C Quantitative kinetics analysis by SPR . All analytes were run at increasing concentrations of 0 . 625 , 1 . 25 , 2 . 5 , 5 , and 10 nM , at 37 ° C and pH 7 . 4 . The positive controls - rhAng1 , BowAng1 , COMP - Ang1 - all bound strongly to immobilized hTie2 - Fc . There was no detectable binding of VT , or the negative control , rhVEGF , to immobilized hTie2 - Fc .

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C Quantitative kinetics analysis by SPR. All analytes were run at increasing concentrations of 0.625, 1.25, 2.5, 5, and 10 nM, at 37°C and pH 7.4. The positive controls-rhAng1, BowAng1, COMP-Ang1-all bound strongly to immobilized hTie2-Fc. There was no detectable binding of VT, or the negative control, rhVEGF, to immobilized hTie2-Fc.


F . A physical 650 - kb window of the genomic distribution of genes surrounding SNP rs4971059 , such as EFNA4 , KRTCAP2 , MUC1 , MIR92B , THBS3 , and DAP3 ( top ) . Total RNAs were extracted from [ GG ] , [ GA ] , or [ AA ] MCF - 10A cells and analyzed for the expression of EFNA4 , KRTCAP2 , TRIM46 , MUC1 , THBS3 , MIR92B , or DAP3 by RT - qPCR . TRIM28 was examined as a negative control ( bottom ) .

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F. A physical 650-kb window of the genomic distribution of genes surrounding SNP rs4971059, such as EFNA4, KRTCAP2, MUC1, MIR92B, THBS3,and DAP3 (top). Total RNAs were extracted from [GG], [GA], or [AA] MCF-10A cells and analyzed for the expression of EFNA4, KRTCAP2, TRIM46, MUC1, THBS3, MIR92B, or DAP3 by RT-qPCR. TRIM28 was examined as a negative control (bottom).


B . MCF - 7 cells were transfected with a control vector or FLAG - TRIM46 for 40 h and treated with DMSO or MG132 ( 2μM ) for 10h . Cellular lysates were then collected for western blotting with antibodies against the indicated proteins .

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B. MCF-7 cells were transfected with a control vector or FLAG-TRIM46 for 40 h and treated with DMSO or MG132 (2μM) for 10h. Cellular lysates were then collected for western blotting with antibodies against the indicated proteins.


F . Schematic diagrams of HDAC1 deletion mutants . GST pull - down experiments were performed with bacterially expressed GST - fused proteins as indicated and in vitro transcribed / translated TRIM46 .

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F. Schematic diagrams of HDAC1 deletion mutants. GST pull-down experiments were performed with bacterially expressed GST-fused proteins as indicated and in vitro transcribed/translated TRIM46.


F . MCF - 7 cells were transfected with empty vector or FLAG - TRIM46 for forty - eight hours and then treated with 50 μg / mL cycloheximide ( CHX ) for the indicated hours . DMSO was added to cells 10 h before cellular proteins were collected for western blotting ( left ) . On the right panel , quantitation was done by densitometry analysis of the immunoblots and expressed as signals of HDAC1 . G . MCF - 7 cells were treated as described in F , except that MG132 ( 10 μM ) was added to cells 10 h before cellular proteins were collected for western blotting ( left ) . Quantitation was performed on the right panel as in F . H . MCF - 7 cells were treated with control siRNA or TRIM46 siRNA . Forty - eight hours after transfection , cells were treated with 50 μg / mL CHX for the indicated hours before cellular proteins were extracted for western blotting analysis ( left ) . Quantitation was performed on the right panel as in F .

O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O 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. MCF-7 cells were transfected with empty vector or FLAG-TRIM46 for forty-eight hours and then treated with 50 μg/mL cycloheximide (CHX) for the indicated hours. DMSO was added to cells 10 h before cellular proteins were collected for western blotting (left). On the right panel, quantitation was done by densitometry analysis of the immunoblots and expressed as signals of HDAC1. G. MCF-7 cells were treated as described in F, except that MG132 (10 μM) was added to cells 10 h before cellular proteins were collected for western blotting (left). Quantitation was performed on the right panel as in F. H. MCF-7 cells were treated with control siRNA or TRIM46 siRNA. Forty-eight hours after transfection, cells were treated with 50 μg/mL CHX for the indicated hours before cellular proteins were extracted for western blotting analysis (left). Quantitation was performed on the right panel as in F.


F . MCF - 7 cells were transfected with empty vector or FLAG - TRIM46 for forty - eight hours and then treated with 50 μg / mL cycloheximide ( CHX ) for the indicated hours . DMSO was added to cells 10 h before cellular proteins were collected for western blotting ( left ) . On the right panel , quantitation was done by densitometry analysis of the immunoblots and expressed as signals of HDAC1 .

