Figure 2 ( A - B ) A . thaliana plants were inoculated with wild - type , XccbphP , pXccBphP or pC13S bacterial strains cultured prior to inoculation under ( A ) normal laboratory illumination or ( B ) under light or dark conditions . After 1 , 2 or 3 d . p . i . , CFU per plant mg were determined ( n = 3 replicates ) . Data presented here derive from more than 4 independent experiments . ( C ) Stomatal closure in the presence of bacterial strains or not ( untreated ) after 1 h under light conditions . ( D ) Stomatal opening after 3 h of incubation with bacterial strains or not ( untreated ) in the dark . A control treatment without bacteria and illuminated was included ( Light ) . ( C - D ) Stomatal apertures were recorded ( n = 80 replicates ) . Data are representative of two independent experiments . ( E ) A . thalianaleaves were inoculated with wild - type , XccbphP , pXccBphP and pC13S strains , stained for callose deposits and observed by fluorescence microscopy . MgCl2 buffer ( untreated ) and flg22 peptide were used as negative and positive controls , respectively . Top panel : representative pictures of three independent experiments . Scale bar represents 200 µm . Bottom panel : the number of callose deposits per field of view ( 0 . 45 mm2 ) were determined ( n = 8 replicates ) . Data are representative of two independent experiments . ( A - E ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a two - tailed Mann - Whitney test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 )

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

Figure 2(A-B) A. thaliana plants were inoculated with wild-type, XccbphP, pXccBphP or pC13S bacterial strains cultured prior to inoculation under (A) normal laboratory illumination or (B) under light or dark conditions. After 1, 2 or 3 d.p.i., CFU per plant mg were determined (n = 3 replicates). Data presented here derive from more than 4 independent experiments.(C) Stomatal closure in the presence of bacterial strains or not (untreated) after 1 h under light conditions. (D) Stomatal opening after 3 h of incubation with bacterial strains or not (untreated) in the dark. A control treatment without bacteria and illuminated was included (Light). (C-D) Stomatal apertures were recorded (n = 80 replicates). Data are representative of two independent experiments.(E) A. thalianaleaves were inoculated with wild-type, XccbphP, pXccBphP and pC13S strains, stained for callose deposits and observed by fluorescence microscopy. MgCl2 buffer (untreated) and flg22 peptide were used as negative and positive controls, respectively. Top panel: representative pictures of three independent experiments. Scale bar represents 200 µm. Bottom panel: the number of callose deposits per field of view (0.45 mm2) were determined (n = 8 replicates). Data are representative of two independent experiments. (A-E) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a two-tailed Mann-Whitney test (*P < 0.05, **P < 0.01, ***P < 0.001)


Figure 3 ( A ) Five µL supernatants from wild - type , XccbphP , pXccBphP and pC13 strains bacterial cultures ( OD600 = 1 ) grown under light or dark conditions were plated onto PYM - carboxymethyl cellulose ( CMC ) agar plates and revealed with Congo redstaining ( n = 2 replicates ) . The extracellular β - 1 , 4 - endoglucanase production levels correlated with CMC degradation halo radiuses ( top panel ) . Halo measurements are presented in the bottom panel . Data derive from 10 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( B ) The extracellular β - 1 , 4 - endoglucanase activity from bacterial cultures supernatants ( OD600 = 1 ) was determined by a colorimetric assay in solution ( n = 2 replicates ) . Wild - type , XccbphP , pXccBphP and pC13 strains were assayed under normal laboratory conditions . Data derive from 3 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

Figure 3(A) Five µL supernatants from wild-type, XccbphP, pXccBphP and pC13 strains bacterial cultures (OD600 = 1) grown under light or dark conditions were plated onto PYM-carboxymethyl cellulose (CMC) agar plates and revealed with Congo redstaining (n = 2 replicates). The extracellular β-1,4-endoglucanase production levels correlated with CMC degradation halo radiuses (top panel). Halo measurements are presented in the bottom panel. Data derive from 10 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).(B) The extracellular β-1,4-endoglucanase activity from bacterial cultures supernatants (OD600 = 1) was determined by a colorimetric assay in solution (n = 2 replicates). Wild-type, XccbphP, pXccBphP and pC13 strains were assayed under normal laboratory conditions. Data derive from 3 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).


