurkat 15 ng ml−1) for 24 h. Cell viability was determined by MTS (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 2.4×10−5, ***P = 0.0024). Cell lysates were blotted and probed with the indicated antibodies (b).c-e) BJAB cells expressing control or Fap-1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand (12.5 ng ml−1) or TRAIL (25 ng ml−1) for 24 h; cell viability was then determined by MTS (c; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 4.8×10−6, **P = 8.1×10−5, ***P = 8.0×10−4, §P = 0.013, NS P>0.05).(d) Immunoblots of lysates collected following chloroquine treatment. Long-term growth was assessed in cells from c by re-plating them at low density and allowing them to recover for 5 days, followed by viability assay (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 0.043; e). Fap-1 blots (b,d) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.


figf6a , b ) Cell viability was determined by MTS assay following Fas ligand ( 4 ng ml − 1 ) treatment in BJAB cells expressing control or p62 lentiviral shRNAs ( a ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 3 . 2 × 10 − 5 , * * P = 1 . 8 × 10 − 6 ) . Immunoblots confirm p62 depletion and Fap - 1 levels ( b ) . ( c ) Co - immunoprecipitation of endogenous p62 and Fap - 1 in BJAB cells treated with 20 μM chloroquine for 16 h , followed by treatment with Fas ligand ( 50 ng ml − 1 ) for 2 h at 4 ° C .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6a,b) Cell viability was determined by MTS assay following Fas ligand (4 ng ml−1) treatment in BJAB cells expressing control or p62 lentiviral shRNAs (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 3.2×10−5, **P = 1.8×10−6). Immunoblots confirm p62 depletion and Fap-1 levels (b).(c) Co-immunoprecipitation of endogenous p62 and Fap-1 in BJAB cells treated with 20 μM chloroquine for 16 h, followed by treatment with Fas ligand (50 ng ml−1) for 2 h at 4°C.


Figure 1 ( A ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) the TAK1 inhibitor NG25 ( 2 μM ) , the IKKβ inhibitor BI605906 ( 5 μM ) or the IKKβ inhibitor PS1145 ( 10 μM ) . The cells were then co - stimulated for 30 min with 100 ng / ml LPS and 4 mM ATP . Cell lysates ( 10 μg protein ) were denatured in SDS , subjected to SDS - PAGE and immunoblotted with the antibodies indicated . ( B ) As in A , except that the cells were co - stimulated for 1 h with 100 ng / ml LPS and 5 μM nigericin . Similar results were obtained in three independent experiments in A and B . ( C , D ) As in A , B , except that primary human monocyte - derived macrophages were used instead of mouse BMDM . Similar results were obtained in two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) WT BMDM were incubated for 1 h without (-) or with (+) the TAK1 inhibitor NG25 (2 μM), the IKKβ inhibitor BI605906 (5 μM) or the IKKβ inhibitor PS1145 (10 μM). The cells were then co-stimulated for 30 min with 100 ng/ml LPS and 4 mM ATP. Cell lysates (10 μg protein) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. (B) As in A, except that the cells were co-stimulated for 1 h with 100 ng/ml LPS and 5 μM nigericin. Similar results were obtained in three independent experiments in A and B. (C, D) As in A, B, except that primary human monocyte-derived macrophages were used instead of mouse BMDM. Similar results were obtained in two independent experiments.


Figure 2 ( A , B ) The Mouse macrophage J774A . 1 cell line was transfected with control siRNA or siRNA against IKKβ ( A ) or IKKα ( B ) . 72 h post - transfection , the cells were co - stimulated for 60 min without ( - ) or with ( + ) 100 ng / ml LPS and / or 5 μM nigericin . Cell lysates ( 10 μg ) were denatured in SDS , subjected to SDS - PAGE and immunoblotted with the antibodies indicated . Similar results were obtained in three ( A ) or two ( B ) independent experiments . ( C , D ) As in ( A , B ) except that BMDM from IKKβ - LysM - Cre ( flox / flox ) ( IKKβ ( fl / fl ) ) or WT control mice were co - stimulated without ( - ) or with ( + ) 100 ng / ml LPS and / or 4 mM ATP ( C ) or with 100 ng / ml LPS and / or 5 μM nigericin ( D ) . Similar results were obtained in two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A, B) The Mouse macrophage J774A.1 cell line was transfected with control siRNA or siRNA against IKKβ (A) or IKKα (B). 72 h post-transfection, the cells were co-stimulated for 60 min without (-) or with (+) 100 ng/ml LPS and/or 5 μM nigericin. Cell lysates (10 μg) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. Similar results were obtained in three (A) or two (B) independent experiments.(C, D) As in (A, B) except that BMDM from IKKβ-LysM-Cre (flox/flox) (IKKβ (fl/fl)) or WT control mice were co-stimulated without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (C) or with 100 ng/ml LPS and/or 5 μM nigericin (D). Similar results were obtained in two independent experiments.