O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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. MCF-7 cells were transfected with empty vector or FLAG-TRIM46 for forty-eight hours and then treated with 50 μg/mL cycloheximide (CHX) for the indicated hours. DMSO was added to cells 10 h before cellular proteins were collected for western blotting (left). On the right panel, quantitation was done by densitometry analysis of the immunoblots and expressed as signals of HDAC1.


In vivo ubiquitination assays performed in MCF - 7 cells . ( B ) Stable shCTR - or shTRIM46 - expressing MCF7 cells were transfected with His - Ub for 40 h . Cells were then treated with MG132 ( 10 μM ) for 12 h before Ni - NTA bead precipitation followed by IB with anti - HDAC1 .

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In vivo ubiquitination assays performed in MCF-7 cells. (B) Stable shCTR- or shTRIM46-expressing MCF7 cells were transfected with His-Ub for 40 h. Cells were then treated with MG132 (10 μM) for 12 h before Ni-NTA bead precipitation followed by IB with anti-HDAC1.


( C ) and ( D ) MCF - 7 cells were co - transfected with the indicated plasmids . Forty - eight hours after transfection , cells were treated with MG132 for 10 h before cellular extracts were harvested for Ni - NTA bead precipitation followed by IB with anti - HDAC1 .

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(C) and (D) MCF-7 cells were co-transfected with the indicated plasmids. Forty-eight hours after transfection, cells were treated with MG132 for 10 h before cellular extracts were harvested for Ni-NTA bead precipitation followed by IB with anti-HDAC1.


E - G . Verification of representative genes targeted by the TRIM46 - HDAC1 axis . ( E ) MCF - 7 cells were transfected with empty vector , FLAG - TRIM46 , or FLAG - TRIM46 together with FLAG - HDAC1 for 48 h . ( F ) MCF - 7 cells were transfected with control siRNA , TRIM46 siRNA , or TRIM46 siRNA together with HDAC1 siRNA for 72 h . ( G ) MCF - 7 cells were transfected with control siRNA , TRIM46 siRNA , or a siRNA - resistant TRIM46 construct together with TRIM46 siRNA for 72 h . Total RNAs were extracted from cells and RT - qPCR was performed with primers specific for indicted genes .

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E-G. Verification of representative genes targeted by the TRIM46-HDAC1 axis. (E) MCF-7 cells were transfected with empty vector, FLAG-TRIM46, or FLAG-TRIM46 together with FLAG-HDAC1 for 48 h. (F) MCF-7 cells were transfected with control siRNA, TRIM46 siRNA, or TRIM46 siRNA together with HDAC1 siRNA for 72 h. (G) MCF-7 cells were transfected with control siRNA, TRIM46 siRNA, or a siRNA-resistant TRIM46 construct together with TRIM46 siRNA for 72 h. Total RNAs were extracted from cells and RT-qPCR was performed with primers specific for indicted genes.


B . EdU assays were performed in MCF - 7 cells treated with control siRNA , TRIM46 siRNA or TRIM46 siRNA and HDAC1 siRNA for 72 h , or MCF - 10A cells treated with vector , FLAG - TRIM46 or FLAG - TRIM46 and FLAG - HDAC1 for 48 h . Representative images from FACS analysis are shown on the left panel , the percentage numbers of cells in S phase was quantified by FlowJo software on the right panel .

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B. EdU assays were performed in MCF-7 cells treated with control siRNA, TRIM46 siRNA or TRIM46 siRNA and HDAC1 siRNA for 72 h, or MCF-10A cells treated with vector, FLAG-TRIM46 or FLAG-TRIM46 and FLAG-HDAC1 for 48 h. Representative images from FACS analysis are shown on the left panel, the percentage numbers of cells in S phase was quantified by FlowJo software on the right panel.


G . Accumulation of γH2AX in cells depleted of TRIM46 or HDAC1 after cisplatin stimulation . MCF - 7 cells were treated with 4 μM cisplatin for 8 h before harvesting . Cell lysates were analyzed by western blotting .

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G. Accumulation of γH2AX in cells depleted of TRIM46 or HDAC1 after cisplatin stimulation. MCF-7 cells were treated with 4 μM cisplatin for 8 h before harvesting. Cell lysates were analyzed by western blotting.


B . MCF - 7 cells stably transfected with indicated shRNAs or expression plasmids or MCF - 10A cells stably transfected with expression plasmids were tested for colony formation assay .

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B. MCF-7 cells stably transfected with indicated shRNAs or expression plasmids or MCF-10A cells stably transfected with expression plasmids were tested for colony formation assay.


D . Control MCF - 7 cells or MCF - 7 cells depleted with TRIM46 were treated with different concentrations of cisplatin ( cis ) , VP16 , or adriamycin ( ADM ) for 72 h , and the proportions of viable cells were examined . E . Control MCF - 10A cells or MCF - 10A cells overexpressed TRIM46 were treated with different concentrations of cisplatin , VP16 , or adriamycin for 72 h , and the proportions of viable cells were examined .

