Figure 4 ( A ) Monocytes were cultured with M - CSF , IL - 4 and TNFα for 5 days . Protein quantification by Western Blot . Actin was used as loading control . Representative results are shown ( n = 6 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor . Monocytes were cultured with M - CSF for 5 days . IRF1 expression was silenced using a lentivirus containing shRNA . ( B ) Protein quantification by Western Blot after 5 days . Actin was used as loading control . Representative results are shown ( n = 8 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor . Monocytes were cultured with RPMI medium ( R10 ) , or combinations of M - CSF , IL - 4 and TNFα in the presence or absence of B18R ( type I interferon inhibitor ) . ( G ) ISG expression after 9 hours was analyzed by RTqPCR . Each symbol represents an individual donor ( n = 5 biological replicates ) . Monocytes were cultured with combinations of M - CSF , IL - 4 and TNFα in the presence or absence of B18R ( type I interferon inhibitor ) . ( H ) Monocyte differentiation after 5 days . One representative donor is shown ( n = 5 biological replicates ) . Proportion of mo - Mac and mo - DC after 5 days . Each symbol represents an individual donor ( n = 5 biological replicates ) . Paired t - 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 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 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

Figure 4(A) Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. Protein quantification by Western Blot. Actin was used as loading control. Representative results are shown (n=6 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor.Monocytes were cultured with M-CSF for 5 days. IRF1 expression was silenced using a lentivirus containing shRNA. (B) Protein quantification by Western Blot after 5 days. Actin was used as loading control. Representative results are shown (n=8 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor.Monocytes were cultured with RPMI medium (R10), or combinations of M-CSF, IL-4 and TNFα in the presence or absence of B18R (type I interferon inhibitor). (G) ISG expression after 9 hours was analyzed by RTqPCR. Each symbol represents an individual donor (n=5 biological replicates).Monocytes were cultured with combinations of M-CSF, IL-4 and TNFα in the presence or absence of B18R (type I interferon inhibitor). (H) Monocyte differentiation after 5 days. One representative donor is shown (n=5 biological replicates). Proportion of mo-Mac and mo-DC after 5 days. Each symbol represents an individual donor (n=5 biological replicates). Paired t-test.


Figure 5 ( B ) ZNF366 expression across clusters . Monocytes were cultured with M - CSF , IL - 4 and TNFα for 5 days . ( C ) Protein quantification by Western Blot . Actin was used as loading control . Representative results are shown ( n = 6 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor . Paired one - way Anova . Monocytes were cultured with M - CSF , IL - 4 and TNFα for 5 days . ZNF366 expression was silenced using a lentivirus containing shRNA . ( D ) Protein quantification by Western Blot after 5 days . Actin was used as loading control . Representative results are shown ( n = 4 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor . Paired one - way Anova . Monocytes were cultured with M - CSF , IL - 4 and TNFα for 5 days . ( G ) Protein quantification by Western Blot . Actin was used as loading control . Representative results are shown ( n = 6 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor . Paired one - way Anova . MAFF expression was silenced using a lentivirus containing shRNA . ( H ) Protein quantification by Western Blot after 5 days . Actin was used as loading control . Representative results are shown ( n = 4 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor . Paired one - way Anova . MAFF expression was silenced using a lentivirus containing shRNA . ( I ) Mo - mac and mo - DC differentiation after 5 days was assessed by flow cytometry . One representative donor is shown ( n = 7 biological replicates ) . DN = double negative . Proportion of mo - DC , mo - Mac and DN cells after 5 days . Median is shown ( n = 7 biological replicates ) . Paired one - way Anova .

O O B-PANEL_START 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 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 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 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

Figure 5(B) ZNF366 expression across clusters.Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. (C) Protein quantification by Western Blot. Actin was used as loading control. Representative results are shown (n=6 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. ZNF366 expression was silenced using a lentivirus containing shRNA. (D) Protein quantification by Western Blot after 5 days. Actin was used as loading control. Representative results are shown (n=4 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. (G) Protein quantification by Western Blot. Actin was used as loading control. Representative results are shown (n=6 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.MAFF expression was silenced using a lentivirus containing shRNA. (H) Protein quantification by Western Blot after 5 days. Actin was used as loading control. Representative results are shown (n=4 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.MAFF expression was silenced using a lentivirus containing shRNA. (I) Mo-mac and mo-DC differentiation after 5 days was assessed by flow cytometry. One representative donor is shown (n=7 biological replicates). DN = double negative. Proportion of mo-DC, mo-Mac and DN cells after 5 days. Median is shown (n=7 biological replicates). Paired one-way Anova.


Figure 1 ( A ) BECN1 expression of microglia from Becn1 + / - and wild type mouse pups in full medium without Bafilomycin A1 ( Baf ) was quantified by Western blotting . Microglia from Becn1 + / - mice show a signifcant reduction of BECN1 ( 48 % ) compared to microglia from wild type mice ; mean ± SEM , wild type n = 3 , Becn1 + / - n = 7 ; two - tailed t - test * * : p < 0 . 01 . Blot shows representative amounts of BECN1 after various treatments . ( B ) Microglia from newborn Becn1 + / - and wild type mouse pups were kept for 2 h either in full medium or HBSS with or without Bafilomycin A1 ( Baf ) . LC3 and ACTIN levels were determined by Western blot . LC3 - II was significantly reduced in microglia from Becn1 + / - mice after Baf treatment in full medium compared to wild type microglia ; mean ± SEM , wild type n = 3 , Becn1 + / - n = 4 ; two - tailed t - test * : p < 0 . 05 . ( C ) Microglia from newborn Becn1 + / - and wild type mouse pups were kept for 2 h either in full medium or HBSS with Bafilomycin A1 ( Baf ) and stained for endogenous LC3 . Exemplary images and the quantification of LC3 - positive vesicles / µm2 cell area show reduced presence of LC3 - positive vesicles in microglia from Becn1 + / - and induction of autophagy by HBSS ; mean ± SEM , wild type n = 4 , Becn1 + / - n = 4 , 3 - 5 fields per view / n and condition and 73 - 271 cells / n and condition ; two - tailed t - test * : p < 0 . 05 ; scale bar : 20

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

Figure 1(A) BECN1 expression of microglia from Becn1+/- and wild type mouse pups in full medium without Bafilomycin A1 (Baf) was quantified by Western blotting. Microglia from Becn1+/- mice show a signifcant reduction of BECN1 (48%) compared to microglia from wild type mice; mean ± SEM, wild type n = 3, Becn1+/- n = 7; two-tailed t-test **: p < 0.01. Blot shows representative amounts of BECN1 after various treatments.(B) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with or without Bafilomycin A1 (Baf). LC3 and ACTIN levels were determined by Western blot. LC3-II was significantly reduced in microglia from Becn1+/- mice after Baf treatment in full medium compared to wild type microglia; mean ± SEM, wild type n = 3, Becn1+/- n = 4; two-tailed t-test *: p < 0.05.(C) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with Bafilomycin A1 (Baf) and stained for endogenous LC3. Exemplary images and the quantification of LC3-positive vesicles/µm2 cell area show reduced presence of LC3-positive vesicles in microglia from Becn1+/- and induction of autophagy by HBSS; mean ± SEM, wild type n = 4, Becn1+/- n = 4, 3-5 fields per view/n and condition and 73-271 cells/n and condition; two-tailed t-test *: p < 0.05; scale bar: 20 µm


Figure 2 ( A , B ) Microglia isolated from newborn Becn1 + / - and wild type mouse pups were either non treated or treated with a pro - inflammatory stimulus ( LPS followed by ATP ) . As additional controls cells treated only with LPS or ATP were used in ( A ) . The concentration of the pro - inflammatory cytokines IL - 1beta ( A ) and IL - 18 ( B ) in the cell supernatant was determined by ELISA . Reduction of BECN1 significantly enhanced release of both cytokines after stimulation with LPS / ATP ; mean ± SEM , wild type n ( A / B ) = 9 / 2 , wild type LPS n = 3 / 0 , wild type ATP n = 3 / 0 , wild type LPS / ATP n = 9 / 9 , Becn1 + / - n = 23 / 5 , Becn1 + / - LPS / ATP n = 23 / 23 , two - tailed t - test * : p < 0 . 05 . ( C ) Microglia isolated from wild type mouse pups were pre - incubated in full medium or starvation medium HBSS before addition of a pro - inflammatory stimulus ( LPS followed by ATP ) . The autophagy blocker 3 - MA was added for the final 1 h 45 min of the experiment . The concentration of the pro - inflammatory cytokine IL - 1beta in the cell supernatant was determined by ELISA . Incubation with HBSS reduced IL - 1beta release significantly while 3 - MA treatment increased it ; mean ± SEM , full medium n = 6 , full medium LPS / ATP n = 6 , full medium LPS / ATP + 3MA n = 3 , HBSS n = 6 , HBSS LPS / ATP n = 6 , HBSS LPS / ATP + 3MA n = 6 ; two - tailed t - test * : p < 0 . 05 ; * * : p < 0 . 01 . ( D ) Microglia isolated from wild type mouse pups were pre - incubated in starvation medium HBSS before addition of a pro - inflammatory stimulus ( LPS followed by ATP ) . The autophagy blocker 3 - MA was added for the final 1 h 45 min of the experiment , microglia were stained for LC3 and images taken by confocal microscopy . Representative images show a reduction of LC3 - positive vesicles in 3 - MA treated cells . Scale bar : 20 µm . ( E ) Proteins were extracted from brains of wild type , Becn1 + / - , APPPS1 and APPPS1 Becn1 + / - mice at the indicated ages . IL - 1beta was measured by electrochemiluminescence ( MesoScale ) and compared within the repective age groups . IL - 1beta was significantly increased in the TBS and TX fraction ; mean ± SEM , wild type : n ( per age group ) = 2 / 6 / 3 , Becn1 + / - : n = 2 / 5 / 2 , APPPS1 : n = 5 / 5 / 6 , APPPS1 Becn1 + / - : n = 7 / 9 / 6 ; ANOVA with Dunnett ´ s post hoc test with WT as control group ; * : p < 0 . 05 ; * * : p < 0 . 01 . ( F ) Proteins were extracted from brains of APPPS1 and of APPPS1 Becn1 + / - mice at the age as indicated . Amyloid beta 1 - 40 and 1 - 42 were measured by electrochemiluminescence ( MesoScale ) and compared within the repective age groups . No difference in the amyloid beta load between the genotypes is apparent ; mean ± SEM , APPPS1 : n = 5 / 5 / 6 , APPPS1 Becn1 + / - : n = 7 / 9 / 6 ; ANOVA with Dunnett ´ s post hoc test with APPPS1 as control group , ns .