Figure 4 ( A ) Five µL from wild - type , XccbphP , pXccBphP and pC13S bacterial cultures ( OD600 = 1 ) were plated onto PYM - glucose agar plates under light or dark conditions . Xanthan production was determined measuring colony diameters ( n = 4 replicates ) for 20 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison test ( * P < 0 . 05 ) . ( B ) Wild - type , XccbphP , pXccBphP and pC13S were grown in 20 ml PYM liquid medium under light or dark conditions . Extracellular xanthan was purified by KCl addition and ethanol precipitation , then dried and weighted ( n = 2 replicates ) for 4 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison test ( * P < 0 . 05 ) . ( C ) Representative confocal laser - scanning microscopy pictures of biofilm development of bacteria cultured for 4 d in minimal medium in chambered cover slides under light or dark conditions . The scale bar represents 30 µm .

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

Figure 4(A) Five µL from wild-type, XccbphP, pXccBphP and pC13S bacterial cultures (OD600 = 1) were plated onto PYM-glucose agar plates under light or dark conditions. Xanthan production was determined measuring colony diameters (n = 4 replicates) for 20 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05).(B) Wild-type, XccbphP, pXccBphP and pC13S were grown in 20 ml PYM liquid medium under light or dark conditions. Extracellular xanthan was purified by KCl addition and ethanol precipitation, then dried and weighted (n = 2 replicates) for 4 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05).(C) Representative confocal laser-scanning microscopy pictures of biofilm development of bacteria cultured for 4 d in minimal medium in chambered cover slides under light or dark conditions. The scale bar represents 30 µm.


Figure 1A - E Microvascular leak and tumor cell extravasation were modeled in vitro using modified Boyden chamber experiments where lung HMVECs ( A - C ) and dermal HMVECs ( D - E ) were grown to 100 % confluence over 8 - μm - pore insert membranes . ECs were first treated with Vasculotide ( VT ) , PBS ( vehicle / negative control ) , Ang1 ( positive control ) , PEG - Cys ( polyethylene glycol backbone ) , T7c ( non - PEGylated CHHHRHSF peptides ) , or VT in the presence of 100 - fold molar excess of T7c . The concentrations used , 10 - 20 ng / mL VT and 200 - 400 ng / mL Ang1 , are estimated molar equivalents ( 0 . 71 - 1 . 43 nM ) . Thirty minutes later , ECs were stimulated with thrombin , 0 . 1 % BSA ( vehicle / negative control ) , or EDTA ( positive control ) . Another 30mins later , the amount of FITC - dextran diffusion into the lower chambers ( A , D ) provides a measure of endothelial permeability . CMTPX - labeled tumor cells ( TCs ) were then dispensed into inserts , and the amount of TC fluorescence emitting from the underside of insert membranes after 20 h ( B - C ) or 28 h ( E ) reflects the efficiency of trans - endothelial TC migration . Representative fluorescent images ( 10 × ) of membrane undersides are shown in ( C ) , where DAPI - stained nuclei of ECs / TCs are shown in blue , ' extravasated ' CMTPX - labeled TCs are shown in red , and the # of CMTPX + pixels are shown numerically in red . Means ± SEM are shown ( A , B , D , E ) . Three experiments ( twice with HMVEC - LBl and once with HMVEC - DBl ) were run with n = 3 - 5 inserts ( independent biological replicates ) per group and analyzed by two - sampled unpaired t - tests .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Microvascular leak and tumor cell extravasation were modeled in vitro using modified Boyden chamber experiments where lung HMVECs (A-C) and dermal HMVECs (D-E) were grown to 100% confluence over 8-μm-pore insert membranes. ECs were first treated with Vasculotide (VT), PBS (vehicle/negative control), Ang1 (positive control), PEG-Cys (polyethylene glycol backbone), T7c (non-PEGylated CHHHRHSF peptides), or VT in the presence of 100-fold molar excess of T7c. The concentrations used, 10-20 ng/mL VT and 200-400 ng/mL Ang1, are estimated molar equivalents (0.71-1.43 nM). Thirty minutes later, ECs were stimulated with thrombin, 0.1% BSA (vehicle/negative control), or EDTA (positive control). Another 30mins later, the amount of FITC-dextran diffusion into the lower chambers (A, D) provides a measure of endothelial permeability. CMTPX-labeled tumor cells (TCs) were then dispensed into inserts, and the amount of TC fluorescence emitting from the underside of insert membranes after 20 h (B-C) or 28 h (E) reflects the efficiency of trans-endothelial TC migration. Representative fluorescent images (10×) of membrane undersides are shown in (C), where DAPI-stained nuclei of ECs/TCs are shown in blue, 'extravasated' CMTPX-labeled TCs are shown in red, and the # of CMTPX+ pixels are shown numerically in red. Means ± SEM are shown (A,B,D,E). Three experiments (twice with HMVEC-LBl and once with HMVEC-DBl) were run with n = 3-5 inserts (independent biological replicates) per group and analyzed by two-sampled unpaired t-tests.


