EMBO/SourceData huggingface dataset:
Figure legend: "(C) A549 cells, stably expressing GFP-LC3, were infected with WT or P. aeruginosa mutants and evaluated by fluorescence microscopy, scale bars: 10 µm. GFP-LC3 puncta representing the autophagosomes was quantified and presented in the graph. Each bar represents mean value from three independent experiments and error bars represent standard deviation. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. *p ≤ 0.05; **p ≤ 0.01. P. aeruginosa mutant strains legend: ΔSTY (mutant deficient for the three cytotoxins), WT (contains all cytotoxin), ΔS (deficient for ExoS but still express ExoT and ExoY), ΔTY (express ExoS but deficient for ExoT and ExoY)."
NCBI gene IDs:
"GFP: 
ExoS: 879837
ExoT: 878350
ExoY: 879421
LC3: 64862"
Figure legend: "(D,E) A549 cells, stably expressing GFP-LC3, were infected for 4 hours with WT or with one of P. aeruginosa mutants (ΔSTY, ΔS and ΔTY) and evaluated by fluorescence microscopy (D). P. aeruginosa were visualized using specific antibody (red) and the colocalized bacteria signals with GFP-LC3 puncta (green) were merged and presented as yellow. Scale bars: 10 µm. (E) Quantitative analysis of co-localization of internalized P. aeruginosa with GFP-LC3 positive puncta. The colocalization of these two signals was quantified with 10 independent visual fields of more than 100 cells. Data represent mean ± SD of three independent experiments. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. *p ≤ 0.05; **p ≤ 0.01."
NCBI gene IDs:
"GFP: 
LC3: 64862"
Figure legend: "(F) Representative transmission electron microscopy images of A549 cells infected with P. aeruginosa containing ExoS (ΔTY) or deficient for the three T3SS effector proteins (ΔSTY) for 4 hours. ΔSTY P. aeruginosa (white star) was engulfed by multiple membrane autophagosomes (black arrow). ΔTY P. aeruginosa (white star) were found in cytoplasm with single membrane surrounded, scale bars: 0.5 µm."
NCBI gene IDs:
"ExoS: 879837"
Figure legend: "(G) A549 cells, stably expressing lentiviral vectors containing random shRNA or Atg7 shRNA, were infected with WT or P. aeruginosa mutants (ΔSTY, ΔTY and ΔS) at MOI of 10. All infections were followed by 1 hours of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: The triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells from three biological replicates. The bars represent the means of CFU of each group (three biological replicates each group), error bars represent standard deviation. The significance of differences between different assay was determined using two-tailed student's t test with Welch's correction, NS: not significant; **p ≤ 0.01."
NCBI gene IDs:
"Atg7: 10533"
Figure legend: "(A) Primary airway epithelial cells were isolated from tracheas of Atg7+/+ and Atg7-/- mouse and were evaluated by immunoblotting."
NCBI gene IDs:
"Atg7: 74244"
Figure legend: "(B) Sections of tracheas from Atg7+/+ and Atg7-/- mouse were evaluated by immunofluorescence microscopy following staining of nuclear DNA with DAPI (Blue), ciliated epithelial cell marker β-tubulin IV (green) and Atg7 (red). Scale bars: 20 µm."
NCBI gene IDs:
"Atg7: 74244"
Figure legend: "(C,D) Atg7+/+ and Atg7-/- mice were intranasally injected with 3 x107 CFU of WT, ΔS, ΔTY or ΔSTY P. aeruginosa. After 24 hours, lungs (C), spleens and livers (D) were harvested, homogenized and diluted for CFU counting. Each data point represents bacterial count (Log10 CFU/g) from individual mouse. The horizontal lines represent the mean value from each group (6 mice each group), error bars represent standard deviation. The significance of differences between different drug treatment was determined using One-way ANOVA and Dunn's multiple comparisons test., NS: not significant; *p ≤ 0.05"
NCBI gene IDs:
"Atg7: 74244"
Figure legend: "(E,F) Atg7+/+ and Atg7-/- mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY (E) ΔSTY (F). Mice survival was monitored for 10 days post infection. Kaplan-Meyer survival curves were made based on the observation of 12-18 mice/group from two independent experiments. The P-values were calculated using the log-rank test (ΔTYAtg7+/+ vs ΔTYAtg7-/-, P = 0.2186; ΔSTYAtg7+/+ vs ΔSTYAtg7-/-, P=0.0145)."
NCBI gene IDs:
"Atg7: 74244"
Figure legend: "(A A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting."
NCBI gene IDs:
"ExoS: 879837"
Figure legend: "(B) A549 cells were transfected, for 24hours, with a plasmid expressing empty vector (EV), WT GFP-ExoS, a GFP-ExoS mutant for ADPRT domain (SG+A-) or a GFP-ExoS mutant deficient in both GAP and ADPRT domains (SG-A-). Cell lysates were evaluated by immunoblotting."