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 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 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 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

Figure 2(A, B) Microglia isolated from newborn Becn1+/- and wild type mouse pups were either non treated or treated with a pro-inflammatory stimulus (LPS followed by ATP). As additional controls cells treated only with LPS or ATP were used in (A). The concentration of the pro-inflammatory cytokines IL-1beta (A) and IL-18 (B) in the cell supernatant was determined by ELISA. Reduction of BECN1 significantly enhanced release of both cytokines after stimulation with LPS/ATP; mean ± SEM, wild type n (A/B) = 9/2, wild type LPS n = 3/0, wild type ATP n = 3/0, wild type LPS/ATP n = 9/9, Becn1+/- n = 23/5, Becn1+/- LPS/ATP n = 23/23, two-tailed t-test *: p < 0.05.(C) Microglia isolated from wild type mouse pups were pre-incubated in full medium or starvation medium HBSS before addition of a pro-inflammatory stimulus (LPS followed by ATP). The autophagy blocker 3-MA was added for the final 1 h 45 min of the experiment. The concentration of the pro-inflammatory cytokine IL-1beta in the cell supernatant was determined by ELISA. Incubation with HBSS reduced IL-1beta release significantly while 3-MA treatment increased it; mean ± SEM, full medium n = 6, full medium LPS/ATP n = 6, full medium LPS/ATP + 3MA n = 3, HBSS n = 6, HBSS LPS/ATP n = 6, HBSS LPS/ATP + 3MA n = 6; two-tailed t-test *: p < 0.05; **: p < 0.01.(D) Microglia isolated from wild type mouse pups were pre-incubated in starvation medium HBSS before addition of a pro-inflammatory stimulus (LPS followed by ATP). The autophagy blocker 3-MA was added for the final 1 h 45 min of the experiment, microglia were stained for LC3 and images taken by confocal microscopy. Representative images show a reduction of LC3-positive vesicles in 3-MA treated cells. Scale bar: 20 µm.(E) Proteins were extracted from brains of wild type, Becn1+/-, APPPS1 and APPPS1 Becn1+/- mice at the indicated ages. IL-1beta was measured by electrochemiluminescence (MesoScale) and compared within the repective age groups. IL-1beta was significantly increased in the TBS and TX fraction; mean ± SEM, wild type: n (per age group) = 2/6/3, Becn1+/-: n = 2/5/2, APPPS1: n = 5/5/6, APPPS1 Becn1+/-: n = 7/9/6; ANOVA with Dunnett´s post hoc test with WT as control group; *: p < 0.05; **: p < 0.01.(F) Proteins were extracted from brains of APPPS1 and of APPPS1 Becn1+/- mice at the age as indicated. Amyloid beta 1-40 and 1-42 were measured by electrochemiluminescence (MesoScale) and compared within the repective age groups. No difference in the amyloid beta load between the genotypes is apparent; mean ± SEM, APPPS1: n = 5/5/6, APPPS1 Becn1+/-: n = 7/9/6; ANOVA with Dunnett´s post hoc test with APPPS1 as control group, ns.


Figure 3 ( A ) The expression of NLRP3 protein in non treated and LPS / ATP treated wild type and Becn1 + / - microglia was determined by Western blot and normalised to ACTIN . Reduction of BECN1 significantly enhanced expression of NLRP3 ; mean ± SEM , wild type n = 4 , wild type LPS / ATP n = 9 , Becn1 + / - n = 4 , Becn1 + / - LPS / ATP n = 23 ; ANOVA with Tukey ´ s post hoc test * : p < 0 . 05 . ( B ) The expression of Nlrp3 mRNA in non treated and LPS / ATP treated wild type and Becn1 + / - microglia was determined by qPCR . No significant differences can be detected between wild type and Becn1 + / - microglia ; mean ± SEM , wild type : n = 7 , Becn1 + / - : n = 3 ; ANOVA with Tukey ´ s post hoc test , ns for LPS / ATP treated wild type versus Becn1 + / - . ( C ) LPS / ATP treated and non treated wild type and Becn1 + / - microglia were immunolabeled for ASC ( red ) and imaged by confocal microscopy to investigate the assembly of inflammasomes . In non stimulated cells ASC is distributed diffusely in the cytoplasm ( upper panels ) . However , after stimulation formation of inflammasomes ( arrowheads ) can be detected in a subpopulation of cells as well as release of inflammasomes after pyroptosis / ejection ( arrow ) . Quantification showed a significantly increased percentage of inflammasomes in or around Becn1 + / - microglia ; mean ± SEM , wild type LPS / ATP n = 4 , Becn1 + / - LPS / ATP n = 7 ; two - tailed t - test * : p < 0 . 05 ; scale bar : 50 µm , insert scale bar : 10 µm . ( D ) The presence of cleaved CASP1 ( p10 ) protein in the supernatant of LPS / ATP treated wild type and Becn1 + / - microglia was determined by Western blot and normalised to ACTIN . Reduction of BECN1 significantly enhanced release of CASP1 ; mean ± SEM , wild type LPS / ATP n = 8 , Becn1 + / - LPS / ATP n = 18 ; two - tailed t - test * : p < 0 . 05 .

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 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 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

Figure 3(A) The expression of NLRP3 protein in non treated and LPS/ATP treated wild type and Becn1+/- microglia was determined by Western blot and normalised to ACTIN. Reduction of BECN1 significantly enhanced expression of NLRP3; mean ± SEM, wild type n = 4, wild type LPS/ATP n = 9, Becn1+/- n = 4, Becn1+/- LPS/ATP n = 23; ANOVA with Tukey´s post hoc test *: p < 0.05.(B) The expression of Nlrp3 mRNA in non treated and LPS/ATP treated wild type and Becn1+/- microglia was determined by qPCR. No significant differences can be detected between wild type and Becn1+/- microglia; mean ± SEM, wild type: n = 7, Becn1+/- : n = 3; ANOVA with Tukey ´s post hoc test, ns for LPS/ATP treated wild type versus Becn1+/-.(C) LPS/ATP treated and non treated wild type and Becn1+/- microglia were immunolabeled for ASC (red) and imaged by confocal microscopy to investigate the assembly of inflammasomes. In non stimulated cells ASC is distributed diffusely in the cytoplasm (upper panels). However, after stimulation formation of inflammasomes (arrowheads) can be detected in a subpopulation of cells as well as release of inflammasomes after pyroptosis/ejection (arrow). Quantification showed a significantly increased percentage of inflammasomes in or around Becn1+/- microglia; mean ± SEM, wild type LPS/ATP n = 4, Becn1+/- LPS/ATP n = 7; two-tailed t-test *: p < 0.05; scale bar: 50 µm, insert scale bar: 10 µm.(D) The presence of cleaved CASP1 (p10) protein in the supernatant of LPS/ATP treated wild type and Becn1+/- microglia was determined by Western blot and normalised to ACTIN. Reduction of BECN1 significantly enhanced release of CASP1; mean ± SEM, wild type LPS/ATP n = 8, Becn1+/- LPS/ATP n = 18; two-tailed t-test *: p < 0.05.


