( E ) Representative western blot showing levels of IKKε , PHGDH , PSAT1 and PSPH in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours .

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(E) Representative western blot showing levels of IKKε, PHGDH, PSAT1 and PSPH in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.


( A ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines . Data are expressed as fold changes , relative to levels in a non - silenced control of each cell line and normalised to β - Actin ( n ≥ 3 biological replicates ) .

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(A) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. Data are expressed as fold changes, relative to levels in a non-silenced control of each cell line and normalised to β-Actin (n≥3 biological replicates).


( B ) Representative western blot showing levels of IKKε and the SBP enzymes in IKBKE ( IKKε ) - silenced ZR - 75 - 1 , T47D , MDA - MB - 468 and MCF7 breast cancer cell lines .

(B) Representative western blot showing levels of IKKε and the SBP enzymes in IKBKE (IKKε)-silenced ZR-75-1, T47D, MDA-MB-468 and MCF7 breast cancer cell lines.


( C - E ) Levels of the SBP enzymes in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines . ( C ) PHGDH , ( D ) PSAT1 and ( E ) PSPH levels in indicated cell lines normalised to Vinculin . Densitometry analysis quantified single sample density as a percentage of total blot density per cell line prior to vinculin normalisation ( n ≥ 3 biological replicates ) .

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(C-E) Levels of the SBP enzymes in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. (C) PHGDH, (D) PSAT1 and (E) PSPH levels in indicated cell lines normalised to Vinculin. Densitometry analysis quantified single sample density as a percentage of total blot density per cell line prior to vinculin normalisation (n≥3 biological replicates).


( F ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in ATF4 - silenced ZR - 75 - 1 , T47D and MDA - MB - 468 breast cancer cell lines . Data are expressed as fold changes , relative to levels in non - silenced control cells and normalised to β - Actin ( n = 3 biological replicates ) .

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(F) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced ZR-75-1, T47D and MDA-MB-468 breast cancer cell lines. Data are expressed as fold changes, relative to levels in non-silenced control cells and normalised to β-Actin (n=3 biological replicates).


( G ) Representative western blot showing levels of PHGDH , PSAT1 and PSPH in ATF4 - silenced ZR - 75 - 1 , MDA - MB - 468 , MDA - MB - 231 , T47D and HCC1143 breast cancer cell lines .

(G) Representative western blot showing levels of PHGDH, PSAT1 and PSPH in ATF4-silenced ZR-75-1, MDA-MB-468, MDA-MB-231, T47D and HCC1143 breast cancer cell lines.


( A ) Correlation of change in OCR ( ΔOCR ) in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines ( from Fig . 3C ) and the change in cell confluency ( Δconfluency ) upon treatment of the panel of cell lines with NCT502 ( from Fig . EV5A ) .

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(A) Correlation of change in OCR (ΔOCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the change in cell confluency (Δconfluency) upon treatment of the panel of cell lines with NCT502 (from Fig. EV5A).


( B ) Correlation of ΔOCR in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines ( from Fig . 3C ) and the Δconfluency upon treatment of the panel of cell lines with 6 - Diazo - 5 - oxo - L - norleucine ( DON ) ( from Fig . EV5C ) .

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(B) Correlation of ΔOCR in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the Δconfluency upon treatment of the panel of cell lines with 6-Diazo-5-oxo-L-norleucine (DON) (from Fig. EV5C).


( C ) Association between IKBKE ( IKKε ) and PSAT1 mRNA overexpression evaluated by a chi - squared independence test . The + sign indicates samples with significant ( p < 0 . 05 ) overexpression of IKBKE or PSAT1 . Number and percentage of samples , as well as the Chi - square values are shown .

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(C) Association between IKBKE (IKKε) and PSAT1 mRNA overexpression evaluated by a chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown.


Expression of IKBKE ( IKKε ) and the SBP enzymes PSAT1 , in the METABRIC dataset . Samples were stratified by Pam50 intrinsic subtypes and ER status . Brown - Forsythe and Welch ANOVA test with unpaired t - test with Welch correction were applied . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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Expression of IKBKE (IKKε) and the SBP enzymes PSAT1, in the METABRIC dataset. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.


Expression of SBP enzymes PHGDH and PSPH in the METABRIC dataset . The expression of a proliferation related gene set and ATF4 are also shown . Samples were stratified by Pam50 intrinsic subtypes and ER status . Brown - Forsythe and Welch ANOVA test with unpaired t - test with Welch correction were applied . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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Expression of SBP enzymes PHGDH and PSPH in the METABRIC dataset. The expression of a proliferation related gene set and ATF4 are also shown. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.


( J ) Association between IKBKE ( IKKε ) and ATF4 mRNA overexpression evaluated by chi - squared independence test . The + sign indicates samples with significant ( p < 0 . 05 ) overexpression of IKBKE or PSAT1 . Number and percentage of samples , as well as the Chi - square values are shown .

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(J) Association between IKBKE (IKKε) and ATF4 mRNA overexpression evaluated by chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown.


( K , L ) Correlation of IKBKE ( IKKε ) and PSAT1 ( K ) or IKBKE ( IKKε ) and ATF4 ( L ) expression in the ER negative sample subset .

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(K, L) Correlation of IKBKE (IKKε) and PSAT1 (K) or IKBKE (IKKε) and ATF4 (L) expression in the ER negative sample subset.


( C ) Adult phenotypes of the ash1 and NSD mutants . In ash122 / ash121 mutants ( designated as ash1 - ) the loss of Abd - B expression results in partial transformation of abdominal segments 6 and 5 toward segments 5 and 4 , which is visible from the partial loss of pigmentation on tergites 5 and 6 ( t5 and t6 ) and appearance of bristles on sternite 6 ( s6 , marked with black arrows ) . The loss of Ubx expression causes transformation of the third thoracic to the second thoracic segment visible as partial haltere ( H ) to wing and third leg ( 3L ) to second leg ( 2L ) transformations . The former is evident from the change in the haltere shape and the appearance of multiple bristles ( black arrowheads ) . The latter is indicated by the apical and pre - apical bristles ( red arrows ) on the tibia of the third leg of ash1 mutants . These are normally present on 2L but absent on 3L ( compare to wild - type ) . Also note the appearance of additional hypopleural bristles on the third thoracic segment of the ash1 - flies ( red arrowheads ) , which indicate its transformation towards the second thoracic segment . Phenotype of the NSDds46 / NSDds46 ( NSD - ) flies is indistinguishable from wild - type and the phenotype of the double ash122 , NSDds46 / ash122 , NSDds46 ( ash1 - , NSD - ) flies is no more severe than that of the single ash122 / ash121 ( ash1 - ) mutants .

O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O B-TISSUE O O O O B-TISSUE O O O O B-TISSUE O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O B-TISSUE O B-GENEPROD O O O 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-TISSUE O O O B-TISSUE O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O

(C) Adult phenotypes of the ash1 and NSD mutants. In ash122/ash121 mutants (designated as ash1-) the loss of Abd-B expression results in partial transformation of abdominal segments 6 and 5 toward segments 5 and 4, which is visible from the partial loss of pigmentation on tergites 5 and 6 (t5 and t6) and appearance of bristles on sternite 6 (s6, marked with black arrows). The loss of Ubx expression causes transformation of the third thoracic to the second thoracic segment visible as partial haltere (H) to wing and third leg (3L) to second leg (2L) transformations. The former is evident from the change in the haltere shape and the appearance of multiple bristles (black arrowheads). The latter is indicated by the apical and pre-apical bristles (red arrows) on the tibia of the third leg of ash1 mutants. These are normally present on 2L but absent on 3L (compare to wild-type). Also note the appearance of additional hypopleural bristles on the third thoracic segment of the ash1- flies (red arrowheads), which indicate its transformation towards the second thoracic segment. Phenotype of the NSDds46/NSDds46 (NSD-) flies is indistinguishable from wild-type and the phenotype of the double ash122,NSDds46/ ash122,NSDds46 (ash1-,NSD-) flies is no more severe than that of the single ash122/ash121 (ash1-) mutants.


( D ) Ubx expression in the haltere imaginal discs . The expression was assayed by immunostaining with antibodies against Ubx ( red ) and acetylated H3K18 ( green , positive control ) . While ash122 / ash121 ( ash1 - ) larvae show stochastic clonal loss of the Ubx immunostaining in haltere discs ( yellow dashed lines ) , Set2 - larvae have uniform expression of Ubx throughout the haltere disc , resembling that in the wild - type larvae . Scale bars indicate 100μm .

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(D) Ubx expression in the haltere imaginal discs. The expression was assayed by immunostaining with antibodies against Ubx (red) and acetylated H3K18 (green, positive control). While ash122/ash121 (ash1-) larvae show stochastic clonal loss of the Ubx immunostaining in haltere discs (yellow dashed lines), Set2- larvae have uniform expression of Ubx throughout the haltere disc, resembling that in the wild-type larvae. Scale bars indicate 100μm.


( A ) Adult phenotypes of the ash122 / ash19011 flies supplemented with transgenic constructs expressing either the full - length Ash1 ( Ash1FL ) or the truncated variants lacking the PHD ( Ash1 ∆ PHD ) or the SET ( Ash1 ∆ SET ) domains . Note the third leg ( L3 ) to second leg ( L2 ) , haltere ( H ) to wing ( black arrows ) , t5 to t4 and t6 to t5 transformations in the Ash1 ∆ PHD and the Ash1 ∆ SET but not in the Ash1FL flies . The latter are evident from the partial loss of pigmentation in t6 and t5 , or the appearance of small bristles ( trichomas ) on t6 of the Ash1 ∆ PHD and the Ash1 ∆ SET flies in the area that is normally naked ( Ash1FL , yellow dashed line ) .

