EMBO/soda-nel huggingface dataset:
BRCA2 promotes R-loop resolution by DDX5 helicase at DNA breaks to facilitate their repair by homologous recombination (2021)
Figure 3

Figure 3. DDX5 and BRCA2 depleted cells accumulate DNA-RNA hybrids at DSBs sites.

(A) Left: Representative images of in situ PLA between S9.6 and γH2AX antibodies in U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC). When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from non-irradiated siC cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Right: Quantification of PLA spots per nucleus in each condition as indicated. At least 500 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot. See also Fig EV4A.

(B) DRIP-qPCR signal values at RBMXL1 and HIST1H2BG loci in U2OS DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1 (RH) pre-immunoprecipitation where indicated. The experiment was performed in both untreated cells (-OHT) and after tamoxifen addition (+OHT). The data represent the mean ± SEM from at least four independent experiments. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant difference. See also Fig EV4B-C.
BRCA2 promotes R-loop resolution by DDX5 helicase at DNA breaks to facilitate their repair by homologous recombination (2021)
Figure 4

Figure 4. BRCA2 enhances DDX5 retention at DNA damage sites.

(A) Top: Scheme showing the experimental set up for laser irradiation in DDX5-GFP transfected U2OS cells depleted of BRCA2 (siBRCA2) or control cells (siC). Middle: Live cell imaging of the recruitment of GFP-53BP1 or DDX5-GFP to DNA damage tracks at different time points as indicated. Exposure and processing were adjusted to best demonstrate stripes and anti-stripes. Scale bar indicates 10 µm. Bottom left: Western blot showing the siRNA mediated knock-down of BRCA2 from U2OS cells transfected with DDX5-GFP. Bottom right: Quantification of the percentage of transfected cells that exhibit DDX5-GFP "anti-stripe" pattern (reduced GFP signal at DNA damage tracks compared to the signal in the nucleus) at the times indicated in cells depleted of BRCA2 (siBRCA2) or treated with control siRNA (siC). The data represent the mean ± SEM from three independent experiments.

(B) DDX5 ChIP-qPCR signal values at RBMXL1 and ASXL1 loci in U2OS DIvA cells transfected with the indicated siRNAs and either untreated cells (-OHT) or after tamoxifen addition (+OHT). The data represent the mean ± SEM from three independent experiments. The green line represents the background levels of DDX5 signal. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant differences between untreated cells (-OHT) and after tamoxifen addition (+OHT). See also Fig EV4D.

(C) Top: Representative images of immunofluorescence of U2OS DIvA cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) and either untreated cells (-OHT) or after tamoxifen addition (+OHT), as indicated. Scale bar indicates 10 µm. Bottom: Quantification of the number of γH2AX foci per nucleus (left) and DDX5 nuclear intensity (right). The data represent at least 800 cells per condition from three independent experiments. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference.
BRCA2 promotes R-loop resolution by DDX5 helicase at DNA breaks to facilitate their repair by homologous recombination (2021)
Figure 5

Figure 5. BRCA2 stimulates the R-loop-unwinding activity of DDX5.

(A) Top: PAGE gel showing a representative unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with 32P-labelled synthetic R-loop substrate in presence or absence of 2 nM purified EGFP-MBP-BRCA2 (top gel left) or 10 nM purified BRCA2LT3 (top gel right) or 50 nM purified BRCA2T1 (bottom gel). Bottom: Quantification of the unwinding experiments showing the percentage of free RNA relative to the R-loop substrate (unwound product) as a function of DDX5 concentration alone (black) or in presence of BRCA2 (red) or BRCA2LT3 (blue) or BRCA2T1 (pink). The data represent the mean ± SD of at least three independent experiments. Right: SDS-PAGE showing 500 ng of purified 2XMBP-BRCA2LT3 and 150 ng of purified BRCA2 used in the unwinding assay. See also Fig EV5A-B.