Figure 4 ( A ) LPS / ATP treated and non treated wild type and Becn1 + / - microglia were immunolabeled for NLRP3 ( green ) and LC3 ( red ) and imaged by confocal microscopy . Stimulation resulted in the appearance of many NLRP3 - containing aggregates of different sizes ( arrows ) . In non treated cells , only a few overlapping signals with LC3 - stained autophagosomes ( arrowheads ) are visible . Stimulated microglia however , showed multiple colocalizations of LC3 positive vesicles and NLRP3 aggregates ; scale bar : 7 . 5 µm . ( B ) LPS / ATP treated wild type microglia were immunolabeled for NLRP3 ( green ) and LC3 ( red ) and analysed by super resolution microscopy ( SIM ) . NLRP3 aggregates of different sizes coclustering with LC3 - positive autophagosomes are clearly visible . 3D volume rendering of large ( I ) and small ( II ) NLRP3 aggregates from the magnified ROIs showed engulfment of NLRP3 by autophagosomes ; scale bar : 10 µm ; magnified ROIs : 1 µm . ( C ) 3D Radial intensity profiles of NLRP3 and LC3 signals derived from SIM images in wild type microglia , centered on the maxima of NLRP3 clusters . The radial profiles confirm that both proteins colocalize to the same organelle ; mean ± SEM , n = 4 .

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 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 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

Figure 4(A) LPS/ATP treated and non treated wild type and Becn1+/- microglia were immunolabeled for NLRP3 (green) and LC3 (red) and imaged by confocal microscopy. Stimulation resulted in the appearance of many NLRP3-containing aggregates of different sizes (arrows). In non treated cells, only a few overlapping signals with LC3-stained autophagosomes (arrowheads) are visible. Stimulated microglia however, showed multiple colocalizations of LC3 positive vesicles and NLRP3 aggregates; scale bar: 7.5 µm.(B) LPS/ATP treated wild type microglia were immunolabeled for NLRP3 (green) and LC3 (red) and analysed by super resolution microscopy (SIM). NLRP3 aggregates of different sizes coclustering with LC3-positive autophagosomes are clearly visible. 3D volume rendering of large (I) and small (II) NLRP3 aggregates from the magnified ROIs showed engulfment of NLRP3 by autophagosomes; scale bar: 10 µm; magnified ROIs: 1 µm.(C) 3D Radial intensity profiles of NLRP3 and LC3 signals derived from SIM images in wild type microglia, centered on the maxima of NLRP3 clusters. The radial profiles confirm that both proteins colocalize to the same organelle; mean ± SEM, n = 4.


Figure 5 ( A ) LPS / ATP treated wild type microglia were stained for NLRP3 ( green ) and CALCOCO2 ( red ) and analysed by confocal microscopy . Colocalizations of NLRP3 aggregates ( arrows ) and CALCOCO2 ( arrowheads ) are apparent ; scale bar : 10 µm . ( B ) LPS / ATP treated wild type microglia were stained for NLRP3 ( green ) and CALCOCO2 ( red ) and analysed by super resolution microscopy ( SIM ) . NLRP3 aggregates coclustering with CALCOCO2 - positive signals are visible . 3D volume rendering of NLRP3 aggregates from the magnified ROIs showed close contact of NLRP3 with the autophagic receptor CALCOCO2 ; scale bar : 10 µm ; magnified ROIs : 1 µm . ( C ) 3D Radial intensity profiles of NLRP3 and CALCOCO2 signals derived from SIM images in wild type microglia , centered on the maxima of NLRP3 clusters . The radial profiles confirm that both proteins colocalize ; mean ± SEM , n = 3 . ( D ) In SIM images from wild type microglia the sub - autophagosomal localization of NLRP3 was analysed in relation to LC3 and CALCOCO2 . LC3 and CALCOCO2 intensities from the center of mass of NLRP3 aggregates were normalised to their maximum within the cluster . While the center of NLRP3 aggregates shows near maximal CALCOCO2 intensity , the relative LC3 intensity there was significantly lower ; mean ± SEM , LC3 : n = 4 , CALCOCO2 : n = 3 ; two - tailed t - test * * : p < 0 . 01 . ( E ) Microglia from Becn1 + / - mice were transfected with scrambled siRNA or with siRNA for CALCOCO2 . 6 d after transfection cells were stimulated with LPS / ATP . The expression of CALCOCO2 protein was determined by Western blot . Application of CALCOCO2 siRNA resulted in significantly reduced expression of CALCOCO2 ; mean ± SEM , n = 3 ; two - tailed t - test * : p < 0 . 05 . ( F ) The amount of IL - 1beta in the supernatant released from the cells described in ( E ) was determined by ELISA . Microglia with a CALCOCO2 knockdown released significantly more IL - 1beta ; mean ± SEM , n = 3 ; two - tailed t - test * : p < 0 . 05 .

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 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 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 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 O O O O O

Figure 5(A) LPS/ATP treated wild type microglia were stained for NLRP3 (green) and CALCOCO2 (red) and analysed by confocal microscopy. Colocalizations of NLRP3 aggregates (arrows) and CALCOCO2 (arrowheads) are apparent; scale bar: 10 µm.(B) LPS/ATP treated wild type microglia were stained for NLRP3 (green) and CALCOCO2 (red) and analysed by super resolution microscopy (SIM). NLRP3 aggregates coclustering with CALCOCO2-positive signals are visible. 3D volume rendering of NLRP3 aggregates from the magnified ROIs showed close contact of NLRP3 with the autophagic receptor CALCOCO2; scale bar: 10 µm; magnified ROIs: 1 µm.(C) 3D Radial intensity profiles of NLRP3 and CALCOCO2 signals derived from SIM images in wild type microglia, centered on the maxima of NLRP3 clusters. The radial profiles confirm that both proteins colocalize; mean ± SEM, n = 3.(D) In SIM images from wild type microglia the sub-autophagosomal localization of NLRP3 was analysed in relation to LC3 and CALCOCO2. LC3 and CALCOCO2 intensities from the center of mass of NLRP3 aggregates were normalised to their maximum within the cluster. While the center of NLRP3 aggregates shows near maximal CALCOCO2 intensity, the relative LC3 intensity there was significantly lower; mean ± SEM, LC3: n = 4, CALCOCO2: n = 3; two-tailed t-test **: p < 0.01.(E) Microglia from Becn1+/- mice were transfected with scrambled siRNA or with siRNA for CALCOCO2. 6 d after transfection cells were stimulated with LPS/ATP. The expression of CALCOCO2 protein was determined by Western blot. Application of CALCOCO2 siRNA resulted in significantly reduced expression of CALCOCO2; mean ± SEM, n = 3; two-tailed t-test *: p < 0.05.(F) The amount of IL-1beta in the supernatant released from the cells described in (E) was determined by ELISA. Microglia with a CALCOCO2 knockdown released significantly more IL-1beta; mean ± SEM, n = 3; two-tailed t-test *: p < 0.05.


Figure 1B . Confocal microscopy images of HeLa , RPE , and U2OS cell lines that were pre - extracted with Triton X - 100 before fixed with formaldehyde and co - stained with DAPI and antibodies against PCNA and FAM111A . Scale bars : 10 μmC . U2OS cells were stained with antibodies against PML and FAM111A and imaged by confocal microscopy . Scale bars : 10 μm .

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 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

Figure 1B. Confocal microscopy images of HeLa, RPE, and U2OS cell lines that were pre-extracted with Triton X-100 before fixed with formaldehyde and co-stained with DAPI and antibodies against PCNA and FAM111A. Scale bars: 10 μmC. U2OS cells were stained with antibodies against PML and FAM111A and imaged by confocal microscopy. Scale bars: 10 μm.


Figure 2HEK293 , RPE , or U2OS / MycBioID - FAM111A cell lines were induced with doxycycline to express wild - type or mutant FAM111A for 24 h ( HEK293 ) , 48 h ( RPE ) , or 72h ( U2OS ) . The colonies were fixed then stained with crystal violet . Cell - cycle profiles of HEK293 , RPE and U2OS FAM111A - expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry . C . HEK293 cell lines were induced with doxycycline to express wild - type or mutant FAM111A for 24 h ( HEK293 ) Cell growth was quantitated as total area on ImageJ . Values are mean ± s . e . m . of independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( two - tailed unpaired t - test ) . D . Cell - cycle profiles of HEK293 FAM111A - expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry .

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 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 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

Figure 2HEK293, RPE, or U2OS/MycBioID-FAM111A cell lines were induced with doxycycline to express wild-type or mutant FAM111A for 24 h (HEK293), 48 h (RPE), or 72h (U2OS). The colonies were fixed then stained with crystal violet.Cell-cycle profiles of HEK293, RPE and U2OS FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry.C. HEK293 cell lines were induced with doxycycline to express wild-type or mutant FAM111A for 24 h (HEK293) Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3). *p<0.05, **p<0.01, ****p<0.0001 (two-tailed unpaired t-test).D. Cell-cycle profiles of HEK293 FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry.


Figure 3A . HEK293 or U2OS / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h . Total protein from 105 cells was resolved by SDS - PAGE , and probed in western blots for both endogenous FAM111A ( 71 kDa ) and MycBioID - FAM111A ( 110 kDa ) as well as full length and cleaved PARP ( 116 and 89 kDa , respectively ) . The Tubulin western blot is a sample loading control . B . HEK293 / MycBioID - FAM111A cell lines with no treatment ( - Dox ) , or treated with doxycycline , along with DMSO or 100 μM Z - VAD - FMK for 24 h ( total inhibition of PARP cleavage was also observed when 50 μM Z - VAD - FMK was used ) . Total protein from ~ 105 cells was resolved by SDS - PAGE , and probed in western blots with Myc , PARP , or Tubulin antibodies . C . HEK293 cell lines were treated with doxycycline and Z - VAD - FMK for 24 h . The colonies were fixed then stained with crystal violet . Cell growth was quantitated as total area on ImageJ . Values are mean ± s . e . m . of independent experiments ( n = 3 ) .