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(A) Adult phenotypes of the ash122/ash19011 flies supplemented with transgenic constructs expressing either the full-length Ash1 (Ash1FL) or the truncated variants lacking the PHD (Ash1∆PHD) or the SET (Ash1∆SET) domains. Note the third leg (L3) to second leg (L2), haltere (H) to wing (black arrows), t5 to t4 and t6 to t5 transformations in the Ash1∆PHD and the Ash1∆SET but not in the Ash1FL flies. The latter are evident from the partial loss of pigmentation in t6 and t5, or the appearance of small bristles (trichomas) on t6 of the Ash1∆PHD and the Ash1∆SET flies in the area that is normally naked (Ash1FL, yellow dashed line).


( B ) Two - fold dilutions of total nuclear protein extracts from the third instar larvae of the ash122 / ash19011 mutants supplemented with various transgenic constructs and wild type flies were analyzed by western blot with anti - Ash1 antibodies . Arrowhead indicates the position of Ash1 protein . Note that transgenic proteins are expressed at comparable level .

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(B) Two-fold dilutions of total nuclear protein extracts from the third instar larvae of the ash122/ash19011 mutants supplemented with various transgenic constructs and wild type flies were analyzed by western blot with anti-Ash1 antibodies. Arrowhead indicates the position of Ash1 protein. Note that transgenic proteins are expressed at comparable level.


( C ) Coomassie staining of SDS - PAGE separated protein extracts from ( B ) was used to control the loading .

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(C) Coomassie staining of SDS-PAGE separated protein extracts from (B) was used to control the loading.


Two - fold serial dilutions of the total protein extracts from the wild - type , ash122 / ash19011 ( ash1 - ) , ash122 , NSDds46 / ash19011 , NSDds46 ( ash1 - , NSD - ) and Set21 ( Set2 - ) larval brains , imaginal discs and salivary glands were analyzed by western blot with antibodies against H3K36me1 ( A ) , H3K36me2 ( B ) and H3K36me3 ( C ) . Note the strong ( > 10 - fold ) reduction of H3K36me3 signal in the Set2 - extract and the slight ( ~ 2 - fold ) reduction of H3K36me1 signal in the ash1 - and ash1 - , NSD - extracts . The protein extracts from the wild - type , double ash1 - , NSD - and single NSD - and Set2 - mutants ( right panels ) were analyzed together on the same membrane , however the images of the H3K36me1 and H3K36me3 western blots were modified to splice out the marker lane between the ash1 - , NSD - and the Set2 - extracts . Western blots with constitutively expressed BEAF - 32 protein were used as loading controls .

O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

Two-fold serial dilutions of the total protein extracts from the wild-type, ash122/ash19011 (ash1-), ash122,NSDds46/ash19011,NSDds46 (ash1-, NSD-) and Set21 (Set2-) larval brains, imaginal discs and salivary glands were analyzed by western blot with antibodies against H3K36me1 (A), H3K36me2 (B) and H3K36me3 (C). Note the strong (>10-fold) reduction of H3K36me3 signal in the Set2- extract and the slight (~2-fold) reduction of H3K36me1 signal in the ash1- and ash1-, NSD- extracts. The protein extracts from the wild-type, double ash1-, NSD- and single NSD- and Set2- mutants (right panels) were analyzed together on the same membrane, however the images of the H3K36me1 and H3K36me3 western blots were modified to splice out the marker lane between the ash1-,NSD- and the Set2- extracts. Western blots with constitutively expressed BEAF-32 protein were used as loading controls.


Chromatin from the wild - type ( dark blue bars ) , ash122 / ash19011 ( ash1 - , red bars ) and transgenic ash122 / ash19011 ( Ash1FL , light blue bars ; Ash1 ∆ PHD , green bars ; Ash1 ∆ SET , orange bars ) larvae was subjected to immunoprecipitation with the antibodies against H3K36me1 , H3K36me2 Histograms show the mean of the two independent experiments ( n = 2 ) with dots indicating individual experimental results . An intergenic region on chromosome 3R ( intergenic ) and the constitutively active TBP - associated factor 4 ( Taf4 ) gene serve as controls . The loss of Ash1 ChIP signal in the ash1 - larvae indicates that the selected genes are the genuine Ash1 binding sites .

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Chromatin from the wild-type (dark blue bars), ash122/ash19011 (ash1-, red bars) and transgenic ash122/ash19011 (Ash1FL, light blue bars; Ash1∆PHD, green bars; Ash1∆SET, orange bars) larvae was subjected to immunoprecipitation with the antibodies against H3K36me1 , H3K36me2 Histograms show the mean of the two independent experiments (n=2) with dots indicating individual experimental results. An intergenic region on chromosome 3R (intergenic) and the constitutively active TBP-associated factor 4 (Taf4) gene serve as controls. The loss of Ash1 ChIP signal in the ash1- larvae indicates that the selected genes are the genuine Ash1 binding sites.


Chromatin from the wild - type ( dark blue bars ) , ash122 / ash19011 ( ash1 - , red bars ) and transgenic ash122 / ash19011 ( Ash1FL , light blue bars ; Ash1 ∆ PHD , green bars ; Ash1 ∆ SET , orange bars ) larvae was subjected to immunoprecipitation with the antibodies against Ash1 ( C ) . Histograms show the mean of the two independent experiments ( n = 2 ) with dots indicating individual experimental results . An intergenic region on chromosome 3R ( intergenic ) and the constitutively active TBP - associated factor 4 ( Taf4 ) gene serve as controls . The loss of Ash1 ChIP signal in the ash1 - larvae indicates that the selected genes are the genuine Ash1 binding sites .

B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O 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-SUBCELLULAR O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O

Chromatin from the wild-type (dark blue bars), ash122/ash19011 (ash1-, red bars) and transgenic ash122/ash19011 (Ash1FL, light blue bars; Ash1∆PHD, green bars; Ash1∆SET, orange bars) larvae was subjected to immunoprecipitation with the antibodies against Ash1 (C). Histograms show the mean of the two independent experiments (n=2) with dots indicating individual experimental results. An intergenic region on chromosome 3R (intergenic) and the constitutively active TBP-associated factor 4 (Taf4) gene serve as controls. The loss of Ash1 ChIP signal in the ash1- larvae indicates that the selected genes are the genuine Ash1 binding sites.


( B ) The ∆ HisC ; 12xH3K36R and control ∆ HisC ; 12xWT flies show no homeotic transformations and are indistinguishable from the wild - type .

O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O

(B) The ∆HisC; 12xH3K36R and control ∆HisC; 12xWT flies show no homeotic transformations and are indistinguishable from the wild-type.


( A ) The Abd - B expression in the Central Nervous System ( CNS ) of stage 16 embryos was assayed by immunostaining with antibodies against Abd - B ( red ) . In the ash122 / TM3 , Sb , e Kr : : GFP or ash19011 / TM3 , Sb , e Kr : : GFP embryos ( heterozygous control ) , Abd - B is expressed in parasegments 14 to 10 in a gradient that recedes from the posterior to anterior parasegment . In the ash122 / ash19011 mutants the Abd - B gradient is much steeper , with reduced staining of parasegments 13 and 12 and at the edge of the detection in parasegments 11 - 10 . Heterozygous control and ash122 / ash19011 mutant embryos were stained together and separated by strong GFP expression ( green ) in the Bolwig ' s organs ( white arrows ) . Here and in B the embryos are oriented with anterior pole facing the top and scaling bars correspond to 50μm .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The Abd-B expression in the Central Nervous System (CNS) of stage 16 embryos was assayed by immunostaining with antibodies against Abd-B (red). In the ash122/TM3, Sb, e Kr::GFP or ash19011/TM3, Sb, e Kr::GFP embryos (heterozygous control), Abd-B is expressed in parasegments 14 to 10 in a gradient that recedes from the posterior to anterior parasegment. In the ash122/ash19011 mutants the Abd-B gradient is much steeper, with reduced staining of parasegments 13 and 12 and at the edge of the detection in parasegments 11-10. Heterozygous control and ash122/ash19011 mutant embryos were stained together and separated by strong GFP expression (green) in the Bolwig's organs (white arrows). Here and in B the embryos are oriented with anterior pole facing the top and scaling bars correspond to 50μm.


( B ) Unlike ash1 mutants , embryos homozygous for His3 . 3A , His3 . 3B , ∆ HisC deletions and supplemented with 12xH3K36R transgene ( His3 . 2 - , His3 . 3 - , H3K36R ) display the wild - type Abd - B immunostaining pattern ( red ) , the same as the control embryos heterozygous for His3 . 3A , and ∆ HisC deletions . In this experiment , immunostaining with antibodies against acetylated H3K18 ( green ) served as positive control .

O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O

(B) Unlike ash1 mutants, embryos homozygous for His3.3A, His3.3B, ∆HisC deletions and supplemented with 12xH3K36R transgene (His3.2-,His3.3-,H3K36R) display the wild-type Abd-B immunostaining pattern (red), the same as the control embryos heterozygous for His3.3A, and ∆HisC deletions. In this experiment, immunostaining with antibodies against acetylated H3K18 (green) served as positive control.