(B) Left: Thin layer chromatography (TLC) plate showing a representative ATP hydrolysis assay in which purified MBP-DDX5-GST (50-200 nM) was incubated with R-loop synthetic substrate and [γ32P] ATP in presence or absence of or 6 nM purified BRCA2LT3. Right: Quantification of the ATP hydrolysed in each condition. No protein control was used as background. The data represent the mean ± SD from three independent experiments.
BRCA2 promotes R-loop resolution by DDX5 helicase at DNA breaks to facilitate their repair by homologous recombination (2021)
Figure 6

Figure 6. Cells bearing BRCA2-T207A show reduced BRCA2 interaction with DDX5 leading to increased DNA-RNA hybrids.

(A) Left: GFP pull-down assay from whole cells extracts of BRCA2 deficient DLD1 expressing BRCA2 (WT) or the variant T207A (T207A). DLD1 BRCA2+/+ (+/+) cell extracts were used as control for the GFP-trap. DDX5 and BRCA2 were detected with specific antibodies against DDX5 and BRCA2, respectively. StainFree images of the gels before transfer was used as loading control (cropped image is shown). Right: Quantification of the GFP-trap pull-down experiments calculated as the co-immunoprecipitated DDX5 with either BRCA2 WT or BRCA2-T207A relative to the input levels of DDX5 and the amount of immunoprecipitated EGFPMBP-BRCA2. Results are presented as the fold change compared to the BRCA2 WT clone. The data represents the mean ± SD of three independent experiments. Statistical significance of the difference was calculated with unpaired t test and the p-values show the significant difference.

(B) Left: Representative images of S9.6 immunofluorescence of DLD1 cells bearing BRCA2 (WT) or BRCA2-T207A (T207A). The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of the average nuclear intensity of S9.6 antibody in DLD1 BRCA2-WT (WT) or BRCA2-T207A (T207A) cells transfected with either a plasmid expressing GFP alone (-RH) or GFP-RNase H1 (+RH), as indicated. The red line in the plot indicates the median and each symbol represents the value of a single cell. Data correspond to at least 170 cells per condition from two independent experiments. The statistical significance of the difference was calculated with Mann-Whitney U-test; p-values show the significant difference. See also Fig EV5C.

(C) Relative DRIP-qPCR signal values (respect to the siC level at each locus) at the MALAT1, RRPH1 and HIST1H2BG loci in DLD1 BRCA2-/- cells bearing BRCA2 (WT) or BRCA2-T207A (T207A) and treated in vitro with RNase H1 (RH) pre-immunoprecipitation where indicated. The data represent the mean ± SEM from seven independent experiments. The statistical significance of the difference was calculated with paired t-Student test; the p-values show the significant difference. See also Fig EV5D.

(D) Top: Representative images of In situ PLA performed between BRCA2 and DDX5 antibodies in cells bearing BRCA2 (WT) or BRCA2-T207A (T207A), as indicated. Cells were fixed directly or 4h post-irradiation (6Gy). Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 750 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.
Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER-Golgi intermediate compartment. (2014)
Figure 1.

Starvation and PI3K-dependent generation of small membranes for LC3 lipidation.

(A–C) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS (Earle's Balanced Salt Solution) for 30 min. Total membranes (mem) from lysed cells were collected and incubated in a lipidation reaction with cytosols prepared from starved HEK293T cells. Reactions contained the indicated concentrations of PI3K inhibitor (PI3KI) 3-methyladenine (3-MA) (B) or FYVE protein (C). A diagram of the experimental scheme is shown in (A). RPN1, Ribophorin 1 (D, E) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS in the absence or presence of 20 nM wortmannin (Wtm) or 10 mM 3-methyladenine (3-MA) for 30 min. Membranes from each treated cell sample were collected and subjected to a differential centrifugation to separate the 3K ×g, 25K ×g and 100K ×g pellet fractions followed by a lipidation assay as above (E). A diagram is shown in (D). (F, G) Atg5 KO MEFs were starved for 30 min. Membranes in the 25K ×g and 100K ×g pellets from a differential centrifugation were collected as described above. A similar lipidation assay was performed in the presence of indicated concentrations (Conc in G) of 3-MA, wortmannin (F) and FYVE protein as well as a PI3P binding-deficient FYVE mutant protein (C/S) (G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).