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 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 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

Figure 3A. HEK293 or U2OS/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from 105 cells was resolved by SDS-PAGE, and probed in western blots for both endogenous FAM111A (71 kDa) and MycBioID-FAM111A (110 kDa) as well as full length and cleaved PARP (116 and 89 kDa, respectively). The Tubulin western blot is a sample loading control.B. HEK293/MycBioID-FAM111A cell lines with no treatment (- Dox), or treated with doxycycline, along with DMSO or 100 μM Z-VAD-FMK for 24 h (total inhibition of PARP cleavage was also observed when 50 μM Z-VAD-FMK was used). Total protein from ~ 105 cells was resolved by SDS-PAGE, and probed in western blots with Myc, PARP, or Tubulin antibodies.C. HEK293 cell lines were treated with doxycycline and Z-VAD-FMK for 24 h. The colonies were fixed then stained with crystal violet. Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3).


Figure 4C . Cells were treated with doxycycline along with DMSO or Z - VAD - FMK for 24 h . They were fixed with formaldehyde , and stained with NPC antibody Mab414 and DAPI . The images were captured by confocal microscopy . Scale bar : 10 μm . D . DMSO or Z - VAD - FMK treated HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h . Total protein from an equal number of cells was incubated with magnetic streptavidin beads , and pull downs were analyzed by western blotting using GANP and Myc antibodies . E . HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h , while growing in the presence or absence of 50 mM supplemental biotin . Total protein from whole cell lysates was incubated with magnetic streptavidin beads . Pulldowns were analyzed by western blotting using GANP and Myc antibodies . F . DMSO or Z - VAD - FMK treated HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h . Total protein from ~ 105 cells was resolved by SDS - PAGE , and probed in western blots using antibodies against NPC ( Mab414 ) , PARP , cleaved Caspase 3 , Myc , or Tubulin .

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 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

Figure 4C. Cells were treated with doxycycline along with DMSO or Z-VAD-FMK for 24 h. They were fixed with formaldehyde, and stained with NPC antibody Mab414 and DAPI. The images were captured by confocal microscopy. Scale bar: 10 μm.D. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from an equal number of cells was incubated with magnetic streptavidin beads, and pull downs were analyzed by western blotting using GANP and Myc antibodies.E. HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h, while growing in the presence or absence of 50 mM supplemental biotin. Total protein from whole cell lysates was incubated with magnetic streptavidin beads. Pulldowns were analyzed by western blotting using GANP and Myc antibodies.F. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from ~ 105 cells was resolved by SDS-PAGE, and probed in western blots using antibodies against NPC (Mab414), PARP, cleaved Caspase 3, Myc, or Tubulin.


Figure 5A . Representative immunofluorescence images of SV40 LT localization 72 h after transfection into U2OS cells infected with control or FAM111A shRNA . Images were taken with a Zeiss Axio Imager . LT is shown in green , and DAPI blue . Scale bar : 100 μm . B . Quantification of LT positive cells described in A and fixed at indicated times post transfection . Five hundred or more cells were counted for each condition . C . Quantification of RFP marker distribution upon co - transfection with SV40 LT variants into U2OS cells infected with control or FAM111A shRNA . Cells were fixed at the indicated times . Five hundred or more cells were counted for each condition . In B & C , values are mean ± s . d . of independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( two - tailed unpaired t - test ) . D . Western blot analysis of control or FAM111A - depleted U2OS cells transfected with SV40 LT variants . Cells were collected and lysed at indicated timepoints after SV40 transfection , immunoblotted with FAM111A , LT , or PARP antibodies . Cdc2 - PSTAIR was used as a loading control .

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 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

Figure 5A. Representative immunofluorescence images of SV40 LT localization 72 h after transfection into U2OS cells infected with control or FAM111A shRNA. Images were taken with a Zeiss Axio Imager. LT is shown in green, and DAPI blue. Scale bar: 100 μm. B. Quantification of LT positive cells described in A and fixed at indicated times post transfection. Five hundred or more cells were counted for each condition. C. Quantification of RFP marker distribution upon co-transfection with SV40 LT variants into U2OS cells infected with control or FAM111A shRNA. Cells were fixed at the indicated times. Five hundred or more cells were counted for each condition. In B & C, values are mean ± s.d. of independent experiments (n = 3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed unpaired t-test). D. Western blot analysis of control or FAM111A-depleted U2OS cells transfected with SV40 LT variants. Cells were collected and lysed at indicated timepoints after SV40 transfection, immunoblotted with FAM111A, LT, or PARP antibodies. Cdc2-PSTAIR was used as a loading control.


Figure 1A Heatmap depiction of the 3 , 731 DNA hypomethylation loci in ago4 ‐ 6 . Each hypomethylated region corresponds to a colored horizontal bar , and the bars are clustered numerically into a column ( y ‐ axis ) . Cytosines were examined as CG , CHG , and CHH . The color ‐ scaled methylation levels indicate the ratios of each type of methylated cytosines over total cytosines of the same type within the examined hypomethylated regions . B Heatmap depiction of the 3 , 678 DNA hypomethylation loci in ago6 ‐ 2 .

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

Figure 1A Heatmap depiction of the 3,731 DNA hypomethylation loci in ago4‐6. Each hypomethylated region corresponds to a colored horizontal bar, and the bars are clustered numerically into a column (y‐axis). Cytosines were examined as CG, CHG, and CHH. The color‐scaled methylation levels indicate the ratios of each type of methylated cytosines over total cytosines of the same type within the examined hypomethylated regions.B Heatmap depiction of the 3,678 DNA hypomethylation loci in ago6‐2.


Figure 2B Box plots showing methylation patterns of different groups of loci as categorized in ( A ) .

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

Figure 2B Box plots showing methylation patterns of different groups of loci as categorized in (A).


Figure 3B DNA methylation levels in different mutants . The 2 , 174 loci where AGO4 and AGO6 are mutually dependent are numerically clustered ( y ‐ axis ) . Cytosines were examined as CG , CHG , and CHH . C DNA methylation levels at TAS1a , TAS1c , and TAS3a loci . Snapshots from whole ‐ genome bisulfite sequencing results are shown . D RT - qPCR measurements of transposon RNA levels . Double : ago4 ‐ 6 ago6 ‐ 2 double mutant . Actin 2 was used as an internal control . RNA levels in the mutants are relative to those in the wild ‐ type ( Col ‐ 0 ) . Means ± SD are shown , n = 3 .

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 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 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

Figure 3B DNA methylation levels in different mutants. The 2,174 loci where AGO4 and AGO6 are mutually dependent are numerically clustered (y‐axis). Cytosines were examined as CG, CHG, and CHH.C DNA methylation levels at TAS1a, TAS1c, and TAS3a loci. Snapshots from whole‐genome bisulfite sequencing results are shown.D RT-qPCR measurements of transposon RNA levels. Double: ago4‐6 ago6‐2 double mutant. Actin 2 was used as an internal control. RNA levels in the mutants are relative to those in the wild‐type (Col‐0). Means ± SD are shown, n = 3.


Figure 4A AGO4 and AGO6 were visualized by immunofluorescence microscopy using their specific antibodies . Yellow signals would be expected in the merged images if the two proteins co ‐ localize , as a result of the overlap of red and green channels . DNA ( blue ) was stained with DAPI . B The largest subunit of Pol IV , NRPD1 ( red ) , was visualized by its specific antibody in cells expressing FLAG ‐ tagged AGO4 or AGO6 ( green ) .

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

Figure 4A AGO4 and AGO6 were visualized by immunofluorescence microscopy using their specific antibodies. Yellow signals would be expected in the merged images if the two proteins co‐localize, as a result of the overlap of red and green channels. DNA (blue) was stained with DAPI.B The largest subunit of Pol IV, NRPD1 (red), was visualized by its specific antibody in cells expressing FLAG‐tagged AGO4 or AGO6 (green).


Figure 5A The largest subunit of Pol V , NRPE1 ( red ) , was visualized by immunofluorescence microscopy using its specific antibody in cells expressing FLAG ‐ tagged AGO4 ( green ) . AGO4 ‐ NRPE1 co ‐ localized to nucleolar dot ( yellow dot , marked with white arrow , upper panel ) but not in nucleoplasmic foci ( lower panel ) . B NRPE1 ( red ) was visualized by its specific antibody in cells expressing FLAG ‐ tagged AGO6 ( green ) . The yellow signals due to the overlap of red and green channels in merged images indicate protein co ‐ localization . DNA ( blue ) was stained with DAPI . AGO6 ‐ NRPE1 co ‐ localized to nucleoplasmic foci ( yellow dots , marked with white arrow ) .