NCBI gene IDs:
"GFP: 
ExoS: 879837"
Figure legend: "(C) A549 cells were treated for 24 hours with 100 µM of the ADPRT activity inhibitor MIBG and then left uninfected (NI) or infected with P. aeruginosa containing ExoS with ADPRT activity only (SG-A+). Cell lysates were evaluated by immunoblotting."
NCBI gene IDs:
"ExoS: 879837"
Figure legend: "A549 cells were infected for 4 hours with WT or P. aeruginosa mutants (ΔpscD Cell lysates were evaluated by immunoblotting."
NCBI gene IDs:
"pscD: 877924"
Figure legend: "F) A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting."
NCBI gene IDs:
"ExoS: 879837"
Figure legend: "(A) A549 cells were transfected with empty vector (VE) and vector contains Ras-G12V for 24 hours, followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting."
NCBI gene IDs:
"Ras: "
Figure legend: "(B) A549 cells were transfected, for 24 hours, with empty vector (VE) and vectors contain Ras-G12V or Ras-G12V&amp;R41K followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting."
NCBI gene IDs:
"Ras: "
Figure legend: "(A) A549 cells were transfected, for 48 hours, with a mCherry-DFCP1 plasmid and then infected, for 4 hours, with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY, SG-A-, SG-A+ or SG+A-) and the mCherry puncta number per cell was analyzed by fluorescence microscopy. Representative immunofluorescence images were showed, and scale bars represent 10 µm. (B) Quantitative analysis of mCherry-DFCP1 puncta by visually count more than 100 cells for each sample. Each bar represents mean value of from three independent experiments and error bars represent standard deviation. The significance of differences between assays was determined using ANOVA with Dunnett multiple-comparison posttest, NS: not significant; *p ≤ 0.05; **p ≤ 0.01. "
NCBI gene IDs:
"mCherry: 
DFCP1: 53349"
Figure legend: "(A A549 cells, transfected for 48 hours with vector encoding Flag-Atg14L, were infected with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY Flag-Atg14L-Vps34 complex was immunoprecipitated (IP) by Flag-antibody magnetic beads and evaluated by immunoblotting."
NCBI gene IDs:
"Flag: 
Atg14L: 22863"
Figure legend: "C) A549 cells, transfected for 48 hours with vector encoding Flag-Atg14L, were infected with P. aeruginosa , ΔSTY SG-A-, SG-A+ or SG+A-). Flag-Atg14L-Vps34 complex was immunoprecipitated (IP) by Flag-antibody magnetic beads and evaluated by immunoblotting."
NCBI gene IDs:
"Flag: 
Atg14L: 22863"
Figure legend: "(A) Amylose pulldown from  benzonase-treated HEK293T cell lysates expressing 2xMBP-BRCA2NT in untreated or irradiated   cells (6Gy; +IR). DDX5 and BRCA2NT (MBP) detected by immunoblot. StainFree  images of the gels before transfer were used as loading control"
NCBI gene IDs:
"MBP: 
BRCA2: 675"
Figure legend: "(C) Left: Representative  images of in situ proximity ligation assay (PLA) between BRCA2  and  DDX5  antibodies in  U2OS cells either left untreated (-) or irradiated (4h post-IR; 6 Gy). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. 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 untreated siC cells are shown. Scale bar indicates 10 µm. Right: Quantification of the  number of PLA  spots per nucleus. 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."
NCBI gene IDs:
"RH: 
RNase H1: "
Figure legend: "(D) Diagram showing the BRCA2 N-terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N-terminal truncations (BRCA2T1, BRCA2LT2, BRCA2LT3) or the 2xMBP tag. DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP, respectively. StainFree images of the gels before transfer were used as loading control"
NCBI gene IDs:
"BRCA2: 675"
Figure legend: "(A) Left: Representative  images  of S9.6  immunofluorescence of U2OS cells depleted of BRCA2 (siBRCA2),  DDX5  (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing  DDX5. The merged  images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of S9.6 average nuclear intensity of U2OS cells depleted of  BRCA2 (siBRCA2), DDX5  (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. 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. The data represent at least 235 cells per condition from three independent experiments."
NCBI gene IDs:
"BRCA2: 675
DDX5: 1655"
Figure legend: "(B) Top: Representative  images of in situ PLA performed with anti-DDX5 and S9.6 antibodies in EdU-labelled U2OS  cells. Where indicated, cells were transfected with a plasmid expressing RNase H1 (RH). Nuclei as defined by auto threshold plugin on the  DAPI image  (Image J) our outlined in yellow. Bottom: Quantification of  PLA spots per nucleus  in each condition as indicated. At least 300 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, the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single  PLA  spot."
NCBI gene IDs:
"RH: 
RNase H1: "
Figure legend: "(C) Representative screenshot of a specific genomic region showing DRIPc-seq  profiles at Watson (W) and Crick (C) strands in K562  cells depleted of DDX5 (siDDX5) or control cells (siC) from two independent experiments."
