Figure 5A Sox2 mRNA expression of pancreatic lysates from KNC , KPNC , and KPC mice , analyzed by real ‐ time PCR in three independent experiments ( means ± SD ) . B , C Western blot analysis shows Sox2 expression in pancreatic lysates of ( B ) KNC mice with indicated tumor grading and in ( C ) KPNC and KPC mice . D Protein expression levels after siRNA ‐ mediated p53 depletion in KNC tumor cells . E , F NFATc1 and Sox2 protein expression after ( E ) NFATc1 depletion or ( F ) upon CsA ( 1 μM , 24 h ) treatment in Panc1 cells . G Sox2 and E ‐ cadherin mRNA expression after NFATc1 depletion in L3 . 6 cells . I , J ChIP assays show NFATc1 binding to Sox2 enhancer and promoter in KNC tumor cells ( I ) I , J ChIP assays show RNA polymerase II binding to Sox2 promoter upon CsA ( 1 μM , 24 h ) treatment in L3 . 6 cells ( J ) . K Co ‐ immunoprecipitation of endogenous HA ‐ NFATc1 and Sox2 was performed in KPNC cells . L ChIP experiments show NFATc1 binding along with H3K27 acetylation ( H3K27ac ) mark at the selected Snai1 enhancer region and RNA polymerase II binding at Snai1 promoter in the presence or absence of Sox2 in KPNC tumor cells . Representative results from at least three independent experiments are shown ( means ± SD ; P 0 . 05 ) . M Relative mRNA expression levels of EMT ‐ related marker genes upon Sox2 depletion in KPNC tumor cells ( P 0 . 05 ) . N Zeb1 and Snail mRNA levels in L3 . 6 cells after shRNA ‐ mediated depletion of Sox2 and transient overexpression of NFATc1 shows that loss of Sox2 significantly rescues NFATc1 ‐ mediated induction of EMT genes ( * P 0 . 05 ) .

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Figure 5A Sox2 mRNA expression of pancreatic lysates from KNC, KPNC, and KPC mice, analyzed by real‐time PCR in three independent experiments (means ± SD).B, C Western blot analysis shows Sox2 expression in pancreatic lysates of (B) KNC mice with indicated tumor grading and in (C) KPNC and KPC mice.D Protein expression levels after siRNA‐mediated p53 depletion in KNC tumor cells.E, F NFATc1 and Sox2 protein expression after (E) NFATc1 depletion or (F) upon CsA (1 μM, 24 h) treatment in Panc1 cells.G Sox2 and E‐cadherin mRNA expression after NFATc1 depletion in L3.6 cells.I, J ChIP assays show NFATc1 binding to Sox2 enhancer and promoter in KNC tumor cells (I)I, J ChIP assays show RNA polymerase II binding to Sox2 promoter upon CsA (1 μM, 24 h) treatment in L3.6 cells (J).K Co‐immunoprecipitation of endogenous HA‐NFATc1 and Sox2 was performed in KPNC cells.L ChIP experiments show NFATc1 binding along with H3K27 acetylation (H3K27ac) mark at the selected Snai1 enhancer region and RNA polymerase II binding at Snai1 promoter in the presence or absence of Sox2 in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05).M Relative mRNA expression levels of EMT‐related marker genes upon Sox2 depletion in KPNC tumor cells (P 0.05).N Zeb1 and Snail mRNA levels in L3.6 cells after shRNA‐mediated depletion of Sox2 and transient overexpression of NFATc1 shows that loss of Sox2 significantly rescues NFATc1‐mediated induction of EMT genes (*P 0.05).


Figure 6A NFATc1 and Sox2 protein expression in L3 . 6 cells ( adherent versus spheres ) . B Relative mRNA expression of NFATc1 and Sox2 in L3 . 6 cells as well as in KPNCmicetumor cells ( adherent versus spheres ) . Graph represents data from three independent experiments ( means ± SD ) . Asterisks display significance ( * P 0 . 05 ) . C Cell sorting was performed to collect CD44 high and low populations in KPNC spheres ( left panel ) , which were analyzed for NFATc1 and Sox2 mRNA expression ( right panel ) . D Sox2 mRNA expression upon CsA ( 1 μM ) treatment in L3 . 6 cells ( adherent versus spheres ) ( * P 0 . 05 ) . E Bright ‐ field light microscopy images as well as quantification of KPNC spheres after transient NFATc1 depletion .

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Figure 6A NFATc1 and Sox2 protein expression in L3.6 cells (adherent versus spheres).B Relative mRNA expression of NFATc1 and Sox2 in L3.6 cells as well as in KPNCmicetumor cells (adherent versus spheres). Graph represents data from three independent experiments (means ± SD). Asterisks display significance (*P 0.05).C Cell sorting was performed to collect CD44 high and low populations in KPNC spheres (left panel), which were analyzed for NFATc1 and Sox2 mRNA expression (right panel).D Sox2 mRNA expression upon CsA (1 μM) treatment in L3.6 cells (adherent versus spheres) (*P 0.05).E Bright‐field light microscopy images as well as quantification of KPNC spheres after transient NFATc1 depletion.