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

Figure 5A The largest subunit of Pol V, NRPE1 (red), was visualized by immunofluorescence microscopy using its specific antibody in cells expressing FLAG‐tagged AGO4 (green). AGO4‐NRPE1 co‐localized to nucleolar dot (yellow dot, marked with white arrow, upper panel) but not in nucleoplasmic foci (lower panel).B NRPE1 (red) was visualized by its specific antibody in cells expressing FLAG‐tagged AGO6 (green). The yellow signals due to the overlap of red and green channels in merged images indicate protein co‐localization. DNA (blue) was stained with DAPI. AGO6‐NRPE1 co‐localized to nucleoplasmic foci (yellow dots, marked with white arrow).


Figure 6A AGO6 dysfunction reduces levels of Pol V ‐ dependent transcripts . RNA levels were measured by RT - qPCR . Means ± SD are shown , n = 3 . B AGO6 dysfunction decreases Pol V occupancy at chromatin of loci where Pol V transcript levels are affected by AGO6 . Anti ‐ NRPE1 antibody was used for ChIP assays . Means ± SD are shown , n = 3 .

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 O O O

Figure 6A AGO6 dysfunction reduces levels of Pol V‐dependent transcripts. RNA levels were measured by RT-qPCR. Means ± SD are shown, n = 3.B AGO6 dysfunction decreases Pol V occupancy at chromatin of loci where Pol V transcript levels are affected by AGO6. Anti‐NRPE1 antibody was used for ChIP assays. Means ± SD are shown, n = 3.


Figure 7A Heatmap depiction of the dependence of AGO4 ‐ and AGO6 ‐ bound siRNAs on Pol IV . Identities of siRNAs were retrieved from Havecker et al ( ) , and siRNAs were grouped into 1 , 210 AGO4 ‐ bound clusters and 1 , 486 AGO6 ‐ bound clusters ( see Materials and Methods for details ) , each of which corresponds to a colored horizontal bar ; the bars are stacked numerically into a column ( y ‐ axis ) . Information on siRNA levels in Pol IV mutant and wild ‐ type plants was retrieved from Zhang et al ( ) . B Heatmap depiction of the dependence of AGO4 ‐ and AGO6 ‐ bound siRNAs on Pol V . C Quantification of individual 24 ‐ nt siRNAs by TaqMan small RNA assays . Double : ago4 ‐ 6 ago6 ‐ 2 double mutant . snoR101 was used as an internal control . RNA levels in the mutants were relative to those of the wild ‐ type ( Col ‐ 0 ) . Error bars indicate SD , n ≥ 3 .

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 B-PANEL_START 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

Figure 7A Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol IV. Identities of siRNAs were retrieved from Havecker et al (), and siRNAs were grouped into 1,210 AGO4‐bound clusters and 1,486 AGO6‐bound clusters (see Materials and Methods for details), each of which corresponds to a colored horizontal bar; the bars are stacked numerically into a column (y‐axis). Information on siRNA levels in Pol IV mutant and wild‐type plants was retrieved from Zhang et al ().B Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol V.C Quantification of individual 24‐nt siRNAs by TaqMan small RNA assays. Double: ago4‐6 ago6‐2 double mutant. snoR101 was used as an internal control. RNA levels in the mutants were relative to those of the wild‐type (Col‐0). Error bars indicate SD, n ≥ 3.


Figure 1 . C - J Bar Chart with data points showing the abundance of indicated bacterial families in the indicated group of mice . Data are expressed as CSS - normalized bacterial

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 1. C-J Bar Chart with data points showing the abundance of indicated bacterial families in the indicated group of mice. Data are expressed as CSS-normalized bacterial counts


Figure 2A - C Bar chart with individual points showing the levels of propionate acetate and butyrate in wt and mdx mice receiving DFZ or vehicle , measured by NMR or GC - MS . Data are expressed as μg / ml or bin intensity ( arbitrary unit - a . u . ) D - F Bar chart with individual points showing the levels of pyruvate , succinate and lactate in wt and mdx mice receiving vehicle or DFZ , measured by NMR .

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

Figure 2A-C Bar chart with individual points showing the levels of propionate acetate and butyrate in wt and mdx mice receiving DFZ or vehicle, measured by NMR or GC-MS. Data are expressed as μg/ml or bin intensity (arbitrary unit - a.u.) D-F Bar chart with individual points showing the levels of pyruvate, succinate and lactate in wt and mdx mice receiving vehicle or DFZ, measured by NMR. G


Figure 3A - B Muscle coordination and strength were measured in 19 weeks old control and mdx mice treated with vehicle ( DMSO ) , NaB ( 100 mg kg − 1 / daily ) or DFZ ( 1 . 2 mg kg − 1 / daily ) for 3 weeks using the rotarod and weight test . Bar charts show the latency to fall or drop the weight of wt and dystrophic mice .

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

Figure 3A-B Muscle coordination and strength were measured in 19 weeks old control and mdx mice treated with vehicle (DMSO), NaB (100 mg kg−1/daily) or DFZ (1.2 mg kg−1/daily) for 3 weeks using the rotarod and weight test. Bar charts show the latency to fall or drop the weight of wt and dystrophic mice.


Figure 4A - I Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in the gastrocnemius of control and mdx mice treated with or without NaB and DFZ . J - K Representative blotting and bar chart with individual points showing the expression and / or phosphorylation of pAKT / AKT and COX2 in the gastrocnemius of the indicated six groups of mice .

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 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 4A-I Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in the gastrocnemius of control and mdx mice treated with or without NaB and DFZ.J-K Representative blotting and bar chart with individual points showing the expression and/or phosphorylation of pAKT/AKT and COX2 in the gastrocnemius of the indicated six groups of mice.


Figure 5A - B Levels of AEA and 2 - AG in plasma samples of wt and mdx mice expressed as pmol mg − 1 of wet tissue weight . C - D Bar charts with individual points showing the mRNA expression levels of CB1 and CB2 measured in the gastrocnemius of the indicated groups of mice . E Representative blots showing the expression levels of CB1 and CB2 proteins in the gastrocnemius of the indicated groups of mice . F Quantification of CB1 and CB2 proteins to the housekeeping protein GAPDH .

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 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 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 5A-B Levels of AEA and 2-AG in plasma samples of wt and mdx mice expressed as pmol mg−1 of wet tissue weight.C-D Bar charts with individual points showing the mRNA expression levels of CB1 and CB2 measured in the gastrocnemius of the indicated groups of mice.E Representative blots showing the expression levels of CB1 and CB2 proteins in the gastrocnemius of the indicated groups of mice. F Quantification of CB1 and CB2 proteins to the housekeeping protein GAPDH. D


Figure 6A - C Bar charts with individual points showing the mRNA expression levels of Ulk , Pink and Becn1 measured in control and mdx mice treated with rimonabant ( 0 . 5 mg kg - 1 ) . D Representative blots showing the expression levels of LC3I and LC3II proteins in the gastrocnemius of the indicated groups of mice . E , F Quantification of LC3I and LC3II proteins to the housekeeping protein GAPDH .

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

Figure 6A-C Bar charts with individual points showing the mRNA expression levels of Ulk, Pink and Becn1 measured in control and mdx mice treated with rimonabant (0.5 mg kg-1).D Representative blots showing the expression levels of LC3I and LC3II proteins in the gastrocnemius of the indicated groups of mice. E, F Quantification of LC3I and LC3II proteins to the housekeeping protein GAPDH. D


Figure 7A - B Bar chart with individual points showing the mRNA expression levels of Il6 and Cox2 in control ( vehicle , DMSO ) and / or GPR109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in the presence or absence of either MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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

Figure 7A-B Bar chart with individual points showing the mRNA expression levels of Il6 and Cox2 in control (vehicle, DMSO) and/or GPR109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either MK1903 (1 μM), rosiglitazone (1 μM) or T007 (1 μM).


Figure 8A - F Bar chart with individual points showing the mRNA expression levels of Cb1 , Daglα , Daglβ , Magl , Napepld , and Faah in control ( vehicle , DMSO ) and / or GPR109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in the presence or absence of either MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) or T007 ( 1 μM ) . G - H Levels of AEA and 2 - AG were measured in C2C12 cells exposed to LPS ( 1 μg / ml ) or NaB ( 3 mM ) for 24 h . I Effect of ACEA ( 1 μM ) and rimonabant ( 1 μM ) on autophagosome formation measured in C2C12 cells .

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 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

Figure 8A-F Bar chart with individual points showing the mRNA expression levels of Cb1, Daglα, Daglβ, Magl, Napepld, and Faah in control (vehicle, DMSO) and/or GPR109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either MK1903 (1 μM), rosiglitazone (1 μM) or T007 (1 μM).G-H Levels of AEA and 2-AG were measured in C2C12 cells exposed to LPS (1 μg/ml) or NaB (3 mM) for 24 h. I Effect of ACEA (1 μM) and rimonabant (1 μM) on autophagosome formation measured in C2C12 cells. D


Figure 9C - J Bar chart with individual points showing the expression of selected miRNAs in control and Gpr109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in the presence or absence of either NaB ( 3 mM ) , MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) . NaB was also tested in the presence or absence of either rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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

Figure 9C-J Bar chart with individual points showing the expression of selected miRNAs in control and Gpr109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either NaB (3 mM), MK1903 (1 μM), rosiglitazone (1 μM). NaB was also tested in the presence or absence of either rosiglitazone (1 μM) or T007 (1 μM).