( D ) Reverse transcription and quantitative PCR ( RT - qPCR ) measurement of Abd - B expression in His3 . 2 - , His3 . 3 - , H3K36R embryos . Expression of Abd - B in stage 16 embryos , which are homozygous for His3 . 3A , His3 . 3B , and ∆ HisC deletions and carry 12xH3K36R transgene ( mutant ) , is not reduced compared to their wild - type counterparts ( control 1 ) or embryos heterozygous for His3 . 3A , and ∆ HisC deletions ( control 2 ) . Histograms show the mean of the two independent experiments ( n = 2 ) with dots indicating individual experimental results .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Reverse transcription and quantitative PCR (RT-qPCR) measurement of Abd-B expression in His3.2-,His3.3-,H3K36R embryos. Expression of Abd-B in stage 16 embryos, which are homozygous for His3.3A, His3.3B, and ∆HisC deletions and carry 12xH3K36R transgene (mutant), is not reduced compared to their wild-type counterparts (control 1) or embryos heterozygous for His3.3A, and ∆HisC deletions (control 2). Histograms show the mean of the two independent experiments (n=2) with dots indicating individual experimental results.


A - C Confocal images of Stage 12 embryos from control , P { GT1 } CG9005BG02278 P - element mutant ( CG9005PBG ) , and CG9005PBG with CG9005 expression restored in macrophages . Macrophage : red . Phalloidin to visualize embryo : green . Germband edge : dotted white line . Data information : Scale bars : 50µm ( A - C )

O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O

A-C Confocal images of Stage 12 embryos from control, P{GT1}CG9005BG02278 P-element mutant (CG9005PBG), and CG9005PBG with CG9005 expression restored in macrophages. Macrophage: red. Phalloidin to visualize embryo: green. Germband edge: dotted white line. Data information: Scale bars: 50µm (A-C)


D Quantification of macrophages that have penetrated the germband from genotypes in ( A - C ) and from CG9005PBG over two deficiencies ( Df ) that remove the gene . n = 35 , 56 , 25 , 9 , 18 embryos respectively , p < 0 . 0001 for control vs CG9005PBG , Df1 , or Df2 ; p = 0 . 98 for control vs mac > CG9005 rescue , p = 0 . 91 , 0 . 90 for CG9005PBG vs Df1 or Df2 . Data information : Throughout paper mac > indicates GAL4 driven expression of a UAS construct specifically in macrophages by srpHemo - GAL4 . N for movies represents imaging from different embryos . ( D ) One - way ANOVA with Tukey .

O O O B-CELL_TYPE O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

D Quantification of macrophages that have penetrated the germband from genotypes in (A-C) and from CG9005PBG over two deficiencies (Df) that remove the gene. n=35, 56, 25, 9, 18 embryos respectively, p<0.0001 for control vs CG9005PBG, Df1, or Df2; p=0.98 for control vs mac>CG9005 rescue, p=0.91, 0.90 for CG9005PBG vs Df1 or Df2. Data information: Throughout paper mac> indicates GAL4 driven expression of a UAS construct specifically in macrophages by srpHemo-GAL4. N for movies represents imaging from different embryos. (D) One-way ANOVA with Tukey.


E Macrophage - specific knockdown of CG9005 by UAS - RNAi lines . n = 22 , 20 , 21 , 23 , 35 , 28 embryos . p < 0 . 0001 for all comparisons . Data information : Throughout paper mac > indicates GAL4 driven expression of a UAS construct specifically in macrophages by srpHemo - GAL4 . N for movies represents imaging from different embryos . Unpaired t - tests . Graphs show mean ± SEM ; ns = p > 0 . 05 , * p < 0 . 05 , * * * * p < 0 . 0001 .

O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O 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-CELL_TYPE O O B-GENEPROD O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Macrophage-specific knockdown of CG9005 by UAS-RNAi lines. n=22, 20, 21, 23, 35, 28 embryos. p<0.0001 for all comparisons. Data information: Throughout paper mac> indicates GAL4 driven expression of a UAS construct specifically in macrophages by srpHemo-GAL4. N for movies represents imaging from different embryos. Unpaired t-tests. Graphs show mean±SEM; ns=p>0.05, *p<0.05, ****p<0.0001.


F Stills from two - photon movies of control and CG9005PBG mutant embryos showing macrophages ( nuclei , red ) migrating starting at Stage 10 from the head towards the germband and invading the germband tissue . Elapsed time indicated in minutes . Germband edge ( white dotted line ) detected by yolk autofluorescence . Data information : Scale bars : 30µm ( F ) . Throughout this work , embryos with stomadeal invagination and germband retraction away from the anterior of less than 29 % were defined as Stage 10 , 29 % - 31 % Stage 11 , and 35 % - 40 % Stage 12 .

O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O B-TISSUE O O B-TISSUE O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Stills from two-photon movies of control and CG9005PBG mutant embryos showing macrophages (nuclei, red) migrating starting at Stage 10 from the head towards the germband and invading the germband tissue. Elapsed time indicated in minutes. Germband edge (white dotted line) detected by yolk autofluorescence. Data information: Scale bars: 30µm (F). Throughout this work, embryos with stomadeal invagination and germband retraction away from the anterior of less than 29% were defined as Stage 10, 29%-31% Stage 11, and 35%-40% Stage 12.


G - H Macrophage migration speed ( G ) in the head or ( H ) between the yolk sac and the germband edge . For ( G ) : control n = 8 movies , CG9005PBG mutant n = 3 ; control n = 360 tracks , mutant n = 450 , p = 0 . 65 . For ( H ) : control n = 7 movies , mutant n = 3 ; control n = 46 tracks , mutant n = 19 , p = 0 . 62 Data information : Unpaired t - tests . Graphs show mean ± SEM ; ns = p > 0 . 05 , * p < 0 . 05 , * * * * p < 0 . 0001 .

O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O B-TISSUE O O O O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-H Macrophage migration speed (G) in the head or (H) between the yolk sac and the germband edge. For (G): control n=8 movies, CG9005PBG mutant n=3; control n=360 tracks, mutant n=450, p=0.65. For (H): control n=7 movies, mutant n=3; control n=46 tracks, mutant n=19, p=0.62 Data information: Unpaired t-tests. Graphs show mean±SEM; ns=p>0.05, *p<0.05, ****p<0.0001.


I The time required for the first macrophage nucleus to enter into the extended germband . Control n = 7 movies , mutant n = 5 . Time to entry : control = 23 min , CG9005PBG = 38 min , p < 0 . 0001 . Data information : Unpaired t - tests . Graphs show mean ± SEM ; ns = p > 0 . 05 , * p < 0 . 05 , * * * * p < 0 . 0001 .

O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I The time required for the first macrophage nucleus to enter into the extended germband. Control n=7 movies, mutant n=5. Time to entry: control=23 min, CG9005PBG=38 min, p<0.0001. Data information: Unpaired t-tests. Graphs show mean±SEM; ns=p>0.05, *p<0.05, ****p<0.0001.


J - L The migration speed of the ( J ) 1st , ( K ) 2nd , or ( L ) 3rd - 5th macrophages along the first 25 - 30µm into the germband between the mesoderm and ectoderm . In schematics , analyzed macrophages - - light blue , other macrophages - - red , ectoderm - - green , mesoderm - - purple , and yolk - - beige . For ( J ) : control n = 6 movies , mutant n = 5 , p = 0 . 012 . For ( K ) : control n = 5 movies , mutant n = 5 , p = 0 . 03 . For ( L ) : control n = 5 movies , mutant n = 4 , p = 0 . 17 . Data information : Unpaired t - tests . Graphs show mean ± SEM ; ns = p > 0 . 05 , * p < 0 . 05 , * * * * p < 0 . 0001 .

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-TISSUE O O B-TISSUE O B-TISSUE O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O B-TISSUE O O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J-L The migration speed of the (J) 1st, (K) 2nd, or (L) 3rd-5th macrophages along the first 25-30µm into the germband between the mesoderm and ectoderm. In schematics, analyzed macrophages--light blue, other macrophages--red, ectoderm--green, mesoderm--purple, and yolk--beige. For (J): control n=6 movies, mutant n=5, p=0.012. For (K): control n=5 movies, mutant n=5, p=0.03. For (L): control n=5 movies, mutant n=4, p=0.17. Data information: Unpaired t-tests. Graphs show mean±SEM; ns=p>0.05, *p<0.05, ****p<0.0001.