Figure 3 ( A , B ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) , the IKKβ inhibitors BI605906 ( 5 μM ) , TPCA - 1 ( 5 μM ) or PS1145 ( 10 μM ) , the TAK1 inhibitor NG25 ( 2 μM ) or the NLRP3 inhibitor MCC950 . The cells were then co - stimulated for 30 min without ( - ) or with ( + ) 100 ng / ml LPS and / or 4 mM ATP ( A ) and / or 5 μM nigericin ( B ) . The cell culture medium was removed and the cells lysed . Protein in the culture medium was precipitated ( see Methods ) , dissolved in SDS . and subjected to SDS - PAGE , along with cell lysates . After transfer to PVDF membranes , immunoblotting was performed with antibodies recognizing both full length ( FL ) and cleaved ( CL ) gasdermin D ( GSDMD ) , the p20 and p10 fragments of caspase - 1 and IL - 18 . Similar results were obtained in two independent experiments . ( C ) As in A , except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) and control WT cells were used . Similar results were obtained in three independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A, B) WT BMDM were incubated for 1 h without (-) or with (+), the IKKβ inhibitors BI605906 (5 μM), TPCA-1 (5 μM) or PS1145 (10 μM), the TAK1 inhibitor NG25 (2 μM) or the NLRP3 inhibitor MCC950. The cells were then co-stimulated for 30 min without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (A) and/or 5 μM nigericin (B). The cell culture medium was removed and the cells lysed. Protein in the culture medium was precipitated (see Methods), dissolved in SDS. and subjected to SDS-PAGE, along with cell lysates. After transfer to PVDF membranes, immunoblotting was performed with antibodies recognizing both full length (FL) and cleaved (CL) gasdermin D (GSDMD), the p20 and p10 fragments of caspase-1 and IL-18. Similar results were obtained in two independent experiments. (C) As in A, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) and control WT cells were used. Similar results were obtained in three independent experiments.


Figure 4 ( A ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) 5 μM BI605906 , then stimulated for 1 h with ( + ) 100 ng / ml LPS and / or 5 μM nigericin or left unstimulated . The cells were lysed in buffer containing 1 % ( v / v ) Triton - X - 100 , and the Triton - X - 100 - soluble and Triton - X - 100 - insoluble fractions were prepared as in Methods . The Triton - X - 100 - insoluble fraction was dissolved SDS subjected to SDS - PAGE and immunoblotted with the antibodies indicated . The nuclear protein Histone H3B ( H3B ) was used as a loading control in the Tritin X - 100 insoluble fraction . Similar results were obtained in three independent experiments . ( B ) As in A , except that the Triton - X - 100 - insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2 . 0 mM DSS . ( C , As in A , B except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) mice ( IKKβ ( fl / fl ) ) were used . The U2 Small Nuclear RNA Auxiliary Factor 1 ( U2AF1 ) was used as a loading control . Similar results were obtained in two independent experiments . ( E , F ) As in A , B except that BMDM from caspase - 1 KO mice were used . Similar results were obtained in two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) WT BMDM were incubated for 1 h without (-) or with (+) 5 μM BI605906, then stimulated for 1 h with (+) 100 ng/ml LPS and/or 5 μM nigericin or left unstimulated. The cells were lysed in buffer containing 1% (v/v) Triton-X-100, and the Triton-X-100-soluble and Triton-X-100-insoluble fractions were prepared as in Methods. The Triton-X-100-insoluble fraction was dissolved SDS subjected to SDS-PAGE and immunoblotted with the antibodies indicated. The nuclear protein Histone H3B (H3B) was used as a loading control in the Tritin X-100 insoluble fraction. Similar results were obtained in three independent experiments. (B) As in A, except that the Triton-X-100-insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2.0 mM DSS. (C, As in A, B except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) mice (IKKβ (fl/fl)) were used. The U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) was used as a loading control. Similar results were obtained in two independent experiments.(E, F) As in A, B except that BMDM from caspase-1 KO mice were used. Similar results were obtained in two independent experiments.