Figure 10A - E Bar chart showing the mRNA expression levels of IL6 , COX2 , ULK 1 , ATG13 and ATG4 mRNA in primary human myoblasts isolated from one healthy donor ( HD ) and five DMD donors ( D1 - D5 ) .

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 10A-E Bar chart showing the mRNA expression levels of IL6, COX2, ULK 1, ATG13 and ATG4 mRNA in primary human myoblasts isolated from one healthy donor (HD) and five DMD donors (D1- D5).


Figure 1 ( C ) Streptavidin Western blotting of total biotinylated proteins in APEX2 : RAB or APEX2 only Flp - In / T - REx HeLa cells . ( D ) APEX2 : RAB biotinylated proteins ( streptavidin ) are partially colocalized with EEA1 in HeLa cells , n = 2 independent experiments . Scale bars : 10µm or 5µm in enlarged views .

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 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

Figure 1(C) Streptavidin Western blotting of total biotinylated proteins in APEX2:RAB or APEX2 only Flp-In/T-REx HeLa cells.(D) APEX2:RAB biotinylated proteins (streptavidin) are partially colocalized with EEA1 in HeLa cells, n=2 independent experiments. Scale bars: 10µm or 5µm in enlarged views.


Figure 2 ( C ) ProHits - viz generated dotblots of RAB effectors , GEFs , GAPs and sorting complexes . Green , blue and beige shadings along with RAB names refer to previously identified interactions between RABs and the depicted proteins .

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

Figure 2(C) ProHits-viz generated dotblots of RAB effectors, GEFs, GAPs and sorting complexes. Green, blue and beige shadings along with RAB names refer to previously identified interactions between RABs and the depicted proteins.


Figure 3Strumpellin and VPS35 are in close proximity to RAB5 and RAB21 . ( A ) Anti - biotin immunoprecipitation and immunoblot of endogenous Strumpellin and VPS35 . Lysates correspond to 1 % of input , n = 3 independent experiments . RAB21 can be seen interacting with various WASH and retromer complexes subunits . GFP trap IP of WT , Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and endogenous ( B ) VPS26 immunoblot Lysates correspond to 5 % of input , n ≥ 3 independent experiments . GFP trap IP of WT , Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and ( C ) FAM21 , Strumpellin and VPS35 immunoblots . Lysates correspond to 5 % of input , n ≥ 3 independent experiments . RAB21 actively pull - downs WASH and retromer complex subunits ( D ) Bacterially - purified and GTP - loaded GST : RAB21 pull - down of HeLa cell lysates followed by Strumpellin , FAM21 , CAPZα and VPS35 immunoblots . Ponceau staining reveals the purity of the GST and GST : RAB21 used for the pull - downs . n = 3 independent experiments . ( E ) RAB21 colocalizes with the WASH and retromer complexes . Transiently expressing GFP : RAB21 HeLa cells were fixed and stained for endogenous EEA1 , FAM21 , WASH and VPS26 . Boxed region is magnified and single channels are depicted . Scale bars : 10µm or 5µm in enlarged view . ( F ) Per cell Pearson Correlation between RAB21 and the various markers . Error bars are SEM , n = 2 independent experiments .

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

Figure 3Strumpellin and VPS35 are in close proximity to RAB5 and RAB21. (A) Anti-biotin immunoprecipitation and immunoblot of endogenous Strumpellin and VPS35. Lysates correspond to 1% of input, n=3 independent experiments. RAB21 can be seen interacting with various WASH and retromer complexes subunits.GFP trap IP of WT, Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and endogenous (B) VPS26 immunoblot Lysates correspond to 5% of input, n≥ 3 independent experiments.GFP trap IP of WT, Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and (C) FAM21, Strumpellin and VPS35 immunoblots. Lysates correspond to 5% of input, n≥ 3 independent experiments.RAB21 actively pull-downs WASH and retromer complex subunits (D) Bacterially-purified and GTP-loaded GST:RAB21 pull-down of HeLa cell lysates followed by Strumpellin, FAM21, CAPZα and VPS35 immunoblots. Ponceau staining reveals the purity of the GST and GST:RAB21 used for the pull-downs. n=3 independent experiments.(E) RAB21 colocalizes with the WASH and retromer complexes. Transiently expressing GFP:RAB21 HeLa cells were fixed and stained for endogenous EEA1, FAM21, WASH and VPS26. Boxed region is magnified and single channels are depicted. Scale bars: 10µm or 5µm in enlarged view.(F) Per cell Pearson Correlation between RAB21 and the various markers. Error bars are SEM, n=2 independent experiments.


Figure 4 ( A ) Low RAB21 expression levels in two independent RAB21 KO cell populations . Immunoblot analysis of endogenous RAB21 , RAB5 , RAB7 and GAPDH . ( B ) Endo - lysosomal compartments are unaffected by the loss of RAB21 . Immunofluorescences of APPL1 , TfR , RAB7 and LAMP1 in parental HeLa cells and in the two RAB21 knockout cell populations . Scale bars : 10µm , n = 3 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 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

Figure 4(A) Low RAB21 expression levels in two independent RAB21 KO cell populations. Immunoblot analysis of endogenous RAB21, RAB5, RAB7 and GAPDH.(B) Endo-lysosomal compartments are unaffected by the loss of RAB21. Immunofluorescences of APPL1, TfR, RAB7 and LAMP1 in parental HeLa cells and in the two RAB21 knockout cell populations. Scale bars :10µm, n=3 independent experiments.


Figure 5The stability of the retromer cargo sorting complex is affected in RAB21 KO cells . Immunoblot analysis of VPS35 , VPS26 , VPS29 , GAPDH and RAB21 in parental and the two RAB21 KO cell populations . Ratio of VPS35 , VPS26 and VPS29 integrated densities to GAPDH for four independent experiments ; SEM . * represents p < 0 . 05 Statistical tests used : One sample t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats . ( B ) WASH complex proteins are not affected by RAB21 deletion . Immunoblot of Strumpellin , WASH , FAM21 , CAPZα , GAPDH and RAB21 . Ratio of WASH , FAM21 , Strumpellin and CAPZα integrated densities to GAPDH for four independent experiments ; SEM . Data information Statistical tests used : One sample t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats . RAB21 knockout decreases VPS35 localization at endosomes . ( C ) Immunofluorescence of endogenous VPS35 and EEA1 in RAB21 - depleted cells . Boxed region is magnified and single channel images are depicted . ( D ) Per cell Pearson Correlation between VPS35 and EEA1 ; SEM , n = 3 independent experiments . Data information scale bars represent 10µm or 2 . 5µm in enlarged views . Statistical tests used : ( D ) - Unpaired t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats . RAB21 knockout decreases VPS35 localization at endosomes . ( E ) Average number of VPS35 puncta per cell ; SEM , n = 3 independent experiments . Data information Statistical tests used : Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats . ( F - H ) RAB21 loss impairs endosomal recruitment of the WASH complex . ( F ) Immunofluorescence of endogenous WASH and EEA1 in RAB21 - depleted cells . Boxed region is magnified and single channel images are depicted . ( G ) Per cell Pearson Correlation between VPS35 and EEA1 and ( H ) FAM21 and EEA1 ; SEM , n = 2 independent experiments . Data information : scale bars represent 10µm or 2 . 5µm in enlarged views . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats . ( I - J ) RAB21 knockout reduces endosomal F - actin levels . ( I ) Immunofluorescence of endogenous F - actin using Alexa - 488 conjugated phalloidin and EEA1 in RAB21 - depleted cells . Boxed region is magnified and single channel images are depicted . ( J ) Per cell Pearson Correlation between internal F - actin and EEA1 ; SEM , n = 3 independent experiments . Data information scale bars represent 10µm or 2 . 5µm in enlarged views . Statistical tests used : Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

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 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 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 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 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 O O O 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 5The stability of the retromer cargo sorting complex is affected in RAB21 KO cells. Immunoblot analysis of VPS35, VPS26, VPS29, GAPDH and RAB21 in parental and the two RAB21 KO cell populations. Ratio of VPS35, VPS26 and VPS29 integrated densities to GAPDH for four independent experiments; SEM. * represents p <0.05 Statistical tests used: One sample t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.(B) WASH complex proteins are not affected by RAB21 deletion. Immunoblot of Strumpellin, WASH, FAM21, CAPZα, GAPDH and RAB21. Ratio of WASH, FAM21, Strumpellin and CAPZα integrated densities to GAPDH for four independent experiments; SEM. Data information Statistical tests used: One sample t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.RAB21 knockout decreases VPS35 localization at endosomes. (C) Immunofluorescence of endogenous VPS35 and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (D) Per cell Pearson Correlation between VPS35 and EEA1; SEM, n=3 independent experiments. Data information scale bars represent 10µm or 2.5µm in enlarged views. Statistical tests used: (D) - Unpaired t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.RAB21 knockout decreases VPS35 localization at endosomes. (E) Average number of VPS35 puncta per cell; SEM, n=3 independent experiments. Data information Statistical tests used: Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.(F-H) RAB21 loss impairs endosomal recruitment of the WASH complex. (F) Immunofluorescence of endogenous WASH and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (G) Per cell Pearson Correlation between VPS35 and EEA1 and (H) FAM21 and EEA1; SEM, n=2 independent experiments. Data information: scale bars represent 10µm or 2.5µm in enlarged views. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.(I-J) RAB21 knockout reduces endosomal F-actin levels. (I) Immunofluorescence of endogenous F-actin using Alexa-488 conjugated phalloidin and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (J) Per cell Pearson Correlation between internal F-actin and EEA1; SEM, n=3 independent experiments. Data information scale bars represent 10µm or 2.5µm in enlarged views. Statistical tests used: Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