Figure 2A - H LM2 - 4luctumor cells were implanted intravenously into SCIDmice , generating predominantly lung - specific metastases in one experiment ( n = 5 - 6 ; A - D ) and predominantly lymphatic - specific metastases in another experiment ( n = 12 ; E - H ) . ( C and G ) Photographs of typical metastases in the lungs and caudal lymphatics , respectively . Kaplan - Meier survival analysis showed that Vasculotide ( VT ) significantly delayed mortality due to lungmetastases ( A ) but not lymphaticmetastases ( E ) . Quantitative analysis of in vivo bioluminescent images taken at 24DPI ( B ) showed that concurrent VT treatment effectively reversed the sunitinib ( SU ) - induced acceleration of lungmetastases ( D ) . With regard to lymphaticmetastases ( F , H ) , SU treatment did not accelerate their progression . Geometric means ± 95 % CI are depicted in ( D and H ) ; P values were derived by one - way ANOVA ( D ) and Kruskal - Wallis test ( H ) . The same trends were reproduced in confirmatory experiments summarized in Supplementary Fig S5 .

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

Figure 2A-H LM2-4luctumor cells were implanted intravenously into SCIDmice, generating predominantly lung-specific metastases in one experiment (n = 5-6; A-D) and predominantly lymphatic-specific metastases in another experiment (n = 12; E-H). (C and G) Photographs of typical metastases in the lungs and caudal lymphatics, respectively. Kaplan-Meier survival analysis showed that Vasculotide (VT) significantly delayed mortality due to lungmetastases (A) but not lymphaticmetastases (E). Quantitative analysis of in vivo bioluminescent images taken at 24DPI (B) showed that concurrent VT treatment effectively reversed the sunitinib (SU)-induced acceleration of lungmetastases (D). With regard to lymphaticmetastases (F, H), SU treatment did not accelerate their progression. Geometric means ± 95%CI are depicted in (D and H); P values were derived by one-way ANOVA (D) and Kruskal-Wallis test (H). The same trends were reproduced in confirmatory experiments summarized in Supplementary Fig S5.


Figure 3A - D An experiment where HT29luc tumor cells were implanted intravenously into YFP - SCID mice . ( A ) Kaplan - Meier survival analysis . ( B ) Quantification of total metastatic burden in the liver and lymphatics , at 35 days post - implantation ( DPI ) , from whole - body bioluminescent imaging ( C ) . ( D ) Photograph of typical liver metastases . E - G An experiment where SN12luc tumor cells were implanted intravenously into YFP - SCID mice . ( E ) Kaplan - Meier survival analysis . ( F ) Quantification of total metastatic burden at 50DPI , mostly within the lungs , from whole - body bioluminescent imaging ( G ) .

