A : Mutant allele fraction ( MAF ) measured in tumor , CSF , plasma and urine samples ( on the y axis ) for the detected mutations , in the 52 genes that are most frequently mutated in gliomas based on the TCGA databases ( on the x axis ) , for the 8 patients analysed by targeted sequencing panels . MAF from tumor are derived from exome sequencing . MAF from CSF , plasma and urine samples are derived from the capture panels sequencing .

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A: Mutant allele fraction (MAF) measured in tumor, CSF, plasma and urine samples (on the y axis) for the detected mutations, in the 52 genes that are most frequently mutated in gliomas based on the TCGA databases (on the x axis), for the 8 patients analysed by targeted sequencing panels. MAF from tumor are derived from exome sequencing. MAF from CSF, plasma and urine samples are derived from the capture panels sequencing.


A - C : Fragment size distributions for mutant ( blue ) and non - mutant ( red ) cfDNA reads , determined from the capture sequencing data for CSF samples ( A ) , plasma samples ( B ) and urine samples ( C ) .

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A-C: Fragment size distributions for mutant (blue) and non-mutant (red) cfDNA reads, determined from the capture sequencing data for CSF samples (A), plasma samples (B) and urine samples (C).


( B ) SEC - MALLS traces of different TRIM25

(B) SEC-MALLS traces of different TRIM25 and


( C ) TRIM32 constructs . The proteins are colour coded and the domain architecture is reported next to the respective MALLS curve . The constructs were analysed over different concentration ranges and the data are reported for : T25R , 4 mg / mL ; T25RB1 , 3 . 2 mg / mL ; T25RB1B2 , 3 . 6 mg / mL ; T25RBCC , 0 . 5 mg / mL ; T32R , 3 mg / mL ; T32RB , 4 mg / mL ; T32RBCC , 4 mg / mL .

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(C) TRIM32 constructs. The proteins are colour coded and the domain architecture is reported next to the respective MALLS curve. The constructs were analysed over different concentration ranges and the data are reported for: T25R, 4 mg/mL; T25RB1, 3.2 mg/mL; T25RB1B2, 3.6 mg/mL; T25RBCC, 0.5 mg/mL; T32R, 3 mg/mL; T32RB, 4 mg/mL; T32RBCC, 4 mg/mL.


( A , C ) UBE2D3 ~ Ub discharge assays with different TRIM25 and TRIM32 constructs , respectively . Assays were carried out with TRIM constructs as indicated and the reaction was monitored over 30 min . The asterisk indicates the band for the TRIM construct .

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(A, C) UBE2D3~Ub discharge assays with different TRIM25 and TRIM32 constructs, respectively. Assays were carried out with TRIM constructs as indicated and the reaction was monitored over 30 min. The asterisk indicates the band for the TRIM construct.


( B , D ) Quantification of the discharge assays using UBE2D1 ~ UbAtto and TRIM25 or TRIM32 , respectively . The loss of UBE2D1 ~ UbAtto is plotted as the average of experimental triplicates ( ± s . d . ) .

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(B, D) Quantification of the discharge assays using UBE2D1~UbAtto and TRIM25 or TRIM32, respectively. The loss of UBE2D1~UbAtto is plotted as the average of experimental triplicates (± s.d.).


( E , G ) K63 poly - ubiquitination assays using UBE2N / UBE2V1 and TRIM25 and TRIM32 , respectively . Reactions were incubated for 30 minutes and samples taken at indicated times . The asterisk indicates the band for the TRIM construct .

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(E, G) K63 poly-ubiquitination assays using UBE2N/UBE2V1 and TRIM25 and TRIM32, respectively. Reactions were incubated for 30 minutes and samples taken at indicated times. The asterisk indicates the band for the TRIM construct.


( F , H ) Assays were quantified ( see Materials and Methods ) by supplementing the reaction with 1 uM UbAtto and integrating the loss of free UbAtto for TRIM25 and TRIM32 , respectively . Assays were carried out in triplicate .

(F, H) Assays were quantified (see Materials and Methods) by supplementing the reaction with 1 uM UbAtto and integrating the loss of free UbAtto for TRIM25 and TRIM32, respectively. Assays were carried out in triplicate.


( A ) Structure of the TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan and blue and the Zn2 + ions as grey spheres .

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(A) Structure of the TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan and blue and the Zn2+ ions as grey spheres.