Figure 6 ( A - C ) RAB21 is not required for CI - MPR or GLUT1 trafficking . ( A ) Immunofluorescence of endogenous CI - MPR and TGN46 or GLUT1 with LAMP1 in RAB21 - knockout cells . Boxed region is magnified . ( B ) Per cell Pearson Correlation between CI - MPR and TGN46 or ( C ) GLUT1 and LAMP1 ; SEM , n = 3 independent experiments . Data information : In ( A ) , scale bars represent 20µm or 5µm in enlarged views for CI - MPR , while they represent 10µm in enlarged views for GLUT1 . Statistical tests used : ( B ) - Unpaired t - tests ( C Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeatsMCT1 trafficking requires RAB21 . ( D ) Immunofluorescence of endogenous MCT1 in wild type or RAB21 - depleted cells . ( E ) Per cell integrated MCT1 intensity ( in Relative Fluorescence Unit ) ; SEM , n = 2 independent experiments . Data information : scale bars represent 20µm or 5µm in enlarged views . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats ( F ) RAB21 interacts with SLC3A2 . FLAG immunoprecipitation of FLAG : RAB21 and immunoblot of co - expressed SLC3A2 : HA . Lysates correspond to 5 % input , n = 3 independent experiments . ( G - H ) RAB21 is required for appropriate SLC3A2 , Basigin and CD44 trafficking . ( G ) Antibody - uptake assays in parental or knockout RAB21 cells . Cells were stained with an Alexa488 conjugated anti - mouse antibody . Arrowheads point to endosomal tubules . Note that non - internalized antibodies were removed by an acid wash , which was not fully efficient for CD147 and CD44 , explaining plasma membrane labeling . ( H ) Percentage of cells with tubules in parental or RAB21 knockout cells ; SEM , n = 3 independent experiments . Data information scale bars represent 20µm or 5µm in enlarged views . Statistical tests used Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all

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 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 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 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

Figure 6(A-C) RAB21 is not required for CI-MPR or GLUT1 trafficking. (A) Immunofluorescence of endogenous CI-MPR and TGN46 or GLUT1 with LAMP1 in RAB21-knockout cells. Boxed region is magnified. (B) Per cell Pearson Correlation between CI-MPR and TGN46 or (C) GLUT1 and LAMP1; SEM, n=3 independent experiments. Data information: In (A), scale bars represent 20µm or 5µm in enlarged views for CI-MPR, while they represent 10µm in enlarged views for GLUT1. Statistical tests used: (B) - Unpaired t-tests (C Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeatsMCT1 trafficking requires RAB21. (D) Immunofluorescence of endogenous MCT1 in wild type or RAB21-depleted cells. (E) Per cell integrated MCT1 intensity (in Relative Fluorescence Unit); SEM, n=2 independent experiments. Data information: scale bars represent 20µm or 5µm in enlarged views. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats(F) RAB21 interacts with SLC3A2. FLAG immunoprecipitation of FLAG:RAB21 and immunoblot of co-expressed SLC3A2:HA. Lysates correspond to 5% input, n=3 independent experiments.(G-H) RAB21 is required for appropriate SLC3A2, Basigin and CD44 trafficking. (G) Antibody-uptake assays in parental or knockout RAB21 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Note that non-internalized antibodies were removed by an acid wash, which was not fully efficient for CD147 and CD44, explaining plasma membrane labeling. (H) Percentage of cells with tubules in parental or RAB21 knockout cells; SEM, n=3 independent experiments. Data information scale bars represent 20µm or 5µm in enlarged views. Statistical tests used Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats


Figure 7 ( A ) Validation of WASH and retromer knockouts and SLC3A2 protein levels . Immunoblots of parental or FAM21 , VARP and VPS29 knockout cell populations . ( B ) Antibody - uptake assays in parental or knockout FAM21 , VARP and VPS29 cells . Cells were stained with an Alexa488 conjugated anti - mouse antibody . Arrowheads point to endosomal tubules . Data information : ( B ) scale bars represent 20µm ( C ) Percentage of cells with tubules in parental or in various knockout cells ; SEM , n = 3 independent experiments . Statistical tests used : ( C ) - Unpaired t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats . ( D ) Ratio of SLC3A2 integrated densities to GAPDH ; SEM , n = 3 independent experiments . Statistical tests used : ( D ) One sample T - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats . Retromer is required for endosomal localization of RAB21 . Immunofluorescence of transiently expressed GFP : RAB21 with endogenous ( E ) EEA1 in parental or VARP - and VPS29 - deleted cells . Boxed region is magnified underneath . Data information scale bars represent 10µm or 2 . 5µm in enlarged views . Retromer is required for endosomal localization of RAB21 . Immunofluorescence of transiently expressed GFP : RAB21 with endogenous ( F ) VPS26 in parental or VARP - and VPS29 - deleted cells . Boxed region is magnified underneath . Data information : scale bars represent 10µm or 2 . 5µm in enlarged views . ( G ) Per cell Pearson Correlation between GFP : RAB21 and EEA1 or VPS26 ; SEM , n = 3 independent experiments . Statistical tests used : ( G ) - Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

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 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 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 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

Figure 7(A) Validation of WASH and retromer knockouts and SLC3A2 protein levels. Immunoblots of parental or FAM21, VARP and VPS29 knockout cell populations.(B) Antibody-uptake assays in parental or knockout FAM21, VARP and VPS29 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Data information: (B) scale bars represent 20µm(C) Percentage of cells with tubules in parental or in various knockout cells; SEM, n=3 independent experiments. Statistical tests used: (C) - Unpaired t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.(D) Ratio of SLC3A2 integrated densities to GAPDH; SEM, n=3 independent experiments. Statistical tests used: (D) One sample T-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.Retromer is required for endosomal localization of RAB21. Immunofluorescence of transiently expressed GFP:RAB21 with endogenous (E) EEA1 in parental or VARP- and VPS29-deleted cells. Boxed region is magnified underneath. Data information scale bars represent 10µm or 2.5µm in enlarged views.Retromer is required for endosomal localization of RAB21. Immunofluorescence of transiently expressed GFP:RAB21 with endogenous (F) VPS26 in parental or VARP- and VPS29-deleted cells. Boxed region is magnified underneath. Data information: scale bars represent 10µm or 2.5µm in enlarged views.(G) Per cell Pearson Correlation between GFP:RAB21 and EEA1 or VPS26; SEM, n=3 independent experiments. Statistical tests used: (G) - Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


Figure 4 ( B ) Drug response curves upon treatment of organoid models with nutlin ‑ 3a , 5 ‑ FU , oxaliplatin , ibrutinib , panobinostat and entinostat ( biological replicates n = 3 , data are shown as mean ± SD ) .

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

Figure 4(B) Drug response curves upon treatment of organoid models with nutlin‑3a, 5‑FU, oxaliplatin, ibrutinib, panobinostat and entinostat (biological replicates n=3, data are shown as mean ±SD).


Figure 5Drug response curves upon treatment with inhibitors for ( B ) EGFR ( afatinib , gefitinib ) , ( biological replicates n = 3 , data are shown as mean ± SD ) . Drug response curves upon treatment with inhibitors for ( C ) B ‑ RAF ( LY3009120 , TAK ‑ 632 , vemurafenib , dabrafenib ) , ( biological replicates n = 3 , data are shown as mean ± SD ) . Drug response curves upon treatment with inhibitors for ( D ) MEK1 / 2 ( selumetinib , binimetinib ) ( biological replicates n = 3 , data are shown as mean ± SD ) .

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 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 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 5Drug response curves upon treatment with inhibitors for (B) EGFR (afatinib, gefitinib), (biological replicates n=3, data are shown as mean ±SD).Drug response curves upon treatment with inhibitors for (C) B‑RAF (LY3009120, TAK‑632, vemurafenib, dabrafenib), (biological replicates n=3, data are shown as mean ±SD).Drug response curves upon treatment with inhibitors for (D) MEK1/2 (selumetinib, binimetinib) (biological replicates n=3, data are shown as mean ±SD).