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D. Control MCF-7 cells or MCF-7 cells depleted with TRIM46 were treated with different concentrations of cisplatin (cis), VP16, or adriamycin (ADM) for 72 h, and the proportions of viable cells were examined. E. Control MCF-10A cells or MCF-10A cells overexpressed TRIM46 were treated with different concentrations of cisplatin, VP16, or adriamycin for 72 h, and the proportions of viable cells were examined.


D . Control MCF - 7 cells or MCF - 7 cells depleted with TRIM46 were treated with different concentrations of cisplatin ( cis ) , VP16 , or adriamycin ( ADM ) for 72 h , and the proportions of viable cells were examined .

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D. Control MCF-7 cells or MCF-7 cells depleted with TRIM46 were treated with different concentrations of cisplatin (cis), VP16, or adriamycin (ADM) for 72 h, and the proportions of viable cells were examined.


A . Immunohistochemical staining of tissue arrays containing 142 breast carcinoma samples ( grade I : n = 29 , grade II : n = 69 , grade III : n = 44 ) and 20 normal mammary tissues for TRIM46 and HDAC1 expression . Representative images are shown on the left panel . The positively stained cells were analyzed , and the mean intensity was scored by Image - Pro Plus software on the right panel . In the boxplot , the central lines , the box limits , and the whiskers represented medians , 25th / 75th percentile , and min / max , respectively . ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA ) .

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A. Immunohistochemical staining of tissue arrays containing 142 breast carcinoma samples (grade I: n=29, grade II: n=69, grade III: n=44) and 20 normal mammary tissues for TRIM46 and HDAC1 expression. Representative images are shown on the left panel. The positively stained cells were analyzed, and the mean intensity was scored by Image-Pro Plus software on the right panel. In the boxplot, the central lines, the box limits, and the whiskers represented medians, 25th/75th percentile, and min/max, respectively. (*P<0.05, **P<0.01, ****P < 0.0001, one-way ANOVA).


B . Total proteins from 20 paired samples of breast cancer " T " versus adjacent normal breast tissues " A " were extracted for western blotting analysis with antibodies against TRIM46 or HDAC1 .

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B. Total proteins from 20 paired samples of breast cancer "T" versus adjacent normal breast tissues "A" were extracted for western blotting analysis with antibodies against TRIM46 or HDAC1.


E . Kaplan - Meier survival analysis of TCGA BRCA dataset for the correlation between overall survival of breast cancer patients and TRIM46 , BRCA1 , or BLM expression .

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E. Kaplan-Meier survival analysis of TCGA BRCA dataset for the correlation between overall survival of breast cancer patients and TRIM46, BRCA1, or BLM expression.


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 )

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 -

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 :

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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 -

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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 :

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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

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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 :

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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 :

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 -

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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 -

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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 -

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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 -

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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


C . Kaplan - Meier Survival plot showing low survival of chd7 mutants after 12 dpf ( N = 5 ) .

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C. Kaplan-Meier Survival plot showing low survival of chd7 mutants after 12 dpf (N=5).


E . Acetylated tubulin staining in 28 hpf controls ( left ) and mutants ( right ) showing severely affected outbranching of the trigeminal nerve ( Vth cranial nerve ) . Notably , chd7 - / - display reduced branching of the Vth cranial nerve ( arrows ) and axonal arborisation in the tectal area . Graphs showing quantitative analyses of percentage ( n = 5 ) and mean total length of peripheral projections ( n = 6 ) per zebrafish in controls and mutants ( * * * p < 0 . 001 ; * * p < 0 . 005 , Student ' s t - test ) .

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E. Acetylated tubulin staining in 28 hpf controls (left) and mutants (right) showing severely affected outbranching of the trigeminal nerve (Vth cranial nerve). Notably, chd7-/- display reduced branching of the Vth cranial nerve (arrows) and axonal arborisation in the tectal area. Graphs showing quantitative analyses of percentage (n=5) and mean total length of peripheral projections (n=6) per zebrafish in controls and mutants (***p<0.001; **p<0.005, Student's t-test).


C - H Total number of GABAergic neurons ( GFP + cells ) in ( C , D ) the optic tectum ( OT ) and cerebellum ( CB ) regions of 5 dpf wild - type and chd7 mutant fish ( n = 16 ; * * * * p < 0 . 0001 ; Student ' s unpaired t - test ) , ( E , F ) the hypothalamus ( hyp ) region ( n = 10 ; * p = 0 . 0182 ; Student ' s t - test ) and ( G , H ) the telencephalon ( tel ) ( n = 7 ; * p = 0 . 0347 ; Student ' s t - test ) .