NCBI gene IDs:
"DDX5: 1655"
Figure legend: "(D) DNA-RNA hybrid distribution along protein-coding genes containing DRIPc-seq peaks in both conditions (siC and siDDX5) and replicates. Gene metaplots represent the mean of antisense or sense mean DRIPc-seq signal from two independent experiments in K562  cells depleted of DDX5 (siDDX5) or control cells (siC) as indicated."
NCBI gene IDs:
"DDX5: 1655"
Figure legend: "(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."
NCBI gene IDs:
"RH: 
RNase H1: 
BRCA2: 675
DDX5: 1655"
Figure legend: "(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."
NCBI gene IDs:
"RH: 
RNase H1: 
HIST1H2BG: 8347
RBMXL1: 494115"
Figure legend: "(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."
NCBI gene IDs:
"GFP: 
BRCA2: 675
DDX5: 1655"
Figure legend: "(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)."
NCBI gene IDs:
"ASXL1: 171023
RBMXL1: 494115"
Figure legend: "(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."
NCBI gene IDs:
"BRCA2: 675
DDX5: 1655"
Figure legend: "(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."
NCBI gene IDs:
"GFP: 
BRCA2: 675"
Figure legend: "(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."
NCBI gene IDs:
"GFP: 
RH: 
RNase H1: 
BRCA2: 675"
Figure legend: "(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."
NCBI gene IDs:
"RH: 
RNase H1: 
RRPH1: 
BRCA2: 675
HIST1H2BG: 8347
MALAT1: 378938"
Figure legend: "(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."
NCBI gene IDs:
"BRCA2: 675"
Figure legend: "(A) Top: Representative images  of In situ PLA performed between  DDX5 and S9.6 (DNA-RNA hybrids) antibodies in BRCA2 deficient  DLD1 cells (BRCA2-/-) or DLD1  stable clones expressing  BRCA2 (WT) or BRCA2-T207A (T207A). Cells were fixed directly or 4h post-irradiation (6Gy). When indicated, cells were transfected/treated with RNase H1 (RH) prior to fixation. 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 500 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. (B) Top: Representative images of In situ PLA performed between DDX5 and γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2 (WT) or BRCA2-T207A (T207A). Cells were fixed directly or 4h post-irradiation (6Gy). When indicated, cells were transfected/treated with RNase H1 (RH) prior to fixation. 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 600 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. "
NCBI gene IDs:
"RH: 
RNase H1: 
BRCA2: 675"
Figure legend: "(C) Left: Representative  images  of the recruitment of transient transfected  GFP-MBP-BRCA2  (WT and T207A) to DNA damage tracks at 5 min post-irradiation in U2OS cells. γH2AX is used as a marker of  DNA  damage. Scale bar indicates 10 µm. Right: Quantification of the  intensity of GFP signal at the laser tracks. At least 130 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the two samples we applied an unpaired t-test. The red line in the plot indicates the median and each symbol represents a single cell intensity  value."
NCBI gene IDs:
"GFP: 
MBP: 
BRCA2: 675"
Figure legend: "(D) Top: Representative images of in situ PLA performed between S9.6 (DNA-RNA hybrids) and anti-γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2  (WT) or BRCA2-T207A (T207A). When indicated, cells were transfected/treated with RNase H1 (RH) or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls in non-irradiated BRCA2  WT 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. Bottom: Quantification of the number of PLA spots per nucleus. At least 600 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."
NCBI gene IDs:
"RH: 
RNase H1: 
BRCA2: 675"
Figure legend: "(A) Top: Representative  immunofluorescence images of cells stained for RAD51 (green) in U2OS cells depleted of  DDX5 (siDDX5) and in control cells (siC) in non-treated (-) or different time points  after exposure to  IR (6Gy), as indicated. Scale bar indicates 10 µm. Bottom: Graph showing the average number of  RAD51 repair foci in both cell lines. The data represent the mean ± SEM of three independent experiments."
NCBI gene IDs:
"DDX5: 1655"
Figure legend: "(B) Top: Representative immunofluorescence images of DLD1 BRCA2  deficient cells (BRCA2-/-) or BRCA2-/- cells expressing BRCA2 WT (WT) or BRCA2-T207A (T207A) in non-treated or at different  time points post-IR (6 Gy), as indicated; hybridized with anti-RAD51 antibody (green). Scale bar indicates 10 µm. Bottom: Graph showing the average  number  of RAD51 repair foci in the three cell lines. The data represent the mean ± SEM of three independent experiments."
NCBI gene IDs:
"BRCA2: 675"
Figure legend: "(C) Left: Representative  immunofluorescence images of DLD1 BRCA2 WT or BRCA2-T207A cells 4h post-irradiation (6Gy) hybridized with anti-γH2AX and anti-RPA antibodies. Right: Quantification of the  number  of γH2AX and RPA foci per  nucleus, as indicated. The data represent at least 400 cells per condition from two 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 focus."
NCBI gene IDs:
"BRCA2: 675"