Figure 5 ( A - H ) WT BMDM were incubated for 1 h without or with the IKKβ inhibitors TPCA - 1 ( 5 μM ) ( C , G ) or BI605906 ( 5 μM ) ( D , H ) . The cells were then stimulated for 1 h with 100 ng / ml LPS ( E ) or 5 μM nigericin ( F , G , H ) , or co - stimulated with 100 ng / ml LPS and 5 μM nigericin ( B , C , D ) or left unstimulated ( control ) ( A ) . The cells were fixed and processed for PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3 . A PLA signal is only if the NLRP3 antibody and the TGN38 antibody come into close proximity . ( A - H ) the panel images show the PLA signal ( red ) and DAPI staining for nuclei ( blue ) . Images were acquired by sequential laser scanning on a confocal microscope . Similar results were obtained in three independent experiments and representative images are shown . Scale bar = 50 μm . ( I - L ) As in A - H , except that IKKβ inhibitors were omitted and iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) ) or WT mice were co - stimulated for 1 h with 100 ng / ml LPS and 10 μM nigericin or left unstimulated ( control ) . Scale bar = 50 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-H) WT BMDM were incubated for 1 h without or with the IKKβ inhibitors TPCA-1 (5 μM) (C, G) or BI605906 (5 μM) (D, H). The cells were then stimulated for 1 h with 100 ng/ml LPS (E) or 5 μM nigericin (F, G, H), or co-stimulated with 100 ng/ml LPS and 5 μM nigericin (B, C, D) or left unstimulated (control) (A). The cells were fixed and processed for PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3. A PLA signal is only if the NLRP3 antibody and the TGN38 antibody come into close proximity. (A-H) the panel images show the PLA signal (red) and DAPI staining for nuclei (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were obtained in three independent experiments and representative images are shown. Scale bar = 50 μm.(I-L) As in A-H, except that IKKβ inhibitors were omitted and iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl)) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated (control). Scale bar = 50 μm.


Figure 6 ( A - H ) The experiments were performed as in Figs 5A - 5H , except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody , which was visualized using a secondary antibody ( red ) . Nuclei were counterstained with DAPI ( blue ) . Images were acquired by sequential laser scanning on a confocal microscope . Similar results were observed in many fields and obtained in three independent experiments . Representative images are shown Data information : In all panels , scale bar = 50 μm . ( I - L ) As in A - H , except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) or WT mice were co - stimulated for 1 h with 100 ng / ml LPS and 10 μM nigericin or left unstimulated . Similar results were obtained in two independent experiments . Data information : In all panels , scale bar = 50 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A-H) The experiments were performed as in Figs 5A-5H, except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody, which was visualized using a secondary antibody (red). Nuclei were counterstained with DAPI (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were observed in many fields and obtained in three independent experiments. Representative images are shown Data information: In all panels, scale bar = 50 μm.(I-L) As in A-H, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated. Similar results were obtained in two independent experiments. Data information: In all panels, scale bar = 50 μm.


Figure 1 ( A ) Western blotting of Mcrs1 at the GV ( 0 h ) , GVBD ( 4 h ) , MI ( 8 h ) , and MII ( 12 h ) stages of mouse oocyte meiosis . The blots were probed with anti - Mcrs1 and anti - actin antibodies . ( G ) Percentages of control ( n = 210 ) , Mcrs1 - KD ( n = 182 ) , and Mcrs1 - rescue ( n = 201 ) oocytes that underwent GVBD and PBE . The bars are representing the mean ± SEM . The P - values were calculated using Student ' s t - test . GVBD % : * * * P = 0 . 0003 ; * P = 0 . 01 , PB1 % : * P = 0 . 03 ; * P = 0 . 02 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Western blotting of Mcrs1 at the GV (0 h), GVBD (4 h), MI (8 h), and MII (12 h) stages of mouse oocyte meiosis. The blots were probed with anti-Mcrs1 and anti-actin antibodies.(G) Percentages of control (n = 210), Mcrs1-KD (n = 182), and Mcrs1-rescue (n = 201) oocytes that underwent GVBD and PBE. The bars are representing the mean ± SEM. The P-values were calculated using Student's t-test. GVBD%: ***P = 0.0003; *P = 0.01, PB1%: *P = 0.03; *P = 0.02.