DOI:http://dx.doi.org/10.7554/eLife.04135.002
Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER-Golgi intermediate compartment. (2014)
Figure 2.

COPII and PI3K-dependent generation of small vesicles from ERGIC for LC3 lipidation.

(A) Diagram showing the cell-free system to generate small vesicles for LC3 lipidation. Briefly, a medium-speed membrane pellet (20K ×g, 20KP) from Atg5 KO MEFs was harvested and incubated with cytosol from Atg5 KO MEFs, GTP and an ATP regeneration system (ATPR), and in the absence or presence of PI3K inhibitors (Figure 2B) or the PI3P blocker FYVE domain protein (Figure 2—figure supplement 1). The slowly-sedimenting membranes generated were separated from the donor membrane by a medium speed centrifugation (20K ×g) and collected by high-speed sedimentation (100K ×g) of the supernatant fraction (20KS). A lipidation assay with HEK293T cytosol was performed to analyze the competency of the small membranes (100KP) to induce LC3 lipidation. (B) A small vesicle generation assay as above was performed with the indicated conditions. Slowly-sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction. 3-MA, 5 mM; Wortmannin, 20 nM; S, starved Atg5 KO MEF cytosol; N, untreated Atg5 KO MEF cytosol; −/AP, in the absence of ATP regeneration and GTP and in the presence of apyrase (AP). (C) Diagram showing the fractionation procedure and fractions collected for the small vesicle generation assay shown in (D). Briefly, Atg5 KO MEF membranes were subjected to differential centrifugation to collect 3K ×g and 25K ×g pellet fractions. Sucrose gradient ultracentrifugation was performed to separate the L and P fraction after which the L was further separated by OptiPrep gradient ultracentrifugtion. Ten fractions from top to bottom were collected and combined to four as indicated (F1-4). (D) A small vesicle generation assay followed by a lipidation reaction was performed with the 3K, P and F1-4 fractions (upper four panels: Small vesicle). SDS-PAGE and immunoblot with indicated antibodies were carried out to probe the organelle enrichment of each fraction (lower five panels: Fraction input). TFR, transferrin receptor (E) A small vesicle generation assay as shown in (A) was performed in the presence of indicated proteins or drugs. Slowly-sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST, H79G (Sar1A (H79G), 0.7 μM), wortmannin (20 nM), or BFA (brefeldin A, 0.5 μg/ml). (F) A small vesicle generation assay as shown in (A) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A (H79G) or 3-MA followed by a lipidation reaction. (G) Atg5 KO MEFs were starved for 30 min. Differential centrifugation as shown in Figure 1D was performed to collect the 25K ×g and 100K ×g pellet fractions. The LC3 lipidation was performed on these two fractions with indicated concentrations of SAR1 (H79G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).

DOI:http://dx.doi.org/10.7554/eLife.04135.004
Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER-Golgi intermediate compartment. (2014)
Figure 3.

PI3K-dependent recruitment of COPII to ERGIC after starvation.

(A) Atg5 KO MEFs were either untreated or starved with or without 20 nM wortmannin for 30 min. Cells were harvested and ERGIC membranes were isolated by pooling fractions 3 and 4 (F2 in Figure 2C,D) of the OptiPrep gradient in the three-step fractionation approach followed by immunoblot to examine the amount of indicated markers on ERGIC membranes. (B) Atg5 KO MEFs were transfected with a plasmid encoding an HA-tagged SEC31A. After transfection (24 hr), the cells were treated as shown in (A). Immunofluorescence was performed with anti-ERGIC53 and anti-HA antibodies and the cells were examined by confocal microscopy. Bar: 10 μM (C) Quantification of SEC31A overlapped with ERGIC53 shown in (B) using the Pearson's correlation coefficient (PCC). Error bars represent standard deviations from ∼20 cells in three independent experiments. p values were calculated by T-test. (D) A proposed model. Starvation activates autophagic PI3K to induce COPII recruitment on ERGIC. COPII vesicles generated from ERGIC serve as templates for efficient LC3 lipidation. These membrane precursors may collaborate with other membranes such as the endoplasmic reticulum (ER), plasma membrane (PM), ATG9 compartment and mitochondria, to form a mature autophagosome.