( B ) Overlap of the RING dimers of TRIM32 ( cyan , 5FEY . pdb ) , TRIM25 ( yellow , 5FER . pdb ) and TRIM5 ( orange , 4TKP . pdb ) . The structures were overlapped on the circled RING domain . This overlap shows that the structures of the RINGs are very similar but that there are differences in the relative orientations of the two RINGs .

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(B) Overlap of the RING dimers of TRIM32 (cyan, 5FEY.pdb), TRIM25 (yellow, 5FER.pdb) and TRIM5 (orange, 4TKP.pdb). The structures were overlapped on the circled RING domain. This overlap shows that the structures of the RINGs are very similar but that there are differences in the relative orientations of the two RINGs.


( C ) Structure of the TRIM25 RING / E2 ~ Ub complex with the RING domains in the same colour scheme as in ( A ) , UBE2D1 in grey and the ubiquitin molecules in salmon and red .

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(C) Structure of the TRIM25 RING/E2~Ub complex with the RING domains in the same colour scheme as in (A), UBE2D1 in grey and the ubiquitin molecules in salmon and red.


( A ) Close - up of the TRIM25 RING dimer interface highlighting the hydrophobic interactions made between the four helices ( left ) . Close - up of the interface between each RING monomer and the proximal ubiquitin ( right ) .

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(A) Close-up of the TRIM25 RING dimer interface highlighting the hydrophobic interactions made between the four helices (left). Close-up of the interface between each RING monomer and the proximal ubiquitin (right).


( B ) UBE2D1 ~ UbAtto discharge assays with TRIM25 wild - type RING , the fused RING constructs and different mutants important for dimerization or the interaction with ubiquitin . Time point zero for the T25 - R Linker and T25 - R Linker V72R samples was taken before the addition of E3 as discharge is very fast . The loss of UBE2D1 ~ UbAtto is plotted as the average of experimental duplicates ( ± s . d . ) .

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(B) UBE2D1~UbAtto discharge assays with TRIM25 wild-type RING, the fused RING constructs and different mutants important for dimerization or the interaction with ubiquitin. Time point zero for the T25-R Linker and T25-R Linker V72R samples was taken before the addition of E3 as discharge is very fast. The loss of UBE2D1~UbAtto is plotted as the average of experimental duplicates (± s.d.).


( C ) UBE2D3 ~ Ub discharge assays with the same mutants as in ( B ) , stained with InstantBlue . ( D ) K63 poly - ubiquitination assays using UBE2N / UBE2V1 . The asterisk indicates the band for the TRIM construct .

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(C) UBE2D3~Ub discharge assays with the same mutants as in (B), stained with InstantBlue. (D) K63 poly-ubiquitination assays using UBE2N/UBE2V1. The asterisk indicates the band for the TRIM construct.


( A ) Role of E10 in the RING of TRIM25 ( blue ) in stabilizing the closed E2 ~ Ub conformation by contacting K11 from the proximal ubiquitin ( salmon ) and N71 of the opposite RING ( cyan ) which in turn contacts K33 of the same ubiquitin .

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(A) Role of E10 in the RING of TRIM25 (blue) in stabilizing the closed E2~Ub conformation by contacting K11 from the proximal ubiquitin (salmon) and N71 of the opposite RING (cyan) which in turn contacts K33 of the same ubiquitin.


( B ) E2 ~ Ub discharge and K63 poly - ubiquitination assays to test the role of E9 and E10 in TRIM25 activity . Substitution of Glu9 with Arg has no significant effect on activity , whereas the E10R mutation almost completely abolishes catalytic activity .

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(B) E2~Ub discharge and K63 poly-ubiquitination assays to test the role of E9 and E10 in TRIM25 activity. Substitution of Glu9 with Arg has no significant effect on activity, whereas the E10R mutation almost completely abolishes catalytic activity.


( C ) Discharge and K63 poly - ubiquitination assays to test the role of the equivalent residue E16 in TRIM32 and the role of I85R . Mutation of E16R abolishes catalytic activity indicating that the role of the glutamate is conserved .

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(C) Discharge and K63 poly-ubiquitination assays to test the role of the equivalent residue E16 in TRIM32 and the role of I85R. Mutation of E16R abolishes catalytic activity indicating that the role of the glutamate is conserved.


( D and E ) Quantification of UBE2D1 ~ UbAtto discharge assays . The loss of E2 ~ Ub is plotted as the average of experimental duplicates ( ± s . d . ) .