Figure 2 ( A ) Representative images of the occurrence of GVBD in control , Mcrs1 - KD , and Mcrs1 - rescue ( Mcrs1 siRNA + Mcrs1 mRNA ) oocytes at 3 h following release from IBMX . Scale bar , 100 μm . ( B ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control ( n = 155 ) , Mcrs1 - KD ( n = 157 ) , and Mcrs1 - rescue ( n = 150 ) oocytes . ( D ) Expression levels of CDK1 in Mcrs1 - KD and control oocytes . The bars represent the mean ( ± SEM ) of 5 biological replicates . The P - values were calculated using Student ' s t - test . * P = 0 . 01 . ( E ) Expression levels of p - CDK1 ( Y15 ) in Mcrs1 - KD and control oocytes at 1 h following release from IBMX ( 200 oocytes per sample ) . * * P = 0 . 006 . ( F ) Expression levels of cyclin B1 in control and Mcrs1 - KD oocytes at 1 h following release from IBMX ( 200 oocytes per sample ) . The bars represent the mean ( ± SEM ) of 3 biological replicates . The P - values were calculated using Student ' s t - test . * * P = 0 . 0042 . ( I ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control oocytes ( n = 112 ) , Mcrs1 - KD oocytes ( n = 98 ) , and Mcrs1 - KD oocytes treated with 5 mM WEE1 - 1N - 4 ( n = 102 ) . ( J ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control ( n = 123 ) , Mcrs1 - KD ( n = 134 ) , and Mcrs1 siRNA + cyclin B1 - GFP ( n = 95 ) oocytes . ( K ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control ( n = 92 ) , Mcrs1 - KD ( n = 97 ) , and Mcrs1 siRNA + cyclin B1 - GFP oocytes treated with 5 mM WEE1 - 1N - 4 ( n = 106 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative images of the occurrence of GVBD in control, Mcrs1-KD, and Mcrs1-rescue (Mcrs1 siRNA + Mcrs1 mRNA) oocytes at 3 h following release from IBMX. Scale bar, 100 μm. (B) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 155), Mcrs1-KD (n = 157), and Mcrs1-rescue (n = 150) oocytes.(D) Expression levels of CDK1 in Mcrs1-KD and control oocytes. The bars represent the mean (± SEM) of 5 biological replicates. The P-values were calculated using Student's t-test. *P = 0.01. (E) Expression levels of p-CDK1(Y15) in Mcrs1-KD and control oocytes at 1 h following release from IBMX (200 oocytes per sample). **P = 0.006. (F) Expression levels of cyclin B1 in control and Mcrs1-KD oocytes at 1 h following release from IBMX (200 oocytes per sample). The bars represent the mean (± SEM) of 3 biological replicates. The P-values were calculated using Student's t-test. **P = 0.0042.(I) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control oocytes (n = 112), Mcrs1-KD oocytes (n = 98), and Mcrs1-KD oocytes treated with 5 mM WEE1-1N-4 (n = 102). (J) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 123), Mcrs1-KD (n = 134), and Mcrs1 siRNA + cyclin B1-GFP (n = 95) oocytes. (K) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 92), Mcrs1-KD (n = 97), and Mcrs1 siRNA + cyclin B1-GFP oocytes treated with 5 mM WEE1-1N-4 (n = 106).


Figure 3 ( B ) Detection of newly synthesized RNAs by EU incorporation in control , Mcrs1 - KD , and Mcrs1 - rescue GV oocytes . The scattergram shows the relative fluorescence intensity of EU in control ( n = 43 ) , Mcrs1 - KD ( n = 52 ) , and Mcrs1 - rescue ( n = 30 ) GV oocytes . Scale bar , 10 μm . * * * P = 0 . 0001 . ( C ) Immunofluorescence analysis of H3K4me2 in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of H3K4me2 signals in control ( n = 26 ) and Mcrs1 - KD ( n = 30 ) GV oocytes . * * * P = 0 . 0001 . ( E ) Immunofluorescence analysis of H3K9me2 in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of H3K9me2 signals in control ( n = 29 ) and Mcrs1 - KD ( n = 22 ) GV oocytes . * * P = 0 . 0035 . ( F ) Immunofluorescence analysis of H4K16ac in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of H4K16ac signals in control ( n = 30 ) and Mcrs1 - KD ( n = 28 ) GV oocytes . * P = 0 . 022 . ( G ) Immunofluorescence analysis of HDAC2 in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of HDAC2 signals in control ( n = 27 ) and Mcrs1 - KD ( n = 24 ) GV oocytes . * * * P = 0 . 0005 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Detection of newly synthesized RNAs by EU incorporation in control, Mcrs1-KD, and Mcrs1-rescue GV oocytes. The scattergram shows the relative fluorescence intensity of EU in control (n = 43), Mcrs1-KD (n = 52), and Mcrs1-rescue (n = 30) GV oocytes. Scale bar, 10 μm. ***P = 0.0001.(C) Immunofluorescence analysis of H3K4me2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H3K4me2 signals in control (n = 26) and Mcrs1-KD (n = 30) GV oocytes. ***P = 0.0001.(E) Immunofluorescence analysis of H3K9me2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H3K9me2 signals in control (n = 29) and Mcrs1-KD (n = 22) GV oocytes. **P = 0.0035. (F) Immunofluorescence analysis of H4K16ac in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H4K16ac signals in control (n = 30) and Mcrs1-KD (n = 28) GV oocytes. *P = 0.022.(G) Immunofluorescence analysis of HDAC2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of HDAC2 signals in control (n = 27) and Mcrs1-KD (n = 24) GV oocytes. ***P = 0.0005.