DOI:http://dx.doi.org/10.7554/eLife.04135.013
Myc-dependent endothelial proliferation is controlled by phosphotyrosine 1212 in VEGF receptor-2 (2019)
Figure 1

Figure 1. Embryonic and postnatal vascular development in Vegfr2Y1212F /Y1212F C57Bl/6 mice

A. Partial embryonic lethality after E11.5. Chi-square, **** p<0.0001. E11.5 n=114 embryos, E14.5 n=70 embryos, Adult n=314 born pups.

B. Hindbrain vessel morphology. IsolectinB4 (IsoB4; white), ERG (red) and EdU incorporation (green). EdU signal was masked with ERG signal. Scale bar, 50 µm.

C. Hindbrain vessel density. IsolectinB4 vessel area normalized to wildtype (WT) mean; one dot is two fields/mouse; error bars: SD; unpaired T test, * p <0.05. n=18-20.

D. Hindbrain EC proliferation. EdU incorporation in ERG-positive ECs (double-positive cells, yellow in B). One dot is two fields/mouse; error bars: SD; unpaired T test, *p < 0.05. n=7-12.

E. Reduced weight gain in the adult. Error bars: SD; 2-way ANOVA p=0.0009; Sidak's multiple comparison test, **p <0.01. n=13-20 mice.

f. P6 retina vessel morphology. IsolectinB4 (IsoB4) in black; the outer 20% of the vascularized area in pink. Scale bar, 250 µm.

G-J. Vessel parameters in the P6 developing retina. G. Radial outgrowth (distance between optic nerve and vascular front). H. Vascularized area. I. Vessel area in the outer 20% of the retinal vasculature; pink in F (vessel area normalized to vascularized area). Each dot represents one retina/mouse; n=3-4. J. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents one retina/mouse; error bars: SD. n=8-9.
Myc-dependent endothelial proliferation is controlled by phosphotyrosine 1212 in VEGF receptor-2 (2019)
Figure 2

Figure 2. Embryonic and postnatal development in Vegfr2Y1212F /Y1212F FVB mice

A. Body weight. P, postnatal day; w, weeks. Error bars: SD. n=5-12 mice.

B-C. Hindbrain vessel parameters. B. Vessel density; isolectinB4 vessel area normalized to WT mean of the litter. C. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents two fields/mouse; error bars: SD; unpaired T test, not significant. n=5-11.

D. P4-P7 retina vessel morphology. IsolectinB4 (IsoB4) in black; outer 20% of the vascularized area in pink. Scale bar, 250 µm. P6 insets; sprout morphology. Scale bar, 50 µm.

E. Radial outgrowth. Distance between optic nerve and vascular front. P, postnatal day; error bars: SD; 2-way ANOVA p<0.0001; Bonferroni's multiple comparison test, *p<0.05. P4 n=3-5 retinas, one retina/mouse, P5 n=4-7, P6 n=9-9, P7 n=4-5.

F. Retina vascularized area. P, postnatal day; error bars: SD; 2-way ANOVA p=0.0013. P4 n=7-9 retinas, one retina/mouse, P5 n=4-9, P6 n=9-9, P7 n=4-5.

G. Retina vessel density in the retinal front. Quantification of the vessel area in the outer 20% of the vasculature (pink in D) (vessel area normalized to vascularized area). P, postnatal day; error bars: SD; 2-way ANOVA p=0.0054; Bonferroni's multiple comparison test, *p<0.05, ** p <0.01. P4 n=3-5 retinas, one retina/mouse, P5 n=4-6, P6 n=3-5, P7 n=4-5.

H-I. Proliferation of retinal ECs. H. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs, at P6. I. IsolectinB4 (IsoB4; white), ERG (red), EdU (green) and double-positive cells (yellow). Lower panel shows ERG/EdU merged staining alone with EdU signal masked by ERG signal. Scale bar, 50 µm; error bars: SD; unpaired T test, *p < 0.05. n=5-8 retinas, one retina/mouse.