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

Figure 3A-D An experiment where HT29luc tumor cells were implanted intravenously into YFP-SCID mice. (A) Kaplan-Meier survival analysis. (B) Quantification of total metastatic burden in the liver and lymphatics, at 35 days post-implantation (DPI), from whole-body bioluminescent imaging (C). (D) Photograph of typical liver metastases.E-G An experiment where SN12luc tumor cells were implanted intravenously into YFP-SCID mice. (E) Kaplan-Meier survival analysis. (F) Quantification of total metastatic burden at 50DPI, mostly within the lungs, from whole-body bioluminescent imaging (G).


Figure 4A , B In modified Boyden chamber experiments , confluent dermal HMVECs were stimulated with tumor cell - conditioned media ( TC - CM ) - that is , media conditioned by either LM2 - 4luc , SN12luc , or HT29luc tumor cells - and treated with 20 ng / mL Vasculotide vs . 400 ng / mL Ang1 ( positive control ) vs . PBS ( negative control ) . Meta - analyses of treatment effects on dextran permeability ( A ) and tumor cell migration ( B ) , where each datapoint represents the mean of an independent experiment , averaged over 2 - 3 biological replicates ( inserts ) per treatment group and normalized internally within that same experiment . Statistical significance of overall Ang1 or VT treatment effects was determined by paired t - tests , n = 11 ( A ) or 6 ( B ) experimental replicates . C Representative fluorescent images ( 10 × ) of ' extravasated ' CMTPX - labeled TCs ( red ) fixed on membrane undersides . D LM2 - 4luc produced the highest levels of VEGF and IL - 8 , but lower levels of IL - 6 , compared to other TC types ; * * * P < 0 . 001 and * P < 0 . 05 ( unpaired t - tests ) .

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

Figure 4A, B In modified Boyden chamber experiments, confluent dermal HMVECs were stimulated with tumor cell-conditioned media (TC-CM)-that is, media conditioned by either LM2-4luc, SN12luc, or HT29luc tumor cells-and treated with 20 ng/mL Vasculotide vs. 400 ng/mL Ang1 (positive control) vs. PBS (negative control). Meta-analyses of treatment effects on dextran permeability (A) and tumor cell migration (B), where each datapoint represents the mean of an independent experiment, averaged over 2-3 biological replicates (inserts) per treatment group and normalized internally within that same experiment. Statistical significance of overall Ang1 or VT treatment effects was determined by paired t-tests, n = 11 (A) or 6 (B) experimental replicates.C Representative fluorescent images (10×) of 'extravasated' CMTPX-labeled TCs (red) fixed on membrane undersides.D LM2-4luc produced the highest levels of VEGF and IL-8, but lower levels of IL-6, compared to other TC types; ***P < 0.001 and *P < 0.05 (unpaired t-tests).


Figure 5A - C Vasculotide ( VT ) did not alter the growth kinetics of orthotopic LM2 - 4luc tumors ; n = 8 mice per group . Sunitinib ( SU ) treatment significantly lowered primary tumor burden as measured by volume ( B : one - way ANOVA , P < 0 . 01 ) or bioluminescent activity ( C : one - way ANOVA , P < 0 . 001 ) . D VT treatment did not alter the mean thickness of the viable peripheral regions of primary tumors , as quantified by H & amp ; amp ; E histological staining . E VT treatment did not alter the mean thickness of the CAIX + hypoxic edge between the viable periphery and necrotic core of a primary tumor ; IHC control and sample images taken at 5 × are shown . F Ex vivo bioluminescent imaging suggests that VT treatment lowered the proportion of mice with metastatic involvement in the lungs , without the corresponding effect on lymphatic metastases . Representative bioluminescent vs . photographic images of dissected lungs in a 24 - well plate are shown . G Human LM2 - 4luc cells can be identified on histological sections of mouse lung tissue by IHC staining of human vimentin , a cytoskeletal protein . Size distribution analysis show VT monotherapy reducing the occurence of bigger metastatic nodules .