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C-H Total number of GABAergic neurons (GFP+ cells) in (C, D) the optic tectum (OT) and cerebellum (CB) regions of 5 dpf wild-type and chd7 mutant fish (n=16; ****p<0.0001; Student's unpaired t-test), (E, F) the hypothalamus (hyp) region (n=10; *p=0.0182; Student's t-test) and (G, H) the telencephalon (tel) (n=7; *p=0.0347; Student's t-test).


F - G . Treatment with the ERK signalling inhibitor U0126 ameliorated the number of GABAergic neuron ( n = 10 - 15 ; * * p < 0 . 05 ; * * * * p < 0 . 0001 , one - way ANOVA ) .

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F-G. Treatment with the ERK signalling inhibitor U0126 ameliorated the number of GABAergic neuron (n=10-15; **p<0.05; ****p<0.0001, one-way ANOVA).


A . qPCR analysis of PAQR3 expression in a CHD7 - mutation positive patient compared to parental controls set ( N = 4 ; * * * * p < 0 . 0001 ; Student ' s t - test ) . Fold change was calculated according to the 2 ( − ΔΔCt ) method , using HPRT1 and RPS1 as housekeeping genes for normalization . All data were expressed as mean fold change ± SD across replicates , relative to control parents set to 1 ( dotted line ) , N is the number of experimental repeats .

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A. qPCR analysis of PAQR3 expression in a CHD7-mutation positive patient compared to parental controls set (N=4; ****p<0.0001; Student's t-test). Fold change was calculated according to the 2(−ΔΔCt) method, using HPRT1 and RPS1 as housekeeping genes for normalization. All data were expressed as mean fold change ± SD across replicates, relative to control parents set to 1 (dotted line), N is the number of experimental repeats.


C . ChIP - qPCR assays in LCLs showing decreased occupation of CHD7 on the PAQR3 proximal promoter in a CHD7 - mutation positive patient ( N = 3 ) compared to parental controls ( N = 6 ) ; * p < 0 . 05 ; Student ' s t - test , All data were expressed as mean fold change ± SD , N is the number of experimental repeats .

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C. ChIP-qPCR assays in LCLs showing decreased occupation of CHD7 on the PAQR3 proximal promoter in a CHD7-mutation positive patient (N=3) compared to parental controls (N=6); *p< 0.05; Student's t-test, All data were expressed as mean fold change ± SD, N is the number of experimental repeats.


A - B . GABAergic neuron defects in wild - type fish treated with ER / Golgi traffic inhibitor BFA ( n = 9 ; * * * * p < 0 . 0001 ; ns , not significant ; One - way ANOVA ) .

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A-B. GABAergic neuron defects in wild-type fish treated with ER/Golgi traffic inhibitor BFA (n=9; ****p<0.0001; ns, not significant; One-way ANOVA).


C - D . Overexpression of paqr3b mRNA improve the number of GABAergic neurons in 3dpf chd7 mutant fish ( n = 7 for chd7 - / - and n = 11 for chd7 - / - + paqr3b mRNA ; * * * * p < 0 . 0001 ; * * p < 0 . 05 ; One - way ANOVA ) . Of note , the rescue experiment was performed at 3 dpf given the transient nature of mRNA .

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C-D. Overexpression of paqr3b mRNA improve the number of GABAergic neurons in 3dpf chd7 mutant fish (n=7 for chd7-/- and n=11 for chd7-/- + paqr3b mRNA; ****p<0.0001; **p<0.05; One-way ANOVA). Of note, the rescue experiment was performed at 3 dpf given the transient nature of mRNA.


B . Lifespan analyses of chd - 7 ( gk290 ) mutants ( n = 339 ; red ) compared to WT ( n = 444 ; black ) . Log - rank test was performed for statistical analyses . * * * p < 0 . 001 .

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B. Lifespan analyses of chd-7(gk290) mutants (n=339; red) compared to WT (n=444; black). Log-rank test was performed for statistical analyses. ***p < 0.001.


D - F . Example pictures of the GABAergic nervous system cell bodies , commissures , axonal gaps and axonal breaks larger than 50 µm per worm in chd - 7 ( gk290 ) mutants expressing unc - 47p : : mCherry .

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D-F. Example pictures of the GABAergic nervous system cell bodies, commissures, axonal gaps and axonal breaks larger than 50 µm per worm in chd-7(gk290) mutants expressing unc-47p::mCherry.


O . Representative images of GABAergic neurons and total number of GABAergic neurons ( GFP + cells ) in the optic tectum ( OT ) and cerebellum ( CB ) regions of wild - type , chd7 mutant and ephedrine - treated fish ( n = 9 ; * * * * p < 0 . 0001 ; One - way ANOVA ) . Treatment with ephedrine ameliorated the number of GABAergic neurons .