J. Retina sprout length. Sprout length from tip to base in P6 retinas; error bars: SD; unpaired T test, *p<0.05; n=3-5 retinas, one retina/mouse.

K-L. Vessel stability. K. IsolectinB4 (IsoB4, green) and collagen IV (red) in P4 wildtype (WT) and Vegfr2Y1212F /Y1212F retinas. Arrows; collagen IV-positive empty sleeves. A; artery. Scale bar, 50 µm. L. Collagen IV empty sleeves around main arteries in P4-P7 retinas, three fields/retina/mouse. Error bars: SD; 2-way ANOVA p=0.0580; Bonferroni's multiple comparison test, ** p <0.01. P4 n=7-9, P5 n=6-12, P6 n=3-5, P7 n=4.
Myc-dependent endothelial proliferation is controlled by phosphotyrosine 1212 in VEGF receptor-2 (2019)
Figure 3

Figure 3. Specific binding of VEGFR2 Y1212 to GRB2 and PI3Kp85

A, B. Immunoblot showing VEGFR2 phosphorylation on pY1212, pY949 and pY1173. A. Anti-VEGFR2 immunoprecipitates (IP) from lung lysates from wildtype (WT) and Vegfr2Y1212F /Y1212F FVB mice, tail-vein injected with PBS or VEGF for 1 minute. B. Quantification as percentage of phosphoprotein level normalized to total VEGFR2. Each lane (A) and each dot (B) represent one individual mouse. Sidak's multiple comparison test, * p <0.05, ** p <0.01, *** p <0.001. n=3.

C. Summary plot of 51 proteins enriched in the immobilized pY1212 peptide precipitate. 10 proteins (red oval) were selected for further analysis. PSM: peptide-spectrum matches. Area: average area of spectra for the three most abundant peptides in arbitrary units.

D. Blot for peptide-enriched GRB2 and PI3Kp85. HDMEC total lysate incubated with indicated immobilized peptides followed by immunoblotting for GRB2 and PI3Kp85.

E-G. VEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes in isolated (i)ECs. E. PLA for VEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes (black puncta) in iECs from FVB wildtype (WT) and Vegfr2Y1212F/Y1212F lungs. Hoechst 33342 show nuclei (grey). Scale bar, 20 µm. F, G. Fold-increase over PBS-treated sample; each dot represents the mean of 6 fields. 2-way ANOVA p=0.0206 (F), p=0.0368 (G); Sidak's multiple comparison test, * p <0.05. n=3 experiments.

H. phosphoERK1/2 and phosphoAkt immunostaining (green) of iECs from FVB wildtype (WT) and Vegfr2Y1212F/Y1212F lungs. Hoechst 33342 (blue) shows nuclei. Arrows indicate nuclear accumulation of phosphoERK1/2 and phosphoAkt, respectively. Scale bar, 20 µm.

I. Nuclear accumulation expressed as phoshoERK1/2-positive nuclear area normalized to the total phosphoERK1/2 area by field. Each dot represents 1 field. Min, minutes of stimulation. 2-way ANOVA p=0.0248; Sidak's multiple comparison test, * p <0.05, ** p <0.01. n=3 experiments.

J. Nuclear accumulation expressed as fold-increase of nuclear phosphoAkt area in Vegfr2Y1212F/Y1212F over PBS-treated wildtype (WT) iECs, normalized to the number of nuclei. Each dot represents 1 field. Min, minutes of stimulation. Unpaired T test, *p < 0.05. n=3 experiments.

K. Immunoblot for phosphoERK1/2, total ERK1/2, phosphoAkt and total Akt on lung lysates from wiltype (WT) and Vegfr2Y1212F /Y1212F FVB mice tail-vein injected with PBS or VEGFA followed by circulation for time points indicated. Each lane represents one individual mouse.

L. Quantification of phosphoERK1/2 /total ERK1/2 expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0025; unpaired T test, *p < 0.05. n=3 experiments.

M. Quantification of phosphoAkt/total Akt expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0003; unpaired T test, *p < 0.05. n=3 experiments.