Figure 6 ( A ) Brigthfield images of human patient derived organoids ( PDOs ) from gastric cancer with and without RTK / MAPK alterations ( scale bar 100µm , zoom in 40µm ) . ( B ) Drug response curves of PDOs upon treatment with the MEK1 / 2 inhibitor trametinib ( biological replicates n = 3 , data are presented as mean , SD values are shown in Table EV2 ) . ( C ) Drug response curves upon HDAC inhibition with panobinostat ( PAN ) and entinostat ( ENT ) ( biological replicates n = 3 , data are presented as mean , SD values are shown in Table EV2 ) . ( D ) Comparison of the relative area under the curve ( AUCrel ) upon trametinib treatment in RTK / MAPK altered ( mean AUCrel : 0 . 5308 ; biological replicate n = 7 ) vs . non ‑ altered ( mean AUCrel : 0 . 7101 ; biological replicates n = 6 ) human PDOs ( two ‑ tailed Student ' s t ‑ Test ; * p = 0 . 0356 ) .

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 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

Figure 6(A) Brigthfield images of human patient derived organoids (PDOs) from gastric cancer with and without RTK/MAPK alterations (scale bar 100µm, zoom in 40µm).(B) Drug response curves of PDOs upon treatment with the MEK1/2 inhibitor trametinib (biological replicates n=3, data are presented as mean, SD values are shown in Table EV2). (C) Drug response curves upon HDAC inhibition with panobinostat (PAN) and entinostat (ENT) (biological replicates n=3, data are presented as mean, SD values are shown in Table EV2). (D) Comparison of the relative area under the curve (AUCrel) upon trametinib treatment in RTK/MAPK altered (mean AUCrel: 0.5308; biological replicate n=7) vs. non‑altered (mean AUCrel: 0.7101; biological replicates n=6) human PDOs (two‑tailed Student's t‑Test; *p= 0.0356).


Figure 1a , Diagram of the cell cycle composed of G1 , S , G2 , and M phases ( not to scale ) . Phase durations were quantified by time - lapse fluorescence microscopy using a PCNA - mCherry reporter to identify four discrete events during the lifetime of an individual cell Images were acquired every 10 min . b , Mean phase durations in RPE , U2OS , and H9 cell lines . Error bars represent standard deviations . c , Coefficient of variation ( CV ) of phase durations . d , Percentage of the total variation in cell cycle duration contributed by individual phases . e , Correlations between individual cell cycle phase durations . Sample sizes were adequate to detect correlations n = 125 ( RPE ) , 130 ( U2OS ) , 113 ( H9 ) . R2 , square of Pearson correlation coefficient .

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 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 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

Figure 1a, Diagram of the cell cycle composed of G1, S, G2, and M phases (not to scale). Phase durations were quantified by time-lapse fluorescence microscopy using a PCNA-mCherry reporter to identify four discrete events during the lifetime of an individual cell Images were acquired every 10 min.b, Mean phase durations in RPE, U2OS, and H9 cell lines. Error bars represent standard deviations.c, Coefficient of variation (CV) of phase durations.d, Percentage of the total variation in cell cycle duration contributed by individual phases.e, Correlations between individual cell cycle phase durations. Sample sizes were adequate to detect correlations n = 125 (RPE), 130 (U2OS), 113 (H9). R2, square of Pearson correlation coefficient.


Figure 3b , Shift in phase durations of RPE cells overexpressing Myc . boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : Myc , n = 116c , Pairwise correlation between cell cycle phase durations of RPE cells overexpressing Myc . Number of cells : Myc , n = 116e , Shift in phase durations of RPE cells treated with 50 ng / mL aphidicolin . boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : aphidicolin , n = 115f , Pairwise correlation between cell cycle phase durations under aphidicolin treatment . Number of cells : aphidicolin , n = 115h , Shift in phase durations of RPE cells incubated at 34 ° C . boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : 34 ° C , n = 122i , Pairwise correlation between phase durations for cells incubated at 34 ° C . Number of cells : 34 ° C , n = 122k , Shift in phase durations of RPE cells treated with NCS . , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : NCS , n = 119 . l , Pairwise correlation between phase durations for cells treated with NCS . Number of cells : NCS , n = 119 .

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 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 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 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 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 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 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

Figure 3b, Shift in phase durations of RPE cells overexpressing Myc. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: Myc, n = 116c, Pairwise correlation between cell cycle phase durations of RPE cells overexpressing Myc. Number of cells: Myc, n = 116e, Shift in phase durations of RPE cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 115f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 115h, Shift in phase durations of RPE cells incubated at 34°C. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: 34°C, n = 122i, Pairwise correlation between phase durations for cells incubated at 34°C. Number of cells: 34°C, n = 122k, Shift in phase durations of RPE cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 119.l, Pairwise correlation between phase durations for cells treated with NCS. Number of cells: NCS, n = 119.


Figure 4g , Shifts in phase durations for RPE cells treated with 2 μM CVT - 313 . A boxplot representing the distributions of durations in untreated cells is underlaid for comparison . * , P < 1 × 10 - 5 , 2 - sided Kolmogorov - Smirnov test . ( n = 117 cells ) . h , Pairwise correlation between cell cycle phase durations upon treatment with CVT - 313 . j , Shifts in phase durations for RPE cells overexpressing cyclin D . A boxplot representing the distributions of durations in untreated cells is underlaid for comparison . * , P < 1 × 10 - 5 , 2 - sided Kolmogorov - Smirnov test . ( n = 113 cells ) . k , Pairwise correlation between cell cycle phase durations upon overexpression of cyclin D . P indicates P - value .

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 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

Figure 4g, Shifts in phase durations for RPE cells treated with 2 μM CVT-313. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 117 cells).h, Pairwise correlation between cell cycle phase durations upon treatment with CVT-313.j, Shifts in phase durations for RPE cells overexpressing cyclin D. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 113 cells).k, Pairwise correlation between cell cycle phase durations upon overexpression of cyclin D. P indicates P-value.


Figure 5b , Shift in phase durations of U2OS cells treated with NCS . , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : NCS , n = 114c , Pairwise correlation between phase durations for U2OS cells treated with NCS . Number of cells : NCS , n = 114e , Shift in phase durations of U2OS cells treated with 50 ng / mL aphidicolin . boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : aphidicolin , n = 153 . f , Pairwise correlation between cell cycle phase durations under aphidicolin treatment . Number of cells : aphidicolin , n = 153 .

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 B-PANEL_START 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 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

Figure 5b, Shift in phase durations of U2OS cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 114c, Pairwise correlation between phase durations for U2OS cells treated with NCS. Number of cells: NCS, n = 114e, Shift in phase durations of U2OS cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 153.f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 153.


Figure 1C . MA plot highlighting genes differentially expressed between state independent of strain ( glm state term , false discovery rate - adjusted P value ( FDR ) < 0 . 05 and log2FC > 1 ) . Core pluripotent TFs highlighted for each state ( maroon = naive ESCs , teal = formative EpiLCs ) . H . Heatmap of motif deviations from 3 biological replicates of B6 and D2 ESCs for TFs highlighted in G .

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

Figure 1C. MA plot highlighting genes differentially expressed between state independent of strain (glm state term, false discovery rate-adjusted P value (FDR) < 0.05 and log2FC >1). Core pluripotent TFs highlighted for each state (maroon = naive ESCs, teal = formative EpiLCs).H. Heatmap of motif deviations from 3 biological replicates of B6 and D2 ESCs for TFs highlighted in G.


Figure 2A - D . Example expression patterns for select genes identified by applying a general linear model ( glm ) including state , genotype , and interaction terms . Dots represent individual biological replicates ( N = 3 ) ; lines highlight changes in mean values for replicates between each state ( log2FC > 1 and FDR < 0 . 05 ) . A . State dependent expression is exemplified by Nanog which showed no difference between strains . B . Expression of Zfp277 exemplifies strain - dependence being consistently higher in B6 . C & D . A significant genotype x state ( GXS ) interaction was identified for Nr5a2 ( C , a marker for pluripotency ) and Sox1 ( D , a marker for neuronal differentiation ) .

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

Figure 2A-D. Example expression patterns for select genes identified by applying a general linear model (glm) including state, genotype, and interaction terms. Dots represent individual biological replicates (N = 3); lines highlight changes in mean values for replicates between each state (log2FC >1 and FDR < 0.05). A. State dependent expression is exemplified by Nanog which showed no difference between strains. B. Expression of Zfp277 exemplifies strain-dependence being consistently higher in B6. C & D. A significant genotype x state (GXS) interaction was identified for Nr5a2 (C, a marker for pluripotency) and Sox1 (D, a marker for neuronal differentiation).


Figure 3D . Feature plot of expression gradients for indicated gene overlaid on UMAP from a . Three lineage markers that help to distinguish cellular identity are shown for five cell clusters . E . Pseudocolor plots of FACS analysis on cells from EBs for both B6 and D2 strains . Cells were labeled with anti - EpCAM ( top ) or biological replicates with anti - SOX1 antibody ( bottom ) . Gated population indicates percentages of EpCAM + ( Q3 ) or SOX1 + cells .

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 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 3D. Feature plot of expression gradients for indicated gene overlaid on UMAP from a. Three lineage markers that help to distinguish cellular identity are shown for five cell clusters.E. Pseudocolor plots of FACS analysis on cells from EBs for both B6 and D2 strains. Cells were labeled with anti-EpCAM (top) or biological replicates with anti-SOX1 antibody (bottom). Gated population indicates percentages of EpCAM+ (Q3) or SOX1+ cells.