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O. Representative images of GABAergic neurons and total number of GABAergic neurons (GFP+ cells) in the optic tectum (OT) and cerebellum (CB) regions of wild-type, chd7 mutant and ephedrine-treated fish (n=9; ****p<0.0001; One-way ANOVA). Treatment with ephedrine ameliorated the number of GABAergic neurons.


ChEP analysis using EGFP ( control ) - or Piwi - KD OSCs . Signals with an adjusted p - value ( Bonferroni test ) of less than 0 . 05 are highlighted in red . The signals for Lam , LamC , and H1 are labelled within the plots .

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ChEP analysis using EGFP (control)- or Piwi-KD OSCs. Signals with an adjusted p-value (Bonferroni test) of less than 0.05 are highlighted in red. The signals for Lam, LamC, and H1 are labelled within the plots.


Lam - or LamC - KD followed by qRT - PCR of RNA levels for piRNA target ( mdg1 and gypsy ) or non - target ( roo ) TEs . Error bars indicate SD ( n = 3 ) . * = p - value < 0 . 05 ( t - test ) . Note that the complete depletion of Lam and LamC is impossible since their KD causes severe damage to cells .

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Lam- or LamC-KD followed by qRT-PCR of RNA levels for piRNA target (mdg1 and gypsy) or non-target (roo) TEs. Error bars indicate SD (n=3). * = p-value<0.05 (t-test). Note that the complete depletion of Lam and LamC is impossible since their KD causes severe damage to cells.


Boxplots showing fold changes in the expression of piRNA target or non - target TEs based on RNA - seq upon Piwi - KD ( left ) , and the frequency of piRNAs , which target piRNA target TEs or non - piRNA target TEs ( right ) . Definition of piRNA target and non - target TEs is in Fig EV1E . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated using the Wilcoxon rank - sum test .

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Boxplots showing fold changes in the expression of piRNA target or non-target TEs based on RNA-seq upon Piwi-KD (left), and the frequency of piRNAs, which target piRNA target TEs or non-piRNA target TEs (right). Definition of piRNA target and non-target TEs is in Fig EV1E. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test.


Boxplot showing DamID - seq signals of piRNA target or non - target TEs upon EGFP ( control ) - or Piwi - KD . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated using the Wilcoxon rank - sum test .

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Boxplot showing DamID-seq signals of piRNA target or non-target TEs upon EGFP (control)- or Piwi-KD. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test.


Oligo - FISH images for the representative piRNA target region described in Fig 4A , upon EGFP ( control ) - or Piwi - KD . Gray indicates DAPI staining for DNA , magenta indicates FISH signal , and green indicates Lamin immunofluorescence staining . Scale bar , 5μm . Ratio of distance from FISH signal to the nuclear periphery ( DAPI surface ) is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U test .

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Oligo-FISH images for the representative piRNA target region described in Fig 4A, upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin immunofluorescence staining. Scale bar, 5μm. Ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Oligo - FISH images for changes in the intra - TAD interaction at the TAD described in ( C ) , upon EGFP ( control ) - or Piwi - KD . Gray indicates DAPI staining for DNA , green and magenta indicates FISH signal at the different positions of TADs ( TAD - 1 and TAD - 2 ) . Details of TAD probe design is described in Fig EV2C . Scale bar , 5μm . Distance between two FISH signals is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U test .

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Oligo-FISH images for changes in the intra-TAD interaction at the TAD described in (C), upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, green and magenta indicates FISH signal at the different positions of TADs (TAD-1 and TAD-2). Details of TAD probe design is described in Fig EV2C. Scale bar, 5μm. Distance between two FISH signals is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Boxplots showing fold changes in the histone mark association of piRNA target or non - target TEs based on ChIP - seq upon Piwi - KD ( Definition of piRNA target and non - target TEs are described in Fig EV1E ) . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated by Wilcoxon rank - sum test .

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Boxplots showing fold changes in the histone mark association of piRNA target or non-target TEs based on ChIP-seq upon Piwi-KD (Definition of piRNA target and non-target TEs are described in Fig EV1E). Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated by Wilcoxon rank-sum test.


ChIP ‐ qPCR analysis of H3K27Ac and H3K4me3 occupancy on the reporter gene upon transfection of λN - Nxf2 expression vector or control EGFP expression vector . Bar graph shows the occupancy relative to that of the sample transfected with a control plasmid . Error bars indicate SD ( n = 3 ) , * = p - value < 0 . 01 ( t - test ) . .

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ChIP‐qPCR analysis of H3K27Ac and H3K4me3 occupancy on the reporter gene upon transfection of λN-Nxf2 expression vector or control EGFP expression vector. Bar graph shows the occupancy relative to that of the sample transfected with a control plasmid. Error bars indicate SD (n = 3), * = p-value<0.01 (t-test)..


Immunoprecipitation ( IP ) from OSC lysate using anti - Lam , or myc antibody , followed by WB using indicated antibodies . Mouse immunoglobulin G ( n . i . ) was used for control IP . Note that OSCs used at the right panel is a line which stably expresses myc - Nxf2 .