Figure 4 ( A ) Representative images of spindle assembly at 3 h post - release from IBMX in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . ( G ) Immunofluorescence analysis of H3S10ph and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The scattergram shows the relative fluorescence intensity of H3S10ph signals in control ( n = 21 ) and Mcrs1 - KD ( n = 23 ) oocytes . * P = 0 . 017 . ( H ) Immunofluorescence analysis of γ - tubulin and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The histogram shows the percentages of control ( n = 55 ) and Mcrs1 - KD ( n = 63 ) oocytes with abnormal γ - tubulin . * P = 0 . 014 . ( I ) Immunofluorescence analysis of acetylated tubulin and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The scattergram shows the relative fluorescence intensity of acetylated tubulin signals in control ( n = 83 ) and Mcrs1 - KD ( n = 66 ) oocytes . * * P = 0 . 0046 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Representative images of spindle assembly at 3 h post-release from IBMX in control and Mcrs1-KD oocytes. Scale bar, 20 μm.(G) Immunofluorescence analysis of H3S10ph and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of H3S10ph signals in control (n = 21) and Mcrs1-KD (n = 23) oocytes. *P = 0.017.(H) Immunofluorescence analysis of γ-tubulin and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The histogram shows the percentages of control (n = 55) and Mcrs1-KD (n = 63) oocytes with abnormal γ-tubulin. *P = 0.014. (I) Immunofluorescence analysis of acetylated tubulin and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of acetylated tubulin signals in control (n = 83) and Mcrs1-KD (n = 66) oocytes. **P = 0.0046.


Figure 5 ( C ) Levels of p - Aurka and p - Aurkc in control and Mcrs1 - KD MI oocytes . The blots were probed with anti - p - Aurora A / B / C and anti - actin antibodies . ( D ) Co - IP was performed with an anti - Mcrs1 antibody . The immunoblots of protein precipitates were probed with an anti - Aurka antibody . ( G ) Percentages of control ( n = 92 ) , Mcrs1 - KD ( n = 73 ) , and Aurka - rescue ( n = 67 ) oocytes that underwent PBE . * P = 0 . 033 ; * P = 0 . 018 . ( H ) Percentages of control ( n = 69 ) , Mcrs1 - KD ( n = 76 ) , and Aurka - rescue ( n = 43 ) oocytes with abnormal spindles . * P = 0 . 018 ; * P = 0 . 024 . ( K ) Immunofluorescence analysis of Kif2A in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The histogram shows the percentages of control ( n = 50 ) and Mcrs1 - KD ( n = 61 ) oocytes with abnormal Kif2A . * * P = 0 . 003 . ( L ) Immunofluorescence analysis of TPX2 and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The scattergram shows the relative fluorescence intensity of TPX2 signals in control ( n = 48 ) and Mcrs1 - KD ( n = 52 ) oocytes .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(C) Levels of p-Aurka and p-Aurkc in control and Mcrs1-KD MI oocytes. The blots were probed with anti-p-Aurora A/B/C and anti-actin antibodies. (D) Co-IP was performed with an anti-Mcrs1 antibody. The immunoblots of protein precipitates were probed with an anti-Aurka antibody.(G) Percentages of control (n = 92), Mcrs1-KD (n = 73), and Aurka-rescue (n = 67) oocytes that underwent PBE. *P = 0.033; *P = 0.018.(H) Percentages of control (n = 69), Mcrs1-KD (n = 76), and Aurka-rescue (n = 43) oocytes with abnormal spindles. *P = 0.018; *P = 0.024.(K) Immunofluorescence analysis of Kif2A in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The histogram shows the percentages of control (n = 50) and Mcrs1-KD (n = 61) oocytes with abnormal Kif2A. **P = 0.003.(L) Immunofluorescence analysis of TPX2 and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of TPX2 signals in control (n = 48) and Mcrs1-KD (n = 52) oocytes.


Figure 1 ( B ) The active fragment of mTOR or active Akt2 was incubated with Flag - RagC WT purified from HEK - 293E cells . The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Akt inhibitor GDC - 0068 . ( C ) HEK - 293E Cells were deprived of serum for 2 hr followed by insulin ( 100nM ) stimulation for the indicated time . Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways . Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls . ( D ) HEK - 293E cells were deprived of total amino acids for 1hr followed by addition of amino acids for the indicated time . Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways . Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls . ( E ) Control or iRapKO MEFs were treated with or without 2 μM 4 - Hydroxytamoxifen ( 4 - OHT ) for 4 days to induce Raptor knockout . Treated cells were deprived of serum for 2 hr , followed by insulin ( 100 nM ) stimulation for 10 min , and samples were analysed by Western blot . ( F ) SIN1 WT was expressed in SIN1 KO MEFs , and cells were selected by FACS . Cell lines were deprived of serum for 2 hr , followed by insulin ( 100 nM ) stimulation for 10 min , and samples were analysed by Western blot . The active fragment of mTOR was incubated with Flag - RagC WT purified from HEK - 293E cells . The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin . Samples were analysed by Mass Spectrum for pRagC T394The active fragment of mTOR was incubated with Flag - RagC WT purified from HEK - 293E cells . The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin . Samples were analysed by Western blot for pRagC