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(D and E) Quantification of UBE2D1~UbAtto discharge assays. The loss of E2~Ub is plotted as the average of experimental duplicates (± s.d.).


A subplantar injection of 5 % of formalin ( 20 µl ) was administered in the right hind paw of Balb / c mice and an equal volume of PBS was injected in left hind paw . After 1 hour of formalin injection , Balb / c mice were treated with a single dose of either PBS ( vehicle control ) or Diclofenac ( 10 mg / kg ) or different concentrations of rPPE2 ( 2 mg / kg and 3 mg / kg ) via intraperitoneal route and after three hours of treatment , mice were sacrificed and the paw sections were prepared and stained with Toluidine blue to quantify mast cell population . ( A ) Photographs of representative sections were visualized at 20X magnification ( scale bar = 50 µm ) . ( B ) Counting of mast cells was performed in Toluidine blue stained paw sections using ImageJ software and were normalized per unit area ( mm2 ) . Data information : Data shown are Mean ± SEM of 8 mice . For ( B , unpaired t - test was applied to calculate p values .

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A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice and an equal volume of PBS was injected in left hind paw. After 1 hour of formalin injection, Balb/c mice were treated with a single dose of either PBS (vehicle control) or Diclofenac (10 mg/kg) or different concentrations of rPPE2 (2 mg/kg and 3 mg/kg) via intraperitoneal route and after three hours of treatment, mice were sacrificed and the paw sections were prepared and stained with Toluidine blue to quantify mast cell population. (A) Photographs of representative sections were visualized at 20X magnification (scale bar = 50 µm). (B) Counting of mast cells was performed in Toluidine blue stained paw sections using ImageJ software and were normalized per unit area (mm2). Data information: Data shown are Mean ± SEM of 8 mice. For (B, unpaired t-test was applied to calculate p values.


( B - E ) In another experiment , NIH - 3T3 cells ( B , C ) or MEFs ( D , E ) were incubated with either PBS or rPPE2 ( 3 µg / ml ) . After 45 minutes of rPPE2 treatment , cells were fixed , permeabilized , and stained with anti - PPE2 Ab . DAPI was used to stain the nucleus . Nuclear localization of rPPE2 in NIH - 3T3 ( C ) and MEFs ( E ) was measured by calculating the colocalization coefficient . Data information : Data represent Mean ± SEM of more than 50 cells of three independent experiments . For Figure C , E unpaired t - test was applied to calculate p values .

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(B-E) In another experiment, NIH-3T3 cells (B, C) or MEFs (D, E) were incubated with either PBS or rPPE2 (3 µg/ml). After 45 minutes of rPPE2 treatment, cells were fixed, permeabilized, and stained with anti-PPE2 Ab. DAPI was used to stain the nucleus. Nuclear localization of rPPE2 in NIH-3T3 (C) and MEFs (E) was measured by calculating the colocalization coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure C, E unpaired t-test was applied to calculate p values.


( F - G ) ( F ) NIH - 3T3 cells were transiently transfected with either pEGFP or pEGFP - PPE2 . After 24 hours of transfection , cells were fixed and observed under a confocal microscope . DAPI was used to stain the nucleus . ( G ) Nuclear localization of rPPE2 was measured by calculating colocalization coefficient . Data information : Data represent Mean ± SEM of more than 50 cells of three independent experiments . For Figure G , unpaired t - test was applied to calculate p values .

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(F-G) (F) NIH-3T3 cells were transiently transfected with either pEGFP or pEGFP-PPE2. After 24 hours of transfection, cells were fixed and observed under a confocal microscope. DAPI was used to stain the nucleus. (G) Nuclear localization of rPPE2 was measured by calculating colocalization coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure G, unpaired t-test was applied to calculate p values.


( D ) Lysates prepared from the same samples used in C were resolved on SDS - PAGE and checked for GFP expression by Western blotting using anti - GFP Ab . GAPDH was used as a loading control .

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(D) Lysates prepared from the same samples used in C were resolved on SDS-PAGE and checked for GFP expression by Western blotting using anti-GFP Ab. GAPDH was used as a loading control.


( E ) Varying concentrations of rPPE2 protein were incubated with [ γ - 32P ] - ATP oligonucleotides of 60 bp spanning SCF promoter . DNA - protein complexes were resolved on 7 % native polyacrylamide gel . In cold competition reactions , equimolar cold / unlabeled 60 bp oligonucleotides were used ( arrow represents PPE2 - oligo complex ) . The data shown are representative of 3 independent experiments .