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Immunoprecipitation (IP) from OSC lysate using anti-Lam, or myc antibody, followed by WB using indicated antibodies. Mouse immunoglobulin G (n.i.) was used for control IP. Note that OSCs used at the right panel is a line which stably expresses myc-Nxf2.


Density plots for DamID - seq signals over consensus sequences from piRNA target ( gypsy and mdg1 , orange ) and non - target TEs ( roo , blue ) in EGFP ( control ) - or Nxf2 - KD cells .

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Density plots for DamID-seq signals over consensus sequences from piRNA target (gypsy and mdg1, orange) and non-target TEs (roo, blue) in EGFP (control)- or Nxf2-KD cells.


Oligo - FISH images for the reporter of the tethering system , at 0h ( control ) , 48h hours , 96 hours after transfection of λN - Nxf2 expression vector . Gray indicates DAPI staining for DNA , magenta indicates FISH signal , and green indicates Lamin staining . Scale bar , 5μm . The ratio of distance from FISH signal to the nuclear periphery ( DAPI surface ) is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U test .

Oligo-FISH images for the reporter of the tethering system, at 0h (control), 48h hours, 96 hours after transfection of λN-Nxf2 expression vector. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin staining. Scale bar, 5μm. The ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Dot plot showing the correlation between the normalized density of indicated TEs per TAD ( x - axis ) and changes in intra - TAD interactions upon H1 ( left ) - , Egg ( middle ) - , or HP1a ( right ) - KD ( y - axis ) . Trend lines are in red . Spearman ' s rank correlation rho is indicated along with the p - value ( algorithm AS 89 ) . n . s . ; not significant ( p > 0 . 05 ) Dot plot showing the correlation between the normalized density of indicated TEs per TAD ( x - axis ) and changes in inter - TAD interactions upon H1 ( left ) - , Egg ( middle ) - , or HP1a ( right ) - KD ( y - axis ) . Trend lines are in red . Spearman ' s rank correlation rho is indicated along with the p - value ( algorithm AS 89 ) . n . s . ; not significant ( p > 0 . 05 )

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Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in intra-TAD interactions upon H1(left)-, Egg(middle)-, or HP1a(right)-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). n.s.; not significant (p > 0.05) Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in inter-TAD interactions upon H1(left)-, Egg(middle)-, or HP1a(right)-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). n.s.; not significant (p > 0.05)


Representative 3D microglia reconstructions showing morphological changes upon LPS injection within the first 48 hours . Scale bar : 10µm Morphological parameters quantification within 48 hours after LPS

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Representative 3D microglia reconstructions showing morphological changes upon LPS injection within the first 48 hours. Scale bar: 10µm Morphological parameters quantification within 48 hours after LPS injection


Representative 3D microglia reconstructions from 5 and 15 months old mice showing microglia changes 2 and 10 days post LPS injection . Scale bar :

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Representative 3D microglia reconstructions from 5 and 15 months old mice showing microglia changes 2 and 10 days post LPS injection. Scale bar: 20µm


Morphological parameters quantification for 5 and 15 months old mice after LPS injection


Two - photon representative images of wild - type and Nlrp3 - / - mice ( 5 and 15 months old ) . Scale bar : 20µm . Quantification of morphological parameters for wild - type and Nlpr3 - / - mice after LPS

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Two-photon representative images of wild-type and Nlrp3-/- mice (5 and 15 months old). Scale bar: 20µm. Quantification of morphological parameters for wild-type and Nlpr3-/- mice after LPS injection


Representative cortical images of MXO4 staining for APP and APP / Nlrp3 - / - 15 months old mice . Scale bar : 50µm .

Representative cortical images of MXO4 staining for APP and APP/Nlrp3-/- 15 months old mice. Scale bar: 50µm.


Cortical amyloid plaque number and size quantification , and Amyloid - beta1 - 40 and 1 - 42 ELISA quantification for APP and APP / Nlrp3 - / - mice ( 5 and 15 month old )

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Cortical amyloid plaque number and size quantification, and Amyloid-beta1-40 and 1-42 ELISA quantification for APP and APP/Nlrp3-/- mice (5 and 15 month old)


Two - photon images of microglia ( e - GFP ) cells clustering around amyloid plaque for APP and APP / Nlrp3 - / - ( 5 and 15 months old ) . Scale bar : 20µm b ) Quantification of morphological parameters in ( a ) ( mean of 5 - 6 ± SEM ) .

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Two-photon images of microglia (e-GFP) cells clustering around amyloid plaque for APP and APP/Nlrp3-/- (5 and 15 months old). Scale bar: 20µm b) Quantification of morphological parameters in (a) (mean of 5-6±SEM).