C - D Confocal images or ( D ) quantification of the macrophages in germband in Stage 12 embryos from the control , atosPBG , and atosPBG expressing Atos itself or variants lacking particular domains . Transgene expression directly from macrophage specific promoter ( mac - ) . For control ( n = 32 embryos ) vs atosPBG mutant ( n = 56 ) p < 0 . 0001 ; vs rescue with mac - atos ( n = 18 ) p > 0 . 99 ; vs rescue with mac - atosDUF - ( n = 17 ) p = 0 . 0003 ; vs rescue with mac - atosChrSeg - ( n = 21 ) p = 0 . 0003 ; vs rescue with mac - atosDUF - / ChrSeg - ( n = 19 ) p = 0 , 00014 ; vs rescue with mac - atosTAD1 - / TAD2 - ( n = 25 ) p = 0 . 0009 , atosPBG mutant vs . rescue with mac - atos p = 0 . 0031 . Data information : Germband edge : dotted white line . Mac - indicates direct expression from the srpHemo promoter . ( C , macrophage nuclei ( red ) , actin by Phalloidin staining ( green ) . ( D One - way ANOVA with Tukey . Mean ± SEM , ns = p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Scale bars : 50µm (

O O O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O B-TISSUE O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D Confocal images or (D) quantification of the macrophages in germband in Stage 12 embryos from the control, atosPBG, and atosPBG expressing Atos itself or variants lacking particular domains. Transgene expression directly from macrophage specific promoter (mac-). For control (n=32 embryos) vs atosPBG mutant (n=56) p<0.0001; vs rescue with mac-atos (n=18) p>0.99; vs rescue with mac-atosDUF- (n=17) p=0.0003; vs rescue with mac-atosChrSeg-(n=21) p=0.0003; vs rescue with mac-atosDUF-/ChrSeg-(n=19) p=0,00014; vs rescue with mac-atosTAD1-/ TAD2- (n=25) p=0.0009, atosPBG mutant vs. rescue with mac-atos p=0.0031. Data information: Germband edge: dotted white line. Mac- indicates direct expression from the srpHemo promoter. (C, macrophage nuclei (red), actin by Phalloidin staining (green). (D One-way ANOVA with Tukey. Mean±SEM, ns=p>0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars: 50µm (C


E Confocal images of atosPBG rescued by expressing Atossa ' s murine orthologs , mFAM214A or B ( mFAMA - B ) in macrophages , F Quantification of macrophages in the germband in Stage 12 embryos from the control , atosPBG , and atosPBG embryos expressing mFAM214A or B specifically in macrophages ( mac - ) . For control ( n = 24 embryos ) vs . atosPBG mutant ( n = 56 ) p < 0 . 0001 ; vs mac - atos rescue ( n = 18 ) p = 0 . 7 ; vs mac - mFAMA rescue ( n = 22 ) p = 0 . 6 ; vs mac - mFAMB rescue ( n = 25 ) p = 0 . 086 . For atosPBG mutant vs mac - atos rescue p = 0 . 0006 ; vs mac - mFAMA rescue p = 0 . 0002 ; vs mac - mFAMB rescue p = 0 . 0043 . Data information : Germband edge : dotted white line . Mac - indicates direct expression from the srpHemo promoter . E ) macrophage nuclei ( red ) , actin by Phalloidin staining ( green ) . F ) One - way ANOVA with Tukey . Mean ± SEM , ns = p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Scale bars : 50µm E ) .

O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-TISSUE O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O B-CELL_TYPE B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Confocal images of atosPBG rescued by expressing Atossa's murine orthologs, mFAM214A or B (mFAMA-B) in macrophages, F Quantification of macrophages in the germband in Stage 12 embryos from the control, atosPBG, and atosPBG embryos expressing mFAM214A or B specifically in macrophages (mac-). For control (n=24 embryos) vs. atosPBG mutant (n=56) p<0.0001; vs mac-atos rescue (n=18) p=0.7; vs mac-mFAMA rescue (n=22) p=0.6; vs mac-mFAMB rescue (n=25) p=0.086. For atosPBG mutant vs mac-atos rescue p=0.0006; vs mac-mFAMA rescue p=0.0002; vs mac-mFAMB rescue p=0.0043. Data information: Germband edge: dotted white line. Mac- indicates direct expression from the srpHemo promoter. E) macrophage nuclei (red), actin by Phalloidin staining (green). F) One-way ANOVA with Tukey. Mean±SEM, ns=p>0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars: 50µm E).


B - D Confocal images of early Stage 12 embryos from the control and lines expressing RNAis against ( B ) porthos , ( C ) GR / HPR or ( D ) LKR / SDH , specifically in macrophages ( red ) . Germband edge : dotted white line . Data information : Scale bar :

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O B-TISSUE O O O O O O O O O O O O

B-D Confocal images of early Stage 12 embryos from the control and lines expressing RNAis against (B) porthos, (C) GR/HPR or (D) LKR/SDH, specifically in macrophages (red). Germband edge: dotted white line. Data information: Scale bar: 50µm


Quantification of the number of germband macrophages in embryos from control and upon RNAi knockdown of the genes In ( E ) control ( n = 36 embryos ) vs . porthos RNAi ( n = 28 ) p < 0 . 0001 . In ( F ) сontrol 1 ( n = 18 embryos ) vs . GR / HPR RNAi 1 ( n = 18 ) and control 2 ( n = 21 ) vs . GR / HPR RNAi 2 ( n = 24 ) , both p < 0 . 0001 ; control 3 ( n = 15 embryos ) vs . GR / HPR RNAi 3 ( n = 23 ) p = 0 . 08 . Data information : Mean ± SEM and ns = p > 0 . 05 , * * * * p < 0 . 0001 . One - way ANOVA with

O O O O O B-TISSUE B-CELL_TYPE 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-GENEPROD O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of the number of germband macrophages in embryos from control and upon RNAi knockdown of the genes In (E) control (n=36 embryos) vs. porthos RNAi (n=28) p<0.0001. In (F) сontrol 1 (n=18 embryos) vs. GR/HPR RNAi 1 (n=18) and control 2 (n=21) vs. GR/HPR RNAi 2 (n=24), both p<0.0001; control 3 (n=15 embryos) vs. GR/HPR RNAi 3 (n=23) p=0.08. Data information: Mean±SEM and ns=p>0.05, ****p<0.0001. One-way ANOVA with Tukey


Quantification of the number of germband macrophages in embryos from control and upon RNAi knockdown of the genes In ( G ) сontrol 1 ( n = 18 embryos ) vs . LKR / SDH RNAi 1 ( n = 17 ) and control 2 ( n = 21 ) vs . LKR / SDH RNAi 2 ( n = 23 ) , both p < 0 . 0001 . Data information : Mean ± SEM and ns = p > 0 . 05 , * * * * p < 0 . 0001 . One - way ANOVA with

O O O O O B-TISSUE B-CELL_TYPE O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of the number of germband macrophages in embryos from control and upon RNAi knockdown of the genes In (G) сontrol 1 (n=18 embryos) vs. LKR/SDH RNAi 1 (n=17) and control 2 (n=21) vs. LKR/SDH RNAi 2 (n=23), both p<0.0001. Data information: Mean±SEM and ns=p>0.05, ****p<0.0001. One-way ANOVA with Tukey


B Stills starting at Stage 11 from two - photon movies of control embryos and those expressing porthos ( pths ) - RNAi in macrophages ( nuclei , red ) , migrating from the head mesoderm towards and into the germband at the indicated time points . White dotted line : germband edge . Data information : Movies in each analysis set are from independent embryos . Scale bars :

O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O B-SUBCELLULAR O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O

B Stills starting at Stage 11 from two-photon movies of control embryos and those expressing porthos (pths)-RNAi in macrophages (nuclei, red), migrating from the head mesoderm towards and into the germband at the indicated time points. White dotted line: germband edge. Data information: Movies in each analysis set are from independent embryos. Scale bars: 30µm


C - H Quantification of macrophage migration parameters from movies as in ( B ) . Macrophage migration speed ( C ) in the head or ( D ) between the yolk sac and the germband edge . ( C ) control n = 4 movies , pths RNAi n = 6 ; control n = 507 tracks , pths RNAi n = 859 ; p = 0 . 56 . ( D ) control n = 5 movies , pths RNAi n = 5 ; control n = 40 tracks , pths RNAi n = 51 ; p = 0 . 45 . ( E ) The time required for the first macrophage nucleus to enter into the extended germband : control = 23 min , CG9005PBG = 38 min , p < 0 . 0001 . The migration speed of the ( F ) 1st , ( G ) 2nd , or ( H ) 3rd - 5th macrophages along the first 25 - 30µm into the germband between the mesoderm and ectoderm : control = 21 . 5 min , n = 6 , pths RNAi = 36 . 2 min , n = 4 , p < 0 . 0001 . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Unpaired t -

O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O B-TISSUE O O O O O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-TISSUE O O B-TISSUE O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-H Quantification of macrophage migration parameters from movies as in (B). Macrophage migration speed (C) in the head or (D) between the yolk sac and the germband edge. (C) control n=4 movies, pths RNAi n=6; control n=507 tracks, pths RNAi n=859; p=0.56. (D) control n=5 movies, pths RNAi n=5; control n=40 tracks, pths RNAi n=51; p=0.45. (E) The time required for the first macrophage nucleus to enter into the extended germband: control=23 min, CG9005PBG=38 min, p<0.0001. The migration speed of the (F) 1st, (G) 2nd, or (H) 3rd-5th macrophages along the first 25-30µm into the germband between the mesoderm and ectoderm: control=21.5 min, n=6, pths RNAi=36.2 min, n=4, p<0.0001. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired t-test


I Confocal images of early Stage 12 embryos from control , atosPBG , and atosPBG expressing atos : : FLAG : : HA ( mac > atos ) or pths : : FLAG : : HA ( mac > pths ) in macrophages ( red ) . Embryo detected by phalloidin staining ( green ) . White dotted line : germband edge . . J Quantification of macrophages in the germband of an atosPBG mutant rescued by expressing pths : : FLAG : : HA in macrophages . For control ( n = 15 embryos ) vs atosPBG mutant ( n = 22 ) and mac > atos rescue ( n = 27 ) both p < 0 . 0001 , vs mac > pths rescue ( n = 30 ) p = 0 . 0007 . Data information : Movies in each analysis set are from independent embryos . Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . one - way ANOVA with Tukey Scale bars :