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(E) Varying concentrations of rPPE2 protein were incubated with [γ-32P]-ATP oligonucleotides of 60 bp spanning SCF promoter. DNA-protein complexes were resolved on 7% native polyacrylamide gel. In cold competition reactions, equimolar cold/unlabeled 60 bp oligonucleotides were used (arrow represents PPE2-oligo complex). The data shown are representative of 3 independent experiments.


A subplantar injection of 5 % of formalin ( 20 µl ) was administered in the right hind paw of Balb / c mice , and an equal volume of PBS was injected in left hind paw . After induction of inflammation , Balb / c mice were treated with PBS or peptide ( 8 mg / kg ) via the intraperitoneal route . After 3 hours , paw tissues were harvested and used for cDNA synthesis . B ) ( B ) Next , Toluidine blue staining was done to check the mast cell population . Photographs of representative sections were visualized at 20X magnification ( scale bar = 100 µm ) .

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A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice, and an equal volume of PBS was injected in left hind paw. After induction of inflammation, Balb/c mice were treated with PBS or peptide (8 mg/kg) via the intraperitoneal route. After 3 hours, paw tissues were harvested and used for cDNA synthesis. B) (B) Next, Toluidine blue staining was done to check the mast cell population. Photographs of representative sections were visualized at 20X magnification (scale bar = 100 µm).


A subplantar injection of 5 % of formalin ( 20 µl ) was administered in the right hind paw of Balb / c mice , and an equal volume of PBS was injected in left hind paw . After induction of inflammation , Balb / c mice were treated with PBS or peptide ( 8 mg / kg ) via the intraperitoneal route . After 3 hours , paw tissues were harvested and used for cDNA synthesis . ( C ) Counting of mast cells was performed in Toluidine blue - stained paw sections using ImageJ software and was normalized per unit area ( mm2 ) . Data information : Data shown are Mean ± SEM of 8 mice . unpaired t - test was applied to calculate p values .

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

A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice, and an equal volume of PBS was injected in left hind paw. After induction of inflammation, Balb/c mice were treated with PBS or peptide (8 mg/kg) via the intraperitoneal route. After 3 hours, paw tissues were harvested and used for cDNA synthesis. (C) Counting of mast cells was performed in Toluidine blue-stained paw sections using ImageJ software and was normalized per unit area (mm2). Data information: Data shown are Mean ± SEM of 8 mice. unpaired t-test was applied to calculate p values.


( B ) The confirmative RT - PCRs were resolved in a 2 % agarose gel . Untreated reverse transcriptase RNAs ( - RT ) and genomic DNA served as negative and positive controls , respectively .

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(B) The confirmative RT-PCRs were resolved in a 2% agarose gel. Untreated reverse transcriptase RNAs (-RT) and genomic DNA served as negative and positive controls, respectively.


( D ) Left panel : Absorption spectra of dark - adapted XccBphP after illumination with red , far - red , fluorescent white lamp light , sunlight or sunlight filtered through green leaves ( LF ) . Pr / Pfr equilibriums scheme derived from absorption spectra is depicted in the bottom panel . Right panel : The XccBphP - C13S mutant was purified in the presence of BV and its absorption spectra recorded upon illumination with red , far - red and white light or dark adaptation .

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(D) Left panel: Absorption spectra of dark-adapted XccBphP after illumination with red, far-red, fluorescent white lamp light, sunlight or sunlight filtered through green leaves (LF). Pr/Pfr equilibriums scheme derived from absorption spectra is depicted in the bottom panel. Right panel: The XccBphP-C13S mutant was purified in the presence of BV and its absorption spectra recorded upon illumination with red, far-red and white light or dark adaptation.


( 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 .

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(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 .

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(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 )

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(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)


( 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 ) .

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(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 O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) The 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).


( 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 ) .

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(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 ) .

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(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 .

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(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.


A - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-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.


A - 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 .

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A-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.


A - 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 .

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A-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 ) .

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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).


A , 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 .

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A, 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 ) .

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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).


A - 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 ) .

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A-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 .

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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 .

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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 .

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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 .

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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.


A 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 .

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A 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 .

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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.


A - 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 ) .

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A-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).


Healthy 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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Healthy 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.


A - 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 .

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A-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 .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

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

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


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

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

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