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) The active fragment of mTOR or active Akt2 was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Akt inhibitor GDC-0068.(C) HEK-293E Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.(D) HEK-293E cells were deprived of total amino acids for 1hr followed by addition of amino acids for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.(E) Control or iRapKO MEFs were treated with or without 2 μM 4-Hydroxytamoxifen (4-OHT) for 4 days to induce Raptor knockout. Treated cells were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.(F) SIN1 WT was expressed in SIN1 KO MEFs, and cells were selected by FACS. Cell lines were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.The active fragment of mTOR was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin. Samples were analysed by Mass Spectrum for pRagC T394The active fragment of mTOR was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin. Samples were analysed by Western blot for pRagC S21


Figure 2 ( A ) The HA - RagC HeLa Flp - In T - Rex stable cell lines were created ( See methods for details ) and the expression of RagC WT or mutants was induced by addition of tetracycline overnight . Cells were deprived of serum for 2 hr followed by insulin ( 100nM ) stimulation for 20 min . Lysates were analyzed by Western blot for levels of the specified proteins and the phosphorylation state of S6K1 . Graphs indicate the mean ± S . E . M . of quantitative analyses of the western blots after normalization for total protein ( * p < 0 . 05 , # no significant difference , two tailed Student ' s t test , n = 3 ) . ( B ) Expression of RagC WT , 3A or 3E was induced by addition of tetracycline overnight . Cells were serum starved for 2 hr followed by insulin ( 100nM ) stimulation for 20 min . Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1 . Graphs indicate the mean ± S . E . M . of quantitative analyses of the western blots after normalization for total protein ( * p < 0 . 05 , # no significant difference , two tailed Student ' s t test , n = 3 ) ( C ) Expression of RagC WT and 3A was induced by addition of tetracycline overnight . Cells were serum starved for 2 hr followed by insulin ( 100nM ) stimulation for indicated time . Left : Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1 . Right : Graph shows mean ± S . E . M . of quantitative analyses of the Western blots after normalization for total protein , n = 3 ( D ) HEK - 293E cells stably expressing RagC WT , 3A or 3E were deprived of total amino acids for 1hr followed by addition of amino acids for 10 min . Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1 . Graphs indicate the mean ± S . E . M . of quantitative analyses of the western blots after normalization for total protein ( * p < 0 . 05 , * * p < 0 . 01 , two tailed Student ' s t test , n = 3 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 (A) The HA-RagC HeLa Flp-In T-Rex stable cell lines were created (See methods for details) and the expression of RagC WT or mutants was induced by addition of tetracycline overnight. Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3). (B) Expression of RagC WT, 3A or 3E was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3)  (C) Expression of RagC WT and 3A was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for indicated time. Left: Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Right: Graph shows mean ± S.E.M. of quantitative analyses of the Western blots after normalization for total protein, n=3  (D) HEK-293E cells stably expressing RagC WT, 3A or 3E were deprived of total amino acids for 1hr followed by addition of amino acids for 10 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, ** p<0.01, two tailed Student's t test, n=3)


Figure 3 ( A ) Expression of RagC WT , 3A , or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * p < 0 . 05 , # no significant difference , One - way ANOVA with Bonferroni multiple comparisons , n = 4 ) ( B ) Phospho - RagC positively regulates wing compartment size in Drosophila . Over - expression ( OE ) of dRagC WT , 3A , or 3E in wing posterior compartment using en - Gal4 driver and the posterior compartment sizes were measured in each genotype ( ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , One - way ANOVA with Bonferroni multiple comparisons , n ≥ 63 flies per condition ) ( C ) Empty vector ( EV ) , RagC WT , 3A or 3E were transiently expressed in stable RagC knockdown HEK - 293E cells followed by cell size analysis using a particle size counter ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , # no significant difference , One - way ANOVA with Bonferroni multiple comparisons , n = 2 for C2 and n = 4 for other conditions ) ( D ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were expressed in HEK - 293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr . Lysates were analyzed by Western blot for phosphorylation of ULK1 and total ULK1 ( E ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were expressed in HEK - 293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr . Chloroquine ( 100μM ) was added to media 30 min prior to harvest . Lysates were analyzed by Western blot for levels of LC3 - II and 14 - 3 - 3 . Graphs show mean ± S . E . M . of quantitative analyses of LC3 - II blotting after normalization for 14 - 3 - 3 . * p < 0 . 05 , two tailed Student ' s t test , n = 4 ( F ) GFP - LC3 was co - expressed with empty vector , RagC WT , 3A or 3E in HEK - 293E cells followed 48 hr later by serum deprivation for 4 hr . Chloroquine ( 100μM ) was added to media 30 min prior to harvest . LC3 and RagC were monitored by confocal fluorescence microscopy . Scale bar , 10 µm ( G ) Quantitative analysis of GFP puncta ( F ) ( * * * * p < 0 . 0001 , two tailed Student ' s t test ; n ≥ 21 ) . Mean value is shown as a horizontal bar