Figure 7A Expression analysis of indicated miRNAs upon adenoviral re ‐ expression of p53 in KPΔNC tumor cells , detected by real ‐ time PCR ( P 0 . 05 ) . B Protein expression of EMT marker genes after adenoviral re ‐ expression of GFP ‐ tagged p53 in KPΔNC tumor cells . C , D Protein ( C ) expression levels of EMT and stemness marker genes upon miRNA ‐ 34a and miRNA ‐ 200c overexpression using specific mimics in KPNC tumor cells . Representative results from at least three independent experiments are shown ( means ± SD ; P 0 . 05 ) . C , D mRNA ( D ) expression levels of EMT and stemness marker genes upon miRNA ‐ 34a and miRNA ‐ 200c overexpression using specific mimics in KPNC tumor cells . Representative results from at least three independent experiments are shown ( means ± SD ; P 0 . 05 ) . E Western blot analysis of EMT marker expression in KNC cells following depletion of miR ‐ 200c . F ChIP experiments showing NFATc1 binding along with H3K27 acetylation mark at the selected Snai1 enhancer region as well as Sox2 and RNA polymerase II binding at Snai1 promoter after miR ‐ 200c overexpression in KPNC tumor cells . Means ± SD are shown from one out of three independent experiments . G ChIP analysis in KP ∆ NC tumor cells after adenoviral GFP ‐ tagged p53 overexpression . NFATc1 and H3K27ac binding are illustrated on Snai1 enhancer , and Sox2 and RNA polymerase II binding are demonstrated on Snai1 promoter . Means ± SD are shown from one out of three independent experiments . H Quantification of KP ∆ NC tumour cell - derived spheres after adenoviralGFP ‐ tagged p53 overexpression .

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Figure 7A Expression analysis of indicated miRNAs upon adenoviral re‐expression of p53 in KPΔNC tumor cells, detected by real‐time PCR (P 0.05).B Protein expression of EMT marker genes after adenoviral re‐expression of GFP‐tagged p53 in KPΔNC tumor cells.C, D Protein (C) expression levels of EMT and stemness marker genes upon miRNA‐34a and miRNA‐200c overexpression using specific mimics in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05).C, D mRNA (D) expression levels of EMT and stemness marker genes upon miRNA‐34a and miRNA‐200c overexpression using specific mimics in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05).E Western blot analysis of EMT marker expression in KNC cells following depletion of miR‐200c.F ChIP experiments showing NFATc1 binding along with H3K27 acetylation mark at the selected Snai1 enhancer region as well as Sox2 and RNA polymerase II binding at Snai1 promoter after miR‐200c overexpression in KPNC tumor cells. Means ± SD are shown from one out of three independent experiments.G ChIP analysis in KP∆NC tumor cells after adenoviral GFP‐tagged p53 overexpression. NFATc1 and H3K27ac binding are illustrated on Snai1 enhancer, and Sox2 and RNA polymerase II binding are demonstrated on Snai1 promoter. Means ± SD are shown from one out of three independent experiments.H Quantification of KP∆NC tumour cell-derived spheres after adenoviralGFP‐tagged p53 overexpression.


Figure 1 ( A ) Top panel : Scheme illustrating the tetracycline inducible Flp - In 293 system that controls the expression of HA - IKKε or HA - GFP . Bottom panel : Representative western blot showing induced expression of HA - IKKε in Flp - In 293 cells treated with doxycycline ( Dox , 50 ng / ml ) for 16 hours compared to endogenous IKKε in T47D , MDA - MB - 231 and MDA - MB - 468 breast cancer cell lines . ( B ) Heatmap and hierarchical clustering of metabolite concentrations in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox , 50 ng / ml , 16 hours ) ( n = 5 technical replicates ) . ( C ) Serine production from glucose ( serine m + 3 , 13C6 - glucose labelling , left panel ) and glutamine ( serine m + 1 , 15N2 - glutamine labelling , right panel ) in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) metabolite levels were normalised to the internal standard HEPES . ( E ) Contribution of pyruvate and glucose - derived carbon to TCA cyle metabolites in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) . ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES . ( F ) Fractional enrichment of serine , malate and citrate 13C - isotopologues in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) ( n = 5 technical replicates ) . ( G ) Fractional enrichment of the serine 15 - N - isotopologue in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) . m + 1 shows the naturally occurring 13C isotopologue ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES .