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

Figure 5A-C Vasculotide (VT) did not alter the growth kinetics of orthotopic LM2-4luc tumors; n = 8 mice per group. Sunitinib (SU) treatment significantly lowered primary tumor burden as measured by volume (B: one-way ANOVA, P < 0.01) or bioluminescent activity (C: one-way ANOVA, P < 0.001).D VT treatment did not alter the mean thickness of the viable peripheral regions of primary tumors, as quantified by H&amp;E histological staining.E VT treatment did not alter the mean thickness of the CAIX+ hypoxic edge between the viable periphery and necrotic core of a primary tumor; IHC control and sample images taken at 5× are shown.F Ex vivo bioluminescent imaging suggests that VT treatment lowered the proportion of mice with metastatic involvement in the lungs, without the corresponding effect on lymphatic metastases. Representative bioluminescent vs. photographic images of dissected lungs in a 24-well plate are shown.G Human LM2-4luc cells can be identified on histological sections of mouse lung tissue by IHC staining of human vimentin, a cytoskeletal protein. Size distribution analysis show VT monotherapy reducing the occurence of bigger metastatic nodules.


Figure 6A Kaplan - Meier survival curves . Number of days gained in median survival due to treatments is listed in brackets . Yellow arrows mark the timepoints at which the bioluminescent images were taken . B Bioluminescent images of tumor burden . C - D Total bioluminescent fluxes , at 35 days post - implantation ( while the ' SU _ 3 wk ' group was still receiving 60 mg / kg / day sunitinib treatment ) , were separately quantified from the thoracic ( C ) and abdominal ( D ) regions . Geometric means and P values from Mann - Whitney tests are depicted in ( C and D ) . Percentages listed below the x - axes in panels ( C and D ) refer to treatment - associated changes in the geometric mean bioluminescence , relative to the vehicle group .

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

Figure 6A Kaplan-Meier survival curves. Number of days gained in median survival due to treatments is listed in brackets. Yellow arrows mark the timepoints at which the bioluminescent images were taken.B Bioluminescent images of tumor burden.C-D Total bioluminescent fluxes, at 35 days post-implantation (while the 'SU_3 wk' group was still receiving 60 mg/kg/day sunitinib treatment), were separately quantified from the thoracic (C) and abdominal (D) regions. Geometric means and P values from Mann-Whitney tests are depicted in (C and D). Percentages listed below the x-axes in panels (C and D) refer to treatment-associated changes in the geometric mean bioluminescence, relative to the vehicle group.


Figure 7A - F Primary human umbilical vein endothelial cells ( HUVECs ) or primary human dermal microvascular endothelial cells ( HMVEC - DBls ) were stimulated at 80 % confluency with Vasculotide ( treatment ) , Ang1 ( positive control ; ligand of Tie2 ) , PBS ( vehicle control ) , or VEGF ( negative control ; non - ligand of Tie2 ) . Tie2 was immunoprecipitated from cell lysates and immunoblotted for total Tie2 or phosphotyrosine . Vasculotide treatment did not lead to increases in the pTie2 / Tie2 ratio in HUVECs after 45 - min ( A - D ) or 15 - min ( E ) treatments , with ( E ) or without ( A - D ) serum / supplement starvation . Vasculotide also did not activate Tie2 receptors in HMVEC - DBls , whether in the presence or absence of serum , growth factor , and cytokine supplements ( F ) .

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

Figure 7A-F Primary human umbilical vein endothelial cells (HUVECs) or primary human dermal microvascular endothelial cells (HMVEC-DBls) were stimulated at 80% confluency with Vasculotide (treatment), Ang1 (positive control; ligand of Tie2), PBS (vehicle control), or VEGF (negative control; non-ligand of Tie2). Tie2 was immunoprecipitated from cell lysates and immunoblotted for total Tie2 or phosphotyrosine. Vasculotide treatment did not lead to increases in the pTie2/Tie2 ratio in HUVECs after 45-min (A-D) or 15-min (E) treatments, with (E) or without (A-D) serum/supplement starvation. Vasculotide also did not activate Tie2 receptors in HMVEC-DBls, whether in the presence or absence of serum, growth factor, and cytokine supplements (F).