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 (A) Expression of RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=4)  (B) Phospho-RagC positively regulates wing compartment size in Drosophila. Over-expression (OE) of dRagC WT, 3A, or 3E in wing posterior compartment using en-Gal4 driver and the posterior compartment sizes were measured in each genotype ((Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. ** p<0.01, *** p<0.001, **** p<0.0001, One-way ANOVA with Bonferroni multiple comparisons, n ≥ 63 flies per condition)  (C) Empty vector (EV), RagC WT, 3A or 3E were transiently expressed in stable RagC knockdown HEK-293E cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, ** p<0.01, **** p<0.0001, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=2 for C2 and n=4 for other conditions)  (D) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Lysates were analyzed by Western blot for phosphorylation of ULK1 and total ULK1 (E) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. Lysates were analyzed by Western blot for levels of LC3-II and 14-3-3.Graphs show mean ± S.E.M. of quantitative analyses of LC3-II blotting after normalization for 14-3-3. * p<0.05, two tailed Student's t test, n=4  (F) GFP-LC3 was co-expressed with empty vector, RagC WT, 3A or 3E in HEK-293E cells followed 48 hr later by serum deprivation for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. LC3 and RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm  (G) Quantitative analysis of GFP puncta (F) (**** p<0.0001, two tailed Student's t test; n ≥ 21). Mean value is shown as a horizontal bar


Figure 4 ( A ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were transiently expressed in HEK - 293E cells . RagC and lysosome maker , Lamp1 , were monitored by confocal fluorescence microscopy . Scale bar , 10 μm ( B ) Empty vector ( EV ) , HA - RagC WT or 3A were transiently expressed with scramble or RagC & RagD shRNA in HEK - 293E cells . mTOR and HA - RagC were monitored by confocal fluorescence microscopy . Scale bar , 10 µm ( C ) Empty vector ( EV ) , Flag - RagC WT , 3A or 3E were transiently expressed with HA - RagA in HEK - 293E cells . Total cell lysates and amounts of mTOR , Raptor and HA - RagA in the Flag - RagC immunoprecipitates were analyzed by Western blot . Graphs show mean ± S . E . M . of quantitative analyses of Western blots ( * p < 0 . 05 , two tailed Student ' s t test , n ≥ 3 ) ( D ) Expression of HA - RagC WT , 3A , or 3E was induced by overnight addition of tetracycline in HeLa cells . Total cell lysates ( TCL ) and amount of mTOR in the Raptor immunoprecipitates were analyzed by Western blot . Graphs show mean ± S . E . M . of quantitative analyses of Western blots ( * * * p < 0 . 001 , Student ' s t test , n = 3 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 (A) Empty vector (EV), HA-RagC WT, 3A or 3E were transiently expressed in HEK-293E cells. RagC and lysosome maker, Lamp1, were monitored by confocal fluorescence microscopy. Scale bar, 10 μm  (B) Empty vector (EV), HA-RagC WT or 3A were transiently expressed with scramble or RagC & RagD shRNA in HEK-293E cells. mTOR and HA-RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm  (C) Empty vector (EV), Flag-RagC WT, 3A or 3E were transiently expressed with HA-RagA in HEK-293E cells. Total cell lysates and amounts of mTOR, Raptor and HA-RagA in the Flag-RagC immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (* p<0.05, two tailed Student's t test, n ≥ 3)  (D) Expression of HA-RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells. Total cell lysates (TCL) and amount of mTOR in the Raptor immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (*** p<0.001, Student's t test, n=3)


Figure 1A , Rb1 protein ( RB ) was expressed in uterine luminal epithelium and stroma during early pregnancy in mice . Scale bar = 100μm ; arrowhead , embryo . B , Phosphorylated RB ( pRB ) protein , the inactive form of RB , was expressed in the luminal epithelium on days 2 and 3 of pregnancy . Scale bar = 100μm . C , RB was efficiently deleted in the uterus of mice with uterine deletion of Rb1 ( Rb1d / d mice ) on day 4 of pregnancy . Scale bar = 100μm . D , RB expression was normal in the ovary of Rb1d / d mice on day 4 of pregnancy . Scale bar = 100μm . E , The number of new - born pups was decreased in Rb1d / d dams ( n = 37 different dams ) compared to their littermate controls ( Rb1f / f mice , n = 20 different dams ) ( mean ± SEM , Student ' s t test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, Rb1 protein (RB) was expressed in uterine luminal epithelium and stroma during early pregnancy in mice. Scale bar = 100μm; arrowhead, embryo. B, Phosphorylated RB (pRB) protein, the inactive form of RB, was expressed in the luminal epithelium on days 2 and 3 of pregnancy. Scale bar = 100μm. C, RB was efficiently deleted in the uterus of mice with uterine deletion of Rb1 (Rb1d/d mice) on day 4 of pregnancy. Scale bar = 100μm. D, RB expression was normal in the ovary of Rb1d/d mice on day 4 of pregnancy. Scale bar = 100μm.E, The number of new-born pups was decreased in Rb1d/d dams (n=37 different dams) compared to their littermate controls (Rb1f/f mice, n=20 different dams) (mean ± SEM, Student's t test).