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Figure 1(A) Top panel: Scheme illustrating the tetracycline inducible Flp-In 293 system that controls the expression of HA-IKKε or HA-GFP. Bottom panel: Representative western blot showing induced expression of HA-IKKε in Flp-In 293 cells treated with doxycycline (Dox, 50 ng/ml) for 16 hours compared to endogenous IKKε in T47D, MDA-MB-231 and MDA-MB-468 breast cancer cell lines.(B) Heatmap and hierarchical clustering of metabolite concentrations in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (Dox, 50 ng/ml, 16 hours) (n=5 technical replicates).(C) Serine production from glucose (serine m+3, 13C6-glucose labelling, left panel) and glutamine (serine m+1, 15N2-glutamine labelling, right panel) in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) metabolite levels were normalised to the internal standard HEPES.(E) Contribution of pyruvate and glucose-derived carbon to TCA cyle metabolites in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.(F) Fractional enrichment of serine, malate and citrate 13C-isotopologues in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) (n=5 technical replicates).(G) Fractional enrichment of the serine 15-N-isotopologue in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). m+1 shows the naturally occurring 13C isotopologue (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.


Figure 2Representative western blot showing level of IKKε in IKBKE ( IKKε ) - silenced ( A ) T47D breast cancer cell lines . Representative western blot showing level of IKKε in IKBKE ( IKKε ) - silenced ( B ) MDA - MB - 468 breast cancer cell lines . ( C - D ) Heatmap and hierarchical clustering of metabolite concentrations in ( C ) IKBKE ( IKKε ) - silenced T47D cells and ( D ) IKBKE ( IKKε ) - silenced MDA - MB - 468 cells ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES . ( E ) Glycine production , representative of serine production , from glutamine ( glycine m + 1 , 15N - glutamine labelling ) and glucose ( glycine m + 2 , 13C6 - glucose labelling ) , and contribution of pyruvate and glucose - derived carbon to TCA cycle metabolites ( citrate m + 2 , malate m + 2 , 13C6 - glucose labelling ) in IKBKE ( IKKε ) - silenced T47D cells . ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES . ( F ) Serine production from glutamine ( serine m + 1 , 15N - glutamine labelling ) , and serine and glycine production from glucose ( glycine m + 2 , serine m + 3 , 13C6 - glucose labelling ) as well as contribution of pyruvate and glucose - derived carbon to TCA cycle metabolites ( malate m + 2 , 13C6 - glucose labelling ) in IKBKE ( IKKε ) - silenced MDA - MB - 468 cells .

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Figure 2Representative western blot showing level of IKKε in IKBKE (IKKε)-silenced (A) T47D breast cancer cell lines.Representative western blot showing level of IKKε in IKBKE (IKKε)-silenced (B) MDA-MB-468 breast cancer cell lines.(C-D) Heatmap and hierarchical clustering of metabolite concentrations in (C) IKBKE (IKKε)-silenced T47D cells and (D) IKBKE (IKKε)-silenced MDA-MB-468 cells (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.(E) Glycine production, representative of serine production, from glutamine (glycine m+1, 15N-glutamine labelling) and glucose (glycine m+2, 13C6-glucose labelling), and contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (citrate m+2, malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced T47D cells. (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.(F) Serine production from glutamine (serine m+1, 15N-glutamine labelling), and serine and glycine production from glucose (glycine m+2, serine m+3, 13C6-glucose labelling) as well as contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced MDA-MB-468 cells.


Figure 3 ( A ) Basal oxygen consumption in Flp - In 293 HA - IKKε wt , Flp - In 293 HA - IKKε KD - m and Flp - In 293 HA - IKKε UbLD - m cells following treatment with doxycycline ( Dox , 16 hours ) , measured using Oroboros high resolution respirometry . Data are normalised to non - treated control cells . ( B ) Average TMRM staining intensity in Flp - In 293 HA - IKKε wt , Flp - In 293 HA - IKKε KD - m and Flp - In 293 HA - IKKε UbLD - m expressing cells induced by doxycycline ( Dox , 16 hours ) . Data are normalised to non - treated control cells . ( C ) Basal mitochondrial oxygen consumption rate ( OCR ) in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines , measured using Seahorse XF96e or XF24 analysis . ( D ) OCR in mitochondria isolated from Flp - In 293 HA - GFP or HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) , measured using Oroboros high resolution respirometry . ( F ) Representative western blot showing l evel of IKKε in three independent single cell clones of Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells following treatment with doxycycline ( 100 ng / ml , 16 hours ) . ( G ) Relative pyruvate dehydrogenase ( PDH ) activity in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) . ( H ) Average TMRM staining intensity in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) and DCA ( both for 16 hours ) . Data are normalised to non - treated Flp - In 293 HA - IKKε cells . ( I ) Basal OCR in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml ) in combination with dichloroacetate ( DCA ) for 16 hours , measured using Oroboros high resolution respirometry . ( J ) Basal OCR in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml ) in combination with pyruvate deprivation for 16 hours , measured using Oroboros high resolution respirometry .