O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O B-TISSUE O O O O O O B-CELL_TYPE O O B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Confocal images of early Stage 12 embryos from control, atosPBG, and atosPBG expressing atos::FLAG::HA (mac>atos) or pths::FLAG::HA (mac>pths) in macrophages (red). Embryo detected by phalloidin staining (green). White dotted line: germband edge.. J Quantification of macrophages in the germband of an atosPBG mutant rescued by expressing pths::FLAG::HA in macrophages. For control (n=15 embryos) vs atosPBG mutant (n=22) and mac>atos rescue (n=27) both p<0.0001, vs mac>pths rescue (n=30) p=0.0007. Data information: Movies in each analysis set are from independent embryos. Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. one-way ANOVA with Tukey Scale bars: 50µm


E - F Confocal images of early Stage 12 embryos from the controls , from lines expressing nlacZ and RNAis against atos or pths in macrophages ( mac > ) as a second control , and lines expressing RNAis targeting atos or pths along with individual components of the dTORC1 pathway in macrophages , including Nrpl2 , Iml1 , and TSC1 . Germband edge : dotted white line . Data information : Scale bar :

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-TISSUE O O O O O O O O O O O O

E-F Confocal images of early Stage 12 embryos from the controls, from lines expressing nlacZ and RNAis against atos or pths in macrophages (mac>) as a second control, and lines expressing RNAis targeting atos or pths along with individual components of the dTORC1 pathway in macrophages, including Nrpl2, Iml1, and TSC1. Germband edge: dotted white line. Data information: Scale bar: 30µm


G - H Quantification of the number of germband macrophages in embryos from the controls and upon RNAi knockdown of the genes in E - F . Expression of different RNAis against Nrpl2 , Iml1 , and TSC1 in atos or pths - depleted macrophages rescues macrophage invasion into the germband while expression of lacZ does not . In ( G ) control ( n = 21 embryos ) vs . atos RNAi ( n = 30 ) p < 0 . 0001 ; control vs . atos RNAi rescued with Nrpl2 RNAi ( n = 32 ) p = 0 . 019 , with Iml1 RNAi ( n = 24 ) p = 0 . 03 , and with TSC1 RNAi ( n = 15 ) p = 0 . 04 . In ( H ) control ( n = 29 embryos ) vs pths RNAi ( n = 26 ) p < 0 . 0001 ; control vs . pths RNAi rescued with Nrpl2 RNAi ( n = 27 ) p = 0 . 01 , with Iml1 RNAi ( n = 17 ) p = 0 . 02 , and with TSC1 RNAi ( n = 18 ) p = 0 . 02 . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * * * p < 0 . 0001 . one - way ANOVA with

O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O B-CELL_TYPE O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD 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-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-H Quantification of the number of germband macrophages in embryos from the controls and upon RNAi knockdown of the genes in E-F. Expression of different RNAis against Nrpl2, Iml1, and TSC1 in atos or pths-depleted macrophages rescues macrophage invasion into the germband while expression of lacZ does not. In (G) control (n=21 embryos) vs. atos RNAi (n=30) p<0.0001; control vs. atos RNAi rescued with Nrpl2 RNAi (n=32) p=0.019, with Iml1 RNAi (n=24) p=0.03, and with TSC1 RNAi (n=15) p=0.04. In (H) control (n=29 embryos) vs pths RNAi (n=26) p<0.0001; control vs. pths RNAi rescued with Nrpl2 RNAi (n=27) p=0.01, with Iml1 RNAi (n=17) p=0.02, and with TSC1 RNAi (n=18) p=0.02. Data information: Mean±SEM, ns=p>0.05, *p<0.05, ****p<0.0001. one-way ANOVA with Tukey


I Western blot of protein extracts from control and pths KD S2R + probed with p - 4EBP1 antibody . Tubulin serves as a loading control and quantification normalized to loading control . N = 3 biological replicates for both control and pths KD , p = 0 . 69 . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * * * p < 0 . 0001 . unpaired t -

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Western blot of protein extracts from control and pths KD S2R+ probed with p-4EBP1 antibody. Tubulin serves as a loading control and quantification normalized to loading control. N=3 biological replicates for both control and pths KD, p=0.69. Data information: Mean±SEM, ns=p>0.05, *p<0.05, ****p<0.0001. unpaired t-test


C - F RT - qPCR analysis of mRNA from ribosomal protein fractions for control ( black ) and pths KD ( gray ) transcripts from S2R + cells . RNA was isolated individually from fractions and pooled into five categories : RNP , 40S / 60S , monosome , low polysome ( di - and trisome ) , and high polysome ( remaining fractions ) . The data were plotted relative to the amount present in the monosome fraction for each transcript . The mRNA levels of subunits of mitochondrial complexes ( C ) I , ( D ) III and ( E ) V were reduced in low and high polysome fractions in pths KD cells compared to the control cells , while ( F ) mRNA levels of GAPDH in the same fractions , used as an internal control , were not changed ( n = 3 independent biological replicates for control and pths KD ) . For ( C , E - F ) low polysome fraction for control vs . pths KD : ( C ) p = 0 . 057 , ( D ) p = 0 . 0039 , ( E ) p = 0 . 09 , ( F ) p = 0 . 5 . For ( C , E - F ) high polysome fraction for control vs . pths KD : ( C ) p = 0 . 039 , ( D ) p = 0 . 046 , ( E ) p = 0 . 041 , ( F ) p = 0 . 66 . G - I Western blots and their quantifications of protein extracts from control and pths KD S2R + probed with ( G ) MT - ND1 ( for mitochondrial complex I ) n = 4 , p < 0 . 0001 ) , ( H ) ATPsynt - β ( for mitochondrial complex V ) n = 3 p = 0 . 034 and ( I ) tubulin β antibodies p = 0 . 56 ) ( n = 3 ) . Profilin serves as loading control . N = biological replicates . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . Unpaired t - tests ( C - F , G - I ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O 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-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD 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-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-F RT-qPCR analysis of mRNA from ribosomal protein fractions for control (black) and pths KD (gray) transcripts from S2R+ cells. RNA was isolated individually from fractions and pooled into five categories: RNP, 40S/60S, monosome, low polysome (di- and trisome), and high polysome (remaining fractions). The data were plotted relative to the amount present in the monosome fraction for each transcript. The mRNA levels of subunits of mitochondrial complexes (C) I, (D) III and (E) V were reduced in low and high polysome fractions in pths KD cells compared to the control cells, while (F) mRNA levels of GAPDH in the same fractions, used as an internal control, were not changed (n=3 independent biological replicates for control and pths KD). For (C,E-F) low polysome fraction for control vs. pths KD: (C) p=0.057, (D) p=0.0039, (E) p=0.09, (F) p=0.5. For (C,E-F) high polysome fraction for control vs. pths KD: (C) p=0.039, (D) p=0.046, (E) p=0.041, (F) p=0.66. G-I Western blots and their quantifications of protein extracts from control and pths KD S2R+ probed with (G) MT-ND1 (for mitochondrial complex I) n=4, p<0.0001), (H) ATPsynt-β (for mitochondrial complex V) n=3 p=0.034 and (I) tubulin β antibodies p=0.56) (n=3). Profilin serves as loading control. N=biological replicates. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. Unpaired t-tests (C-F, G-I).


B The Oxygen Consumption Rate ( OCR , pmols O2 / min ) assessed as a representative parameter of OxPhos in control and pths - KD S2R + cells by a Seahorse efflux assay . n ≥ 3 independent biological experiments each with n > 6 technical replicate . Data information : Mean ± SEM , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Unpaired t -

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The Oxygen Consumption Rate (OCR, pmols O2/min) assessed as a representative parameter of OxPhos in control and pths-KD S2R+ cells by a Seahorse efflux assay. n≥3 independent biological experiments each with n>6 technical replicate. Data information: Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired t-tests


C Distinct respiration parameters calculated from relative OCR values obtained in experiments shown in B . p < 0 . 0001 for all comparisons . Data information : Mean ± SEM , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . two - way ANOVA with Sidak ' s

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Distinct respiration parameters calculated from relative OCR values obtained in experiments shown in B. p<0.0001 for all comparisons. Data information: Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. two-way ANOVA with Sidak's test


Confocal images of macrophages from control embryos and those expressing a dominant negative c - ring of ATP synthase ( CV - DN ) in macrophages . from Stage 12 embryos and germband edge shown with dotted white line . Data information : show Stage 12 embryos . Scale bars :

B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O B-CELL_TYPE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O

Confocal images of macrophages from control embryos and those expressing a dominant negative c-ring of ATP synthase (CV-DN) in macrophages. from Stage 12 embryos and germband edge shown with dotted white line. Data information: show Stage 12 embryos. Scale bars: 50µm


quantification of macrophages ( E ) in the germband or ( F ) on the yolk next to the germband from control embryos and those expressing a dominant negative c - ring of ATP synthase ( CV - DN ) in macrophages . from Stage 12 embryos For ( E ) control ( n = 24 embryos ) vs CV - DN ( n = 20 ) p = 0 . 0032 . For ( F ) control ( n = 21 embryos ) vs CV - DN ( n = 17 ) p = 0 . 003 . Data information : show Stage 12 embryos . Mean ± SEM , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Unpaired t -