Flow cytometry plots from APP and APP / Nlrp3 - / - mice ( 15 months old ) , cells were gated on CD11b and MXO4 after microglia isolation . d ) Relative Aβ microglia uptake

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Flow cytometry plots from APP and APP/Nlrp3-/- mice (15 months old), cells were gated on CD11b and MXO4 after microglia isolation. d) Relative Aβ microglia uptake quantification


Two - photon microglia ( e - GFP ) images from APP and APP / Nlrp3 - / - in areas free of plaque ( 5 and 15 months old mice ) . Scale bar : 20µm Quantification of APP and APP / Nlrp3 - / - morphological parameters for microglia in areas free of

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Two-photon microglia (e-GFP) images from APP and APP/Nlrp3-/- in areas free of plaque (5 and 15 months old mice). Scale bar: 20µm Quantification of APP and APP/Nlrp3-/- morphological parameters for microglia in areas free of plaque


Iba - 1 ( green ) , CD169 ( red ) and MXO4 staining in plaque - associated areas ( cortex and hippocampus ) of 5 and 15 months old of APP and APP / Nlrp3 - / - . Note the colocalization between Iba - 1 and CD169 ( white arrows ) in APP 15 - month - old mice 2 days post LPS injection . Scale bar :

Iba-1 (green), CD169 (red) and MXO4 staining in plaque-associated areas (cortex and hippocampus) of 5 and 15 months old of APP and APP/Nlrp3-/-. Note the colocalization between Iba-1 and CD169 (white arrows) in APP 15-month-old mice 2 days post LPS injection. Scale bar: 20µm


Iba - 1 , Ki67 and DAPI staining in the cortex and hippocampus of wild - type and Nlrp3 - / - ( 5 and 15 months old ) . Microglia proliferates upon LPS injection ( white arrows ) . Non - microglia cells proliferation was observed as well ( yellow arrows ) Scale bar : 20µm . Quantification of microglial proliferation in cortex ( left panel ) and hippocampus ( right panel ) ( mean of 5 ± SEM ; two - way ANOVA followed by Tukey ' s post hoc test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

Iba-1, Ki67 and DAPI staining in the cortex and hippocampus of wild-type and Nlrp3-/- (5 and 15 months old). Microglia proliferates upon LPS injection (white arrows). Non-microglia cells proliferation was observed as well (yellow arrows) Scale bar: 20µm. Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel) (mean of 5±SEM; two-way ANOVA followed by Tukey's post hoc test, *p<0.05, **p<0.01, ***p<0.001).


Iba - 1 , Ki67 and MXO4 staining in the cortex and hippocampus of APP and APP / Nlrp3 - / - ( 5 and 15 months old ) . Microglia proliferates upon LPS injection ( white arrows ) . Non - microglia cells proliferation was observed as well ( yellow arrows ) . Scale bar : 20µm Quantification of microglial proliferation in cortex ( left panel ) and hippocampus ( right panel )

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Iba-1, Ki67 and MXO4 staining in the cortex and hippocampus of APP and APP/Nlrp3-/- (5 and 15 months old). Microglia proliferates upon LPS injection (white arrows). Non-microglia cells proliferation was observed as well (yellow arrows). Scale bar: 20µm Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel)


( A ) Summary of HMCES recovery upon TAP - MS analyses performed with 293T cells stably expressing SFB - PCNA . Cells were treated with CPT : camptothecin ( 1 μg / ml for 6 h ) ; HU : hydroxyurea ( 2 mM for 16 h ) , UV : ultraviolet light irradiation ( 10 mJ / cm2 harvested after 6 h post - irradiation ) or Thymidine ( 100 mM for 6 h ) . Recovered peptide - spectrum matches ( PSMs ) for PCNA and HMCES are indicated for each condition .

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(A) Summary of HMCES recovery upon TAP-MS analyses performed with 293T cells stably expressing SFB-PCNA. Cells were treated with CPT: camptothecin (1 μg/ml for 6 h); HU: hydroxyurea (2 mM for 16 h), UV: ultraviolet light irradiation (10 mJ/cm2 harvested after 6 h post-irradiation) or Thymidine (100 mM for 6 h). Recovered peptide-spectrum matches (PSMs) for PCNA and HMCES are indicated for each condition.


( C ) Immunoprecipitation ( IP ) of SFB - tagged full - length ( FL ) HMCES and indicated HMCES deletion mutants was conducted to decipher the region responsible for PCNA interaction .

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(C) Immunoprecipitation (IP) of SFB-tagged full-length (FL) HMCES and indicated HMCES deletion mutants was conducted to decipher the region responsible for PCNA interaction.


( D ) GST - PCNA pulldown assay was used to evaluate the interaction between PCNA and full - length HMCES or indicated HMCES deletion mutants .

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(D) GST-PCNA pulldown assay was used to evaluate the interaction between PCNA and full-length HMCES or indicated HMCES deletion mutants.