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Figure 3(A) Basal oxygen consumption in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m cells following treatment with doxycycline (Dox, 16 hours), measured using Oroboros high resolution respirometry. Data are normalised to non-treated control cells.(B) Average TMRM staining intensity in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m expressing cells induced by doxycycline (Dox, 16 hours). Data are normalised to non-treated control cells.(C) Basal mitochondrial oxygen consumption rate (OCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines, measured using Seahorse XF96e or XF24 analysis.(D) OCR in mitochondria isolated from Flp-In 293 HA-GFP or HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours), measured using Oroboros high resolution respirometry.(F) Representative western blot showing level of IKKε in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells following treatment with doxycycline (100 ng/ml, 16 hours).(G) Relative pyruvate dehydrogenase (PDH) activity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours).(H) Average TMRM staining intensity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) and DCA (both for 16 hours). Data are normalised to non-treated Flp-In 293 HA-IKKε cells.(I) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with dichloroacetate (DCA) for 16 hours, measured using Oroboros high resolution respirometry.(J) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with pyruvate deprivation for 16 hours, measured using Oroboros high resolution respirometry.


Figure 4 ( A ) Representative western blot showing level of ATF4 in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( 16 hours ) . ( B ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours . Data are expressed as fold changes , relative to levels in non - treated Flp - In 293 HA - GFP cells and normalised to β - Actin ( n = 4 biological replicates ) ( C ) Representative western blot showing levels of IKKε , IRF3 , phosphorylated IRF3 ( S396 ) and SBP enzymes in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours . ( D ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours . Data are expressed as fold changes , relative to levels in a non - silenced , non - treated control , and normalised to β - Actin ( n = 5 biological replicates ) . ( 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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Figure 4(A) Representative western blot showing level of ATF4 in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (16 hours).(B) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in non-treated Flp-In 293 HA-GFP cells and normalised to β-Actin (n=4 biological replicates)(C) Representative western blot showing levels of IKKε, IRF3, phosphorylated IRF3 (S396) and SBP enzymes in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.(D) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in a non-silenced, non-treated control, and normalised to β-Actin (n=5 biological replicates).(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.


Figure 5 ( 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 . ( 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 ) . ( 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 .

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

Figure 5(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.(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).(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.


Figure 6 ( 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 ) . ( 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 . 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 . ( K , L ) Correlation of IKBKE ( IKKε ) and PSAT1 ( K ) or IKBKE ( IKKε ) and ATF4 ( L ) expression in the ER negative sample subset .

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

Figure 6(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).(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.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.(K, L) Correlation of IKBKE (IKKε) and PSAT1 (K) or IKBKE (IKKε) and ATF4 (L) expression in the ER negative sample subset.


Figure 1 ( 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 .

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

Figure 1(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.


Figure 2 ( 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 . ( C ) Coomassie staining of SDS - PAGE separated protein extracts from ( B ) was used to control the loading .

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

Figure 2(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.(C) Coomassie staining of SDS-PAGE separated protein extracts from (B) was used to control the loading.


Figure 3Two - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Two-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.


Figure 4Chromatin 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 .

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

Figure 4Chromatin 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.


Figure 5 ( B ) The ∆ HisC ; 12xH3K36R and control ∆ HisC ; 12xWT flies show no homeotic transformations and are indistinguishable from the wild - type .

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

Figure 5(B) The ∆HisC; 12xH3K36R and control ∆HisC; 12xWT flies show no homeotic transformations and are indistinguishable from the wild-type.


Figure 6 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.(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.


Figure 1A - 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A-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.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.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.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.


Figure 2C - 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 ( CE 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2C-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 (CE 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).


Figure 3B - 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 : 50µmQuantification 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 TukeyQuantification 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B-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: 50µmQuantification 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 TukeyQuantification 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


Figure 4B 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 : 30µmC - 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 - testI 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B 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: 30µmC-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-testI 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


Figure 5E - 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 : 30µmG - 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 TukeyI 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5E-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: 30µmG-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 TukeyI 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


Figure 6C - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6C-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).


Figure 7B 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 - testsC 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 testConfocal 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 : 50µmquantification 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 - testsConfocal 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 : 50µmquantification 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 : 10µmJ 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B 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-testsC 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 testConfocal 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: 50µmquantification 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-testsConfocal 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: 50µmquantification 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: 10µmJ 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


Figure 8A 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 : 10µmB 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 TukeyC - 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 : 50µmE 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 : 10µmF 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A 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: 10µmB 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 TukeyC-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: 50µmE 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: 10µmF 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