O O B-CELL_TYPE O O O O O B-TISSUE O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

quantification of macrophages (E) in the germband or (F) on the yolk next to the germband from control embryos and those expressing a dominant negative c-ring of ATP synthase (CV-DN) in macrophages. from Stage 12 embryos For (E) control (n=24 embryos) vs CV-DN (n=20) p=0.0032. For (F) control (n=21 embryos) vs CV-DN (n=17) p=0.003. Data information: show Stage 12 embryos. Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired t-tests


Confocal images of germband macrophages in control embryos and those expressing different RNAis against mitochondrial OxPhos Complex III ( or an RNAi against Complex V . Data information : show Stage 12 embryos . Complex III RNAis target Cyt - c1 , UQCR - cp1 - 2 , for Complex V , ATP synthase F1F0 . Scale bars :

B-EXP_ASSAY O O B-TISSUE B-CELL_TYPE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O

Confocal images of germband macrophages in control embryos and those expressing different RNAis against mitochondrial OxPhos Complex III (or an RNAi against Complex V. Data information: show Stage 12 embryos. Complex III RNAis target Cyt-c1, UQCR-cp1-2, for Complex V, ATP synthase F1F0. Scale bars: 50µm


quantification of germband macrophages in control embryos and those expressing different RNAis against mitochondrial OxPhos Complex III ( or an RNAi against Complex V . For ( H ) control ( n = 34 embryos ) vs Complex III RNAi 1 ( n = 20 ) p = 0 . 0001 ; vs . Complex III RNAi 2 ( n = 18 ) p = 0 . 027 ; vs . Complex III RNAi 3 ( n = 16 ) p < 0 . 0001 ; vs . Complex V RNAi ( n = 14 ) p < 0 . 0001 . Data information : show Stage 12 embryos . Complex III RNAis target Cyt - c1 , UQCR - cp1 - 2 , for Complex V , ATP synthase F1F0 . Mean ± SEM , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . one - way ANOVA with Tukey ' s ( H ) .

O O B-TISSUE B-CELL_TYPE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

quantification of germband macrophages in control embryos and those expressing different RNAis against mitochondrial OxPhos Complex III (or an RNAi against Complex V. For (H) control (n=34 embryos) vs Complex III RNAi 1 (n=20) p=0.0001; vs. Complex III RNAi 2 (n=18) p=0.027; vs. Complex III RNAi 3 (n=16) p<0.0001; vs. Complex V RNAi (n=14) p<0.0001. Data information: show Stage 12 embryos. Complex III RNAis target Cyt-c1, UQCR-cp1-2, for Complex V, ATP synthase F1F0. Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. one-way ANOVA with Tukey's (H).


I Single plane confocal microscope image during germband entry from control or atosPBG embryos , or those expressing pths - RNAi or CV - DN in macrophages . Antibodies against S293 - phosphorylated inactivated Pyruvate Dehydrogenase ( pPDH , green ) or total PDH ( magenta ) in macrophages ( red ) . Higher pPDH levels are usually found when ATP / ADP levels are high and input into the TCA cycle is being downregulated Data information : show Stage 12 embryos . Scale bars :

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

I Single plane confocal microscope image during germband entry from control or atosPBG embryos, or those expressing pths-RNAi or CV-DN in macrophages. Antibodies against S293-phosphorylated inactivated Pyruvate Dehydrogenase (pPDH, green) or total PDH (magenta) in macrophages (red). Higher pPDH levels are usually found when ATP/ADP levels are high and input into the TCA cycle is being downregulated Data information: show Stage 12 embryos. Scale bars: 10µm


J Quantification of normalized pPDH / PDH levels calculated from fluorescence intensities in macrophages from the genotypes in ( I ) during initial germband invasion . The pPDH / PDH ratio is significantly reduced , arguing that decreased function of CV , Atos or Pths in macrophages results in lower cellular ATP / ADP ratios compared to the control . N = 3 independent experiments . Ctrl 1 ( n = 10 embryos ) vs CV - DN ( n = 9 ) and vs . atos mutant ( n = 13 ) both p = 0 . 0002 ; Ctrl 2 ( n = 7 embryos ) vs pths RNAi ( n = 8 ) p = 0 . 0001 . * * * shown above columns in J are for comparison to relevant control . Data information : Mean ± SEM , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Unpaired t -

O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O B-TISSUE O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Quantification of normalized pPDH/PDH levels calculated from fluorescence intensities in macrophages from the genotypes in (I) during initial germband invasion. The pPDH/PDH ratio is significantly reduced, arguing that decreased function of CV, Atos or Pths in macrophages results in lower cellular ATP/ADP ratios compared to the control. N=3 independent experiments. Ctrl 1 (n=10 embryos) vs CV-DN (n=9) and vs. atos mutant (n=13) both p=0.0002; Ctrl 2 (n=7 embryos) vs pths RNAi (n=8) p=0.0001.*** shown above columns in J are for comparison to relevant control. Data information: Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired t-tests


A Single plane confocal microscope image during germband entry from control or atosPBG embryos , or those expressing pths , GR / HPR , and LKR / SDH in macrophages ( mac > ) . Embryos were stained for antibodies against S293 - phosphorylated inactivated Pyruvate Dehydrogenase ( pPDH , green ) or total PDH ( magenta ) in macrophages ( red ) . Higher pPDH / PDH ratios are consistent with higher ATP / ADP levels . Data information : Scale bars :

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

A Single plane confocal microscope image during germband entry from control or atosPBG embryos, or those expressing pths, GR/HPR, and LKR/SDH in macrophages (mac>). Embryos were stained for antibodies against S293-phosphorylated inactivated Pyruvate Dehydrogenase (pPDH, green) or total PDH (magenta) in macrophages (red). Higher pPDH/PDH ratios are consistent with higher ATP/ADP levels. Data information: Scale bars: 10µm


B Quantification of normalized pPDH / PDH levels calculated from fluorescence intensities in macrophages from the genotypes in ( A ) during initial germband invasion . The pPDH / PDH ratio is significantly increased in atosPBG embryos expressing either pths , GR / HPR , or LKR / SDH in macrophages compared to the atosPBG embryos . This argues that the decreased Atos function in atosPBG macrophages , resulting in lower cellular ATP / ADP ratios , was restored by expressing either its targets or murine orthologs . N = 3 independent experiments . Control ( n = 9 embryos ) vs . atos mutant ( n = 10 ) p < 0 . 0001 . Control vs . atos mutant rescued with mac > pths ( n = 10 ) p = 0 . 77 ; rescued with mac > GRHPR ( n = 9 ) p = 0 . 48 ; rescued with mac > LKRSDH ( n = 10 ) p = 0 . 012 . For atos mutant vs . atos rescued with mac > pths or mac > GRHPR p < 0 . 0001 . vs . atos rescued with mac > LKRSDH p = 0 . 0014 . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . One - way ANOVA with

O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O B-TISSUE O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O 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-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of normalized pPDH/PDH levels calculated from fluorescence intensities in macrophages from the genotypes in (A) during initial germband invasion. The pPDH/PDH ratio is significantly increased in atosPBG embryos expressing either pths, GR/HPR, or LKR/SDH in macrophages compared to the atosPBG embryos. This argues that the decreased Atos function in atosPBG macrophages, resulting in lower cellular ATP/ADP ratios, was restored by expressing either its targets or murine orthologs. N=3 independent experiments. Control (n=9 embryos) vs. atos mutant (n=10) p<0.0001. Control vs. atos mutant rescued with mac>pths (n=10) p=0.77; rescued with mac>GRHPR (n=9) p=0.48; rescued with mac>LKRSDH (n=10) p=0.012. For atos mutant vs. atos rescued with mac>pths or mac>GRHPR p<0.0001. vs. atos rescued with mac>LKRSDH p=0.0014. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. One-way ANOVA with Tukey


C - D ( C ) Confocal images or ( D ) quantification of the macrophages in germband in Stage 12 embryos from the control , atosPBG , and atosPBG expressing atos itself or GR / HPR , or LKR / SDH in macrophages . Germband edge : dotted white line . For ( D ) control ( n = 29 embryos ) vs . atos mutant ( n = 19 ) p < 0 . 0001 . Control vs . atos mutant rescued with mac > atos ( n = 27 embryos ) p = 0 . 99 ; rescued with mac > GRHPR ( n = 28 ) p = 0 . 29 ; rescued with mac > LKRSDH ( n = 20 ) p = 0 . 036 . atos mutant vs . atos rescued with mac > atos or mac > GRHPR p < 0 . 0001 ; rescued with mac > LKRSDH p = 0 . 0134 . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . One - way ANOVA with Tukey Scale bars :

O O O O O O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O B-TISSUE O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D (C) Confocal images or (D) quantification of the macrophages in germband in Stage 12 embryos from the control, atosPBG, and atosPBG expressing atos itself or GR/HPR, or LKR/SDH in macrophages. Germband edge: dotted white line. For (D) control (n=29 embryos) vs. atos mutant (n=19) p<0.0001. Control vs. atos mutant rescued with mac>atos (n=27 embryos) p=0.99; rescued with mac>GRHPR (n=28) p=0.29; rescued with mac>LKRSDH (n=20) p=0.036. atos mutant vs. atos rescued with mac>atos or mac>GRHPR p<0.0001; rescued with mac>LKRSDH p=0.0134. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. One-way ANOVA with Tukey Scale bars: 50µm