( E ) aniPOND - Western blotting analysis revealed the presence of HMCES on replication fork . Cells were pulsed with EdU for 10 mins , and click - reaction was performed . In thymidine chase samples , cells were incubated with thymidine for another 30 mins prior to click - reaction .

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(E) aniPOND-Western blotting analysis revealed the presence of HMCES on replication fork. Cells were pulsed with EdU for 10 mins, and click-reaction was performed. In thymidine chase samples, cells were incubated with thymidine for another 30 mins prior to click-reaction.


( F ) Analysis of HMCES level throughout the cell cycle . Cells were synchronized by double thymidine block and released for the indicated time points . CYCLIN E and CYCLIN B1 were used as markers for cell cycle phases .

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(F) Analysis of HMCES level throughout the cell cycle. Cells were synchronized by double thymidine block and released for the indicated time points. CYCLIN E and CYCLIN B1 were used as markers for cell cycle phases.


( A ) Western blotting analysis confirmed HMCES knockout in 293A cells ( 293A HMCES KO ) . Blots also included knockout cells reconstituted with WT SFB - HMCES .

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(A) Western blotting analysis confirmed HMCES knockout in 293A cells (293A HMCES KO). Blots also included knockout cells reconstituted with WT SFB- HMCES.


( B , C ) Colony - forming capacity of HMCES KO cells was compared to that of control wild - type 293A cells . ( C ) Box plot showing colony forming efficiencies of 293A wild - type control and HMCES KO cells . Box limits represent 25th and 75th percentile , centerline shows the median and whiskers extend minimum to maximum . Data include more than three independent repeats performed in duplicates . Student ' s t - test were performed for statistical analysis ( * * * * , p < 0 . 0001 ) .

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(B, C) Colony-forming capacity of HMCES KO cells was compared to that of control wild-type 293A cells. (C) Box plot showing colony forming efficiencies of 293A wild-type control and HMCES KO cells. Box limits represent 25th and 75th percentile, centerline shows the median and whiskers extend minimum to maximum. Data include more than three independent repeats performed in duplicates. Student's t-test were performed for statistical analysis (****, p<0.0001).


( D ) Colonogenic survival assay of 293A wild - type control and HMCES KO cells treated with various concentrations of hydroxyurea ( HU ) was shown . Seeding density with respect to control was indicated below each image . The images shown are representative of three biological repeats , each performed in duplicate .

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(D) Colonogenic survival assay of 293A wild-type control and HMCES KO cells treated with various concentrations of hydroxyurea (HU) was shown. Seeding density with respect to control was indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate.


( E ) Quantification showing survival fraction of wild - type control ( WT ) and HMCES KO cells upon HU treatment . Data are presented as mean ± s . e . m ( n = 3 , each performed in duplicates ) and student ' s t - test were performed for statistical analysis ( ns , not significant ; * , p < 0 . 05 ) .

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(E) Quantification showing survival fraction of wild-type control (WT) and HMCES KO cells upon HU treatment. Data are presented as mean±s.e.m (n=3, each performed in duplicates) and student's t-test were performed for statistical analysis (ns, not significant; *, p<0.05).


( F ) Quantification of colonogenic survival assay upon exposure of WT and HMCES KO cells to ionizing radiation ( IR ) . Data are presented as mean ± s . e . m . Quantifications are from three biological repeats performed in duplicates . Student ' s t - test was performed for statistical analysis ( * , p < 0 . 05 ; * * , p < 0 . 01 ) .

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(F) Quantification of colonogenic survival assay upon exposure of WT and HMCES KO cells to ionizing radiation (IR). Data are presented as mean±s.e.m. Quantifications are from three biological repeats performed in duplicates. Student's t-test was performed for statistical analysis (*, p<0.05; **, p<0.01).


( G , H ) Sensitivity assay and quantification of surviving WT control and HMCES KO cells determined by colony - forming assay upon exposure to H2O2 . Experiments were repeated three independent times , each time performed in duplicates and data are presented as mean ± s . e . m . Statistical significance for the corresponding experiment was analyzed by student ' s t - test ( * , p < 0 . 05 ) .

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(G, H) Sensitivity assay and quantification of surviving WT control and HMCES KO cells determined by colony-forming assay upon exposure to H2O2. Experiments were repeated three independent times, each time performed in duplicates and data are presented as mean±s.e.m. Statistical significance for the corresponding experiment was analyzed by student's t-test (*, p<0.05).


( I ) Bar graph showing sensitivity of WT , HMCES KO and stably reconstituted WT cells towards H2O2 treatment . Experiment was repeated two independent times with technical repeats .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(I) Bar graph showing sensitivity of WT, HMCES KO and stably reconstituted WT cells towards H2O2 treatment. Experiment was repeated two independent times with technical repeats.