Figure 8Healthy non - tumor - bearing mice ( n = 8 - 9 per group ) were treated for 1 week with vehicle ( Veh ) ; Vasculotide 250 ng / 2 days IP ( VT ) ; sunitinib 60 mg / kg / day PO ( SU ) ; or VT + SU . Tie2 immunoprecipitates from lung , kidney , or liver homogenates were pooled from two mice per treatment group and immunoblotted with an anti - Tie2 antibody ( ' Tie2 ' ) or an anti - phosphotyrosine antibody ( ' p - Tie2 ' ) . Densitometry analysis of immunoblots from ( A ) . pTie2 / Tie2 ratios were internally normalized to the vehicle group within each blot . Means ± SEM and P values ( unpaired t - tests ) are depicted .

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Figure 8Healthy non-tumor-bearing mice (n = 8-9 per group) were treated for 1 week with vehicle (Veh); Vasculotide 250 ng/2 days IP (VT); sunitinib 60 mg/kg/day PO (SU); or VT + SU. Tie2 immunoprecipitates from lung, kidney, or liver homogenates were pooled from two mice per treatment group and immunoblotted with an anti-Tie2 antibody ('Tie2') or an anti-phosphotyrosine antibody ('p-Tie2').Densitometry analysis of immunoblots from (A). pTie2/Tie2 ratios were internally normalized to the vehicle group within each blot. Means ± SEM and P values (unpaired t-tests) are depicted.


Figure 9A - B Pull - down assay . Input ligands - rhAng1 ( A ) and VT ( B ) - were incubated with either protein A sepharose beads only , bead - conjugated mouse Tie2 - Fc ( mTie2 ) , or bead - conjugated human Tie2 - Fc ( hTie2 ) . The ' bound ' fraction shows the total amount pulled down by beads . The ' unbound ' fraction is 7 % of residual / free ligands remaining in the supernatant . 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 .

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

Figure 9A-B Pull-down assay. Input ligands-rhAng1 (A) and VT (B)-were incubated with either protein A sepharose beads only, bead-conjugated mouse Tie2-Fc (mTie2), or bead-conjugated human Tie2-Fc (hTie2). The 'bound' fraction shows the total amount pulled down by beads. The 'unbound' fraction is 7% of residual/free ligands remaining in the supernatant.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.


Figure 1 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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).


Figure 2 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 .

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

Figure 2 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.


Figure 3 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 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.


Figure 4 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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Figure 4  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.


Figure 5 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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Figure 5 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.


Figure 6 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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Figure 6 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.


Figure 7 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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Figure 7 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.


Figure 8 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 . 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 .

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

Figure 8 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. 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.


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


Figure 9Cellular 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 - testCellular 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 - testCellular 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 - testCellular metabolites were measured by LC - MS - based untargeted metabolomics from extracts of Stage 11 embryos ; biological replicates for control n = 5 , for atosPBG n = 7 . Heatmap of non - targeted metabolites in atosPBG compared to wild - type embryos shown with average log2fold change ( FC ) in the non - targeted LC - MS / MS analysis ( F ) We observe increases in atosPBG in intermediates of mitochondrial fatty acid β - oxidation ( FAO ) , including different carnitine - conjugated lipids , and ( F - G ) a significant increase in ketone body substituents compared to the control . Data information : Values in heat maps are obtained from untargeted metabolomic analysis , unpaired t -

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

Figure 9Cellular 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-testCellular 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-testCellular 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-testCellular 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


Figure 1C . 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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Figure 1C. 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).


Figure 2C - 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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Figure 2C-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).


Figure 3F - 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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Figure 3F-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).


Figure 4A . 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 .

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

Figure 4A. 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.


Figure 5A - 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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Figure 5A-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.