Figure 2A - C , Ovulation , fertilization and development of preimplantation embryos were normal in Rb1d / d mice ( A & C , mean ± SEM , Student ' s t test ; n = 5 mice for each group ; B , Fisher ' s exact test ; n = 5 mice for each group ) . Number of Ki67 - positive cells on day 4 in uterine epithelium of Rb1d / d mice was higher than that of Rb1f / f mice ( mean ± SEM , Student ' s t test ; n = 5 mice for each group ) . Scale bar , 100μm ; le , luminal epithelium ; s , stroma . Number of Ki67 - positive cells on day 4 in uterine epithelium of Rb1d / d mice was higher than that of Rb1f / f mice ( mean ± SEM , Student ' s t test ; n = 5 mice for each group ) . Scale bar , 100μm ; le , luminal epithelium ; s , stroma . F - G , Embryo attachment occurred normally in Rb1d / d mice at 0900h on day 5 ( mean ± SEM , Student ' s t test ; n = 5 mice for each group ) . Arrow , implantation site ; scale bar = 1 cm . H - I , Rb1d / d mice showed higher rate of embryo resorption on day 8 ( * P < 0 . 05 ; Fisher ' s exact test ; n = 45 implantation sites for Rb1f / f mice and n = 46 implantation sites for Rb1d / d mice ) . Arrow , implantation site ; scale bar = 1 cm . J , H & E staining showed embryo resorption in Rb1d / d mice on day 8 of pregnancy . Scale bar = 200 μm ; arrowhead , embryo ; arrow , broken embryo with blood cell infiltration . K , Weight of implantation site was comparable between Rb1f / f and Rb1d / d mice on day 6 , but reduced on day 8 in Rb1d / d mice compared to Rb1f / f mice ( mean ± SEM , Student ' s t test ; n = 6 mice for each group ) . L - M , Expression of Cox2 and Bmp2 , markers of decidualization , was comparable in between Rb1d / d and Rb1f / f mice at 0900h on day 6 in implantation sites ( mean ± SEM , Student ' s t test ; n = 4 mice for each group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-C, Ovulation, fertilization and development of preimplantation embryos were normal in Rb1d/d mice (A&C, mean ± SEM, Student's t test; n=5 mice for each group; B, Fisher's exact test; n=5 mice for each group).Number of Ki67-positive cells on day 4 in uterine epithelium of Rb1d/d mice was higher than that of Rb1f/f mice (mean ± SEM, Student's t test; n=5 mice for each group). Scale bar, 100μm; le, luminal epithelium; s, stroma.Number of Ki67-positive cells on day 4 in uterine epithelium of Rb1d/d mice was higher than that of Rb1f/f mice (mean ± SEM, Student's t test; n=5 mice for each group). Scale bar, 100μm; le, luminal epithelium; s, stroma.F-G, Embryo attachment occurred normally in Rb1d/d mice at 0900h on day 5 (mean ± SEM, Student's t test; n=5 mice for each group). Arrow, implantation site; scale bar = 1 cm.H-I, Rb1d/d mice showed higher rate of embryo resorption on day 8 (*P<0.05; Fisher's exact test; n=45 implantation sites for Rb1f/f mice and n=46 implantation sites for Rb1d/d mice). Arrow, implantation site; scale bar = 1 cm.J, H&E staining showed embryo resorption in Rb1d/d mice on day 8 of pregnancy. Scale bar = 200 μm; arrowhead, embryo; arrow, broken embryo with blood cell infiltration.K, Weight of implantation site was comparable between Rb1f/f and Rb1d/d mice on day 6, but reduced on day 8 in Rb1d/d mice compared to Rb1f/f mice (mean ± SEM, Student's t test; n=6 mice for each group).L-M, Expression of Cox2 and Bmp2, markers of decidualization, was comparable in between Rb1d/d and Rb1f/f mice at 0900h on day 6 in implantation sites (mean ± SEM, Student's t test; n=4 mice for each group).