E Single plane confocal microscope images during germband entry from control or atosPBG embryos , or those expressing mammalian orthologs mFAM214A or B ( FAMA - B ) in macrophages ( mac > ) . Embryos were stained for antibodies against S293 - phosphorylated inactivated Pyruvate Dehydrogenase ( pPDH , green ) or total PDH ( magenta ) in macrophages ( red ) . Data information : Scale bars :

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O B-CELL_TYPE O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O

E Single plane confocal microscope images during germband entry from control or atosPBG embryos, or those expressing mammalian orthologs mFAM214A or B (FAMA-B) in macrophages (mac>). Embryos were stained for antibodies against S293-phosphorylated inactivated Pyruvate Dehydrogenase (pPDH, green) or total PDH (magenta) in macrophages (red). Data information: Scale bars: 10µm


F Quantification of normalized pPDH / PDH levels measured from fluorescence intensities in macrophages from the genotypes in ( E ) during initial germband invasion . The pPDH / PDH ratio is significantly increased in atosPBG embryos expressing either mammalian ortholog mFAM214A or B in macrophages compared to the atosPBG embryos . Results from three independent experiments . Control ( n = 10 embryos ) vs . atos mutant ( n = 11 ) p < 0 . 0001 . Control vs . atos mutant rescued with mac - mFAMA ( n = 14 embryos ) p = 0 . 06 ; rescued with mac - mFAMB ( n = 12 ) p = 0 . 13 . atos mutant vs . atos mutant rescued with mac - mFAMA p = 0 . 0025 or mac - mFAMB p = 0 . 0019 . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . One - way ANOVA with

O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O B-TISSUE O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Quantification of normalized pPDH/PDH levels measured from fluorescence intensities in macrophages from the genotypes in (E) during initial germband invasion. The pPDH/PDH ratio is significantly increased in atosPBG embryos expressing either mammalian ortholog mFAM214A or B in macrophages compared to the atosPBG embryos. Results from three independent experiments. Control (n=10 embryos) vs. atos mutant (n=11) p<0.0001. Control vs. atos mutant rescued with mac-mFAMA (n=14 embryos) p=0.06; rescued with mac-mFAMB (n=12) p=0.13. atos mutant vs. atos mutant rescued with mac-mFAMA p=0.0025 or mac-mFAMB p=0.0019. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. One-way ANOVA with Tukey


Cellular metabolites were measured by LC - MS - based untargeted metabolomics from extracts of Stage 11 embryos ; biological replicates for control n = 5 , for atosPBG n = 7 . ( B ) Normalized ATP / ADP ratio values are decreased in atosPBG compared to control embryos ( p = 0 . 028 ) . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . unpaired t -

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. (B) Normalized ATP/ADP ratio values are decreased in atosPBG compared to control embryos (p=0.028). Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. unpaired t-test


Cellular metabolites were measured by LC - MS - based untargeted metabolomics from extracts of Stage 11 embryos ; biological replicates for control n = 5 , for atosPBG n = 7 . Heatmap of non - targeted metabolites in atosPBG compared to wild - type embryos shown with average log2fold change ( FC ) in the non - targeted LC - MS / MS analysis reveals ( C ) an increase in atosPBG in substrates of the dGR / HPR enzyme , including 4 - hydroxy α - ketoglutarate and hydroxyproline ( HLP ) and a smaller decrease in its products , glycolate and glycerate , ( D ) a significant increase in some dipeptides including those containing hydroxyproline . Data information : Values in heat maps are obtained from untargeted metabolomic analysis , unpaired t -

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O

Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. Heatmap of non-targeted metabolites in atosPBG compared to wild-type embryos shown with average log2fold change (FC) in the non-targeted LC-MS/MS analysis reveals (C) an increase in atosPBG in substrates of the dGR/HPR enzyme, including 4-hydroxy α-ketoglutarate and hydroxyproline (HLP) and a smaller decrease in its products, glycolate and glycerate, (D) a significant increase in some dipeptides including those containing hydroxyproline. Data information: Values in heat maps are obtained from untargeted metabolomic analysis, unpaired t-test


Cellular metabolites were measured by LC - MS - based untargeted metabolomics from extracts of Stage 11 embryos ; biological replicates for control n = 5 , for atosPBG n = 7 . ( E ) Quantification shows an increase in atosPBG in the pyruvate / glucose ratio ( p = 0 . 035 ) , but none for the lactate / glucose ratio ( p = 0 . 65 ) . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . unpaired t -

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. (E) Quantification shows an increase in atosPBG in the pyruvate/glucose ratio (p=0.035), but none for the lactate/glucose ratio (p=0.65). Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. unpaired t-test


Cellular metabolites were measured by LC - MS - based untargeted metabolomics from extracts of Stage 11 embryos ; biological replicates for control n = 5 , for atosPBG n = 7 . Heatmap of non - targeted metabolites in atosPBG compared to wild - type embryos shown with average log2fold change ( FC ) in the non - targeted LC - MS / MS analysis ( F ) We observe increases in atosPBG in intermediates of mitochondrial fatty acid β - oxidation ( FAO ) , including different carnitine - conjugated lipids , and ( F - G ) a significant increase in ketone body substituents compared to the control . Data information : Values in heat maps are obtained from untargeted metabolomic analysis , unpaired t -

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O

Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. Heatmap of non-targeted metabolites in atosPBG compared to wild-type embryos shown with average log2fold change (FC) in the non-targeted LC-MS/MS analysis (F) We observe increases in atosPBG in intermediates of mitochondrial fatty acid β-oxidation (FAO), including different carnitine-conjugated lipids, and (F-G) a significant increase in ketone body substituents compared to the control. Data information: Values in heat maps are obtained from untargeted metabolomic analysis, unpaired t-test


( A ) Immunofluorescence microscopy for POMC ( green ) and Cre ( red ; n = 4 ) , ( B ) POMC ( green ) and Atg7 ( red ; n = 4 ) and ( C ) POMC ( green ) and p62 ( red ; n = 6 ) in MBH sections from Con and Atg7F / F ‐ POMC ‐ Cre ( KO )

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O

(A) Immunofluorescence microscopy for POMC (green) and Cre (red; n=4), (B) POMC (green) and Atg7 (red; n=4) and (C) POMC (green) and p62 (red; n=6) in MBH sections from Con and Atg7F/F‐POMC‐Cre (KO) mice


( D ) TUNEL ‐ positivity ( green ; n = 4 ) and ( E ) number of POMC ‐ positive neurons ( red ) in MBH sections from Con and KO mice ( n = 6 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * * P0 . 001 . Nuclei are blue ( DAPI ) . Scale inset : 10 μm . White arrows indicate Atg7 , Cre , p62 or POMC signal . Yellow arrows indicate merged signal . Con , control ; DAPI , 4 ′ ‐ 6 ‐ diamidino ‐ 2 ‐ phenylindole ; KO , knockout ; MBH , mediobasal hypothalamic ; NS , not statistically significant ; POMC , proopiomelanocortin ; TUNEL , TdT ‐ mediated dUTP nick end labelling .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O

(D) TUNEL‐positivity (green; n=4) and (E) number of POMC‐positive neurons (red) in MBH sections from Con and KO mice (n=6). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. White arrows indicate Atg7, Cre, p62 or POMC signal. Yellow arrows indicate merged signal. Con, control; DAPI, 4′‐6‐diamidino‐2‐phenylindole; KO, knockout; MBH, mediobasal hypothalamic; NS, not statistically significant; POMC, proopiomelanocortin; TUNEL, TdT‐mediated dUTP nick end labelling.


( A ) Body wt of 4 ‐ mo ‐ and 12 ‐ mo ‐ old Con and KO mice on RD ( n = 9 - 13 ) or HFD ( n = 5 - 7 ) . ( B ) Total body fat and ( C ) lean mass of 4 ‐ mo ‐ old Con and KO mice on RD ( n = 7 ) and 12 ‐ mo ‐ old Con and KO mice on HFD ( n = 6 - 8 ) .

O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O

(A) Body wt of 4‐mo‐ and 12‐mo‐old Con and KO mice on RD (n=9-13) or HFD (n=5-7). (B) Total body fat and (C) lean mass of 4‐mo‐old Con and KO mice on RD (n=7) and 12‐mo‐old Con and KO mice on HFD (n=6-8).


( D ) eWAT wt , ( E ) fat pads and ( F ) liver wt of 4 ‐ mo ‐ old Con and KO mice on RD ( n = 6 - 8 ) .

O O O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O O O O O B-ORGANISM O O O O O O O O O O

(D) eWAT wt, (E) fat pads and (F) liver wt of 4‐mo‐old Con and KO mice on RD (n=6-8).


( G ) Immunoblots for POMC and ACTH , and ( H ) NPY in MBH from RD ‐ fed Con and KO mice ( n = 3 - 4 ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O

(G) Immunoblots for POMC and ACTH, and (H) NPY in MBH from RD‐fed Con and KO mice (n=3-4).


