Journal: Science Advances

Article Title: RAD51C-XRCC3 complex regulates FANCM-mediated R-loop resolution to safeguard genome integrity

doi: 10.1126/sciadv.aea5932

Figure Lengend Snippet: ( A ) Quantification of S9.6 staining in control, C/X3/X2-depleted U2OS cells with or without HU (1 mM, 4 hours) treatment. Data are represented as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥242 cells were analyzed per condition). ( B ) Quantification of S9.6 staining in control, C/X3/X2-depleted U2OS cells with or without Aph (0.4 μM, 16 hours) treatment. Data are represented as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥211 cells were analyzed per condition). ( C ) Quantification of γH2AX intensity in the presence or absence of HU (1 mM, 4 hours) treatment in control, C/X3-depleted cells with or without overexpression of RNH1-Flag. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test, ≥241 cells were analyzed per condition). ( D ) Effect of RAD51C or XRCC3 depletion on fork progression/fork symmetry following 50 nM CPT treatment as indicated in the schematic. Top: Experimental schematic; middle: representative images; and bottom: quantification of sister fork ratio. Sister fork ratios were measured by dividing the longer sister fork by the shorter fork (IdU tracts). Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥40 sister fork pairs were analyzed per condition). P values are indicated.

Article Snippet: Human cell lines U2OS (American Type Culture Collection; HTB-96; RRID: CVCL_0042), HeLa Kyoto (Sachin Kotak laboratory; RRID: CVCL_1922), and hTERT RPE-1 (Sachin Kotak laboratory) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Sigma-Aldrich), and 1% Glutamax (Gibco) under standard conditions (5% CO 2 , humidified atmosphere) at 37°C.

Techniques: Staining, Control, Two Tailed Test, Over Expression

Journal: Science Advances

Article Title: RAD51C-XRCC3 complex regulates FANCM-mediated R-loop resolution to safeguard genome integrity

doi: 10.1126/sciadv.aea5932

Figure Lengend Snippet: ( A ) Representative immunoblot indicating levels of WT and R258H RAD51C-Flag after depletion of endogenous RAD51C in U2OS cells. MCM3 levels are shown as loading control. ( B ) Quantification of S9.6 staining after transfection of cells with the indicated DNA constructs, as shown in (A). Relative nuclear S9.6 intensity data are plotted as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥252 cells were analyzed per condition). ( C ) Quantification of RNAPIIS2P + PCNA PLA foci after transfection of cells with the indicated DNA constructs. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥303 cells were analyzed per condition). ( D ) Representative immunofluorescence images (top) and quantification (bottom) of S9.6 + Flag PLA foci in WT and R258H Flag-RAD51C–transfected cells. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥731 cells were analyzed per condition). Scale bar, 5 μm. ( E ) Co-IP of exogenously expressed Flag-tagged WT and R258H RAD51C, followed by immunoblotting with indicated antibodies. Endogenous protein levels were shown in the input. ( F and G ) Representative immunofluorescence images (F) and quantification (G) of S9.6 + FANCM PLA foci in cells transfected with the indicated DNA constructs. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥251 cells were analyzed per condition). Scale bar, 5 μm. P values are indicated. res, shRNA-resistant.

Article Snippet: Human cell lines U2OS (American Type Culture Collection; HTB-96; RRID: CVCL_0042), HeLa Kyoto (Sachin Kotak laboratory; RRID: CVCL_1922), and hTERT RPE-1 (Sachin Kotak laboratory) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Sigma-Aldrich), and 1% Glutamax (Gibco) under standard conditions (5% CO 2 , humidified atmosphere) at 37°C.

Techniques: Western Blot, Control, Staining, Transfection, Construct, Two Tailed Test, Immunofluorescence, Co-Immunoprecipitation Assay, shRNA

Journal: Science Advances

Article Title: RAD51C-XRCC3 complex regulates FANCM-mediated R-loop resolution to safeguard genome integrity

doi: 10.1126/sciadv.aea5932

Figure Lengend Snippet: ( A ) Experimental schematic and representative images, and ( B ) quantification of IdU/CldU ratio for fork protection assay in cells expressing the indicated mutants of RAD51C. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, Mann-Whitney test, ≥203 fibers were analyzed per condition). ( C ) Effect of expressing WT, K131A, and K131R RAD51C mutants on replication restart following the treatment of U2OS cells with 2 mM HU for the indicated time. Top: Experimental schematic; and bottom: quantification of stalled forks. Data show percentage of stalled forks as means ± SD ( n = 3, two-tailed unpaired t test). ( D and E ) Representative immunofluorescence images (D) and quantification (E) of S9.6 staining after transfection of cells with indicated shRNA and RAD51C constructs. Relative nuclear S9.6 intensity data are plotted as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥278 cells were analyzed per condition). Scale bar, 5 μm. P values are indicated. EV, empty vector. h, hours.

Article Snippet: Human cell lines U2OS (American Type Culture Collection; HTB-96; RRID: CVCL_0042), HeLa Kyoto (Sachin Kotak laboratory; RRID: CVCL_1922), and hTERT RPE-1 (Sachin Kotak laboratory) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Sigma-Aldrich), and 1% Glutamax (Gibco) under standard conditions (5% CO 2 , humidified atmosphere) at 37°C.

Techniques: Expressing, MANN-WHITNEY, Two Tailed Test, Immunofluorescence, Staining, Transfection, shRNA, Construct, Plasmid Preparation