( I ) Immunofluorescence microscopy for POMC ( green ) and α ‐ MSH ( red ) , and ( J ) α ‐ MSH ( green ) in MBH from Con and KO mice on RD ( n = 5 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . Nuclei are blue ( DAPI ) . Scale inset : 10 μm . Yellow arrows indicate merged signal . ACTH , adrenocorticotrophic hormone ; Con , control ; DAPI , 4 ′ ‐ 6 ‐ diamidino ‐ 2 ‐ phenylindole ; eWAT , epididymal fat ; HDF , high ‐ fat diet ; KO , knockout ; MBH , mediobasal hypothalamic ; mo , month ; MSH , melanocyte ‐ stimulating hormone ; NPY , neuropeptide Y ; POMC , proopiomelanocortin ; RD , regular chow ; wt , weight .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O 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-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(I) Immunofluorescence microscopy for POMC (green) and α‐MSH (red), and (J) α‐MSH (green) in MBH from Con and KO mice on RD (n=5). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. Yellow arrows indicate merged signal. ACTH, adrenocorticotrophic hormone; Con, control; DAPI, 4′‐6‐diamidino‐2‐phenylindole; eWAT, epididymal fat; HDF, high‐fat diet; KO, knockout; MBH, mediobasal hypothalamic; mo, month; MSH, melanocyte‐stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin; RD, regular chow; wt, weight.


( A ) Percent loss of body fat and ( B ) lean mass in 24 ‐ h ‐ fasted Con and KO mice on RD or HFD ( n = 6 - 8 ) .

O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O

(A) Percent loss of body fat and (B) lean mass in 24‐h‐fasted Con and KO mice on RD or HFD (n=6-8).


( C ) Histology for eWAT from fed or 6 ‐ h ‐ fasted Con and KO mice on RD ( n = 3 - 4 ) .

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(C) Histology for eWAT from fed or 6‐h‐fasted Con and KO mice on RD (n=3-4).


( D ) Serum fatty acids and ( E ) glycerol from fed or 6 ‐ h ‐ fasted Con and KO mice on RD ( n = 7 - 9 ) .

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(D) Serum fatty acids and (E) glycerol from fed or 6‐h‐fasted Con and KO mice on RD (n=7-9).


( F ) Serum fatty acids and ( G ) glycerol after intraperitoneal isoproterenol ( Iso ) in 6 ‐ h ‐ fasted Con and KO mice on RD ( n = 5 - 6 ) .

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(F) Serum fatty acids and (G) glycerol after intraperitoneal isoproterenol (Iso) in 6‐h‐fasted Con and KO mice on RD (n=5-6).


( H ) Immunoblot for oxidized proteins in eWAT from RD ‐ fed Con and KO mice ( n = 3 ) . ( I ) FACS analysis for M1 and M2 macrophage proportions in adipose stromal vascular fractions from fed Con and KO mice on RD ( n = 3 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . Con , control ; eWAT , epididymal fat ; FACS , fluorescence ‐ activated cell sorting ; HDF , high ‐ fat diet ; Iso , isoproterenol ; KO , knockout ; POMC , proopiomelanocortin ; RD , regular chow ; Stv , fasted .

O O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O

(H) Immunoblot for oxidized proteins in eWAT from RD‐fed Con and KO mice (n=3). (I) FACS analysis for M1 and M2 macrophage proportions in adipose stromal vascular fractions from fed Con and KO mice on RD (n=3). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Con, control; eWAT, epididymal fat; FACS, fluorescence‐activated cell sorting; HDF, high‐fat diet; Iso, isoproterenol; KO, knockout; POMC, proopiomelanocortin; RD, regular chow; Stv, fasted.


( A ) Blood glucose levels in 6 mo ( n = 7 - 9 ) and ( B ) 12 mo Con and KO mice on RD ( n = 6 ) , and in ( C ) 12 ‐ mo ‐ old Con and KO mice on HFD ( n = 4 ) .

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(A) Blood glucose levels in 6 mo (n=7-9) and (B) 12 mo Con and KO mice on RD (n=6), and in (C) 12‐mo‐old Con and KO mice on HFD (n=4).


( D ) Serum insulin levels in 6 mo , ( n = 7 - 9 ) and ( E ) 12 ‐ mo ‐ old RD ‐ fed Con and KO mice that were fed or 6 h fasted ( n = 6 ) .

O O O O B-SMALL_MOLECULE O O O 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-ORGANISM O O O O O O O O O O O O O

(D) Serum insulin levels in 6 mo, (n=7-9) and (E) 12‐mo‐old RD‐fed Con and KO mice that were fed or 6 h fasted (n=6).


( F ) HOMA values from 6 ‐ h ‐ fasted 6 ‐ mo ‐ old ( n = 7 - 9 ) and ( G ) 12 ‐ mo ‐ old Con and KO mice ( n = 6 ) on RD . ( H ) Glucose tolerance tests in 6 ‐ h ‐ fasted 6 ‐ mo ‐ old mice on RD ( n = 4 ) and in ( I ) 6 ‐ h ‐ fasted 12 ‐ mo ‐ old Con and KO mice fed HFD for 10 mo ( n = 4 ) .

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(F) HOMA values from 6‐h‐fasted 6‐mo‐old (n=7-9) and (G) 12‐mo‐old Con and KO mice (n=6) on RD. (H) Glucose tolerance tests in 6‐h‐fasted 6‐mo‐old mice on RD (n=4) and in (I) 6‐h‐fasted 12‐mo‐old Con and KO mice fed HFD for 10 mo (n=4).


( J ) Insulin tolerance tests in RD ‐ fed 6 ‐ mo ‐ old ( n = 4 ) and ( K ) HFD ‐ fed 12 ‐ mo ‐ old Con and KO mice ( n = 4 ) .

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(J) Insulin tolerance tests in RD‐fed 6‐mo‐old (n=4) and (K) HFD‐fed 12‐mo‐old Con and KO mice (n=4).


( L ) Insulin tolerance tests and ( M ) body weights of 1 ‐ mo ‐ old Con and KO mice on RD ( n = 3 - 5 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . Con , control ; HDF , high ‐ fat diet ; HOMA , homeostasis model of insulin resistance ; KO , knockout ; mo , month ; POMC , proopiomelanocortin ; RD , regular chow ; Stv , fasted .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(L) Insulin tolerance tests and (M) body weights of 1‐mo‐old Con and KO mice on RD (n=3-5). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Con, control; HDF, high‐fat diet; HOMA, homeostasis model of insulin resistance; KO, knockout; mo, month; POMC, proopiomelanocortin; RD, regular chow; Stv, fasted.


( A ) Immunoblots for Atg7 in MBH from young ( 3 mo ) , middle ‐ aged ( 12 mo ) and aged ( 22 mo ) fed and 6 ‐ h ‐ fasted ( Stv ) mice ( n = 4 ) , and ( B ) LC3 ‐ II in MBH explants from 3 mo and 22 mo fed mice in presence or absence of lysosomal inhibitors ( Inh ) ( n = 11 - 13 ) . Mean ± s . e . m values for ( B ) steady ‐ state LC3 ‐ II and ( C ) LC3 ‐ II flux are shown ( n = 11 - 13 ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE O O O O O O 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-ORGANISM O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O B-ORGANISM O O O O O O O O O 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-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O

(A) Immunoblots for Atg7 in MBH from young (3 mo), middle‐aged (12 mo) and aged (22 mo) fed and 6‐h‐fasted (Stv) mice (n=4), and (B) LC3‐II in MBH explants from 3 mo and 22 mo fed mice in presence or absence of lysosomal inhibitors (Inh) (n=11-13). Mean±s.e.m values for (B) steady‐state LC3‐II and (C) LC3‐II flux are shown (n=11-13).


( D ) Immunoblots for p62 and NBR1 flux in MBH from 3 mo and 22 mo fed mice in presence or absence of Inh ( n = 11 - 13 ) .

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(D) Immunoblots for p62 and NBR1 flux in MBH from 3 mo and 22 mo fed mice in presence or absence of Inh (n=11-13).


( E ) Immunofluorescence microscopy for POMC ( green ) and p62 ( red ) ( n = 4 ) , ( F ) immunoblots for POMC and ACTH ( n = 4 ) , and ( G ) immunofluorescence for α ‐ MSH in MBH from 3 mo and 22 mo mice on RD ( n = 4 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O

(E) Immunofluorescence microscopy for POMC (green) and p62 (red) (n=4), (F) immunoblots for POMC and ACTH (n=4), and (G) immunofluorescence for α‐MSH in MBH from 3 mo and 22 mo mice on RD (n=4).


( H ) Body wt and ( I ) eWAT wt of 3 ‐ mo ‐ and 22 ‐ mo ‐ old mice on RD ( n = 9 - 13 ) .

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(H) Body wt and (I) eWAT wt of 3‐mo‐ and 22‐mo‐old mice on RD (n=9-13).


( J ) Serum fatty acids and ( K ) glycerol from fed or 6 ‐ h ‐ fasted ( Stv ) 3 mo and 22 mo mice ( n = 7 - 9 ) , and ( L , M ) after intraperitoneal Iso in 6 ‐ h ‐ fasted 3 mo and 22 mo mice ( n = 5 - 6 ) .

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(J) Serum fatty acids and (K) glycerol from fed or 6‐h‐fasted (Stv) 3 mo and 22 mo mice (n=7-9), and (L,M) after intraperitoneal Iso in 6‐h‐fasted 3 mo and 22 mo mice (n=5-6).