Fig 1: Depletion of RAD51C and XRCC3, but not RAD51B, RAD51D, and XRCC2, leads to R-loop accumulation in U2OS cells.(A and B) Representative images (A) and quantification (B) of S9.6 staining in control, RAD51, and RAD51 paralog-depleted cells. Relative nuclear signal intensity data are plotted as box plot (n = 3, two-tailed unpaired t test with Welch’s correction on means of biological replicates, ≥253 cells were analyzed per condition). Scale bar, 5 μm. (C) Relative S9.6 nuclear signal intensity in control, C/X3-depleted cells with or without RNH1-Flag overexpression. Data are represented as a box plot (n = 3, two-tailed unpaired t test with Welch’s correction on means of biological replicates, ≥266 cells were analyzed per condition). (D) Relative S9.6 nuclear signal intensity in control, C/X3-depleted cells with or without RNase III/RNase H treatment. Data are represented as a box plot (n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥308 cells were analyzed per condition). (E) S9.6 dot blot analysis of control, C/X3-depleted genomic DNA with or without RNase H treatment. ssDNA levels served as loading controls. (F) Quantification of S9.6 dot blot signal in control, C/X3-depleted cells. Data are plotted as means ± SD (n = 3, two-tailed unpaired t test with Welch’s correction). (G) Chromatin-bound RNH1(D210N)-Flag foci in control, C/X3/X2-depleted 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, ≥328 cells were analyzed per condition). (H) RNH1(D210N)-Flag ChIP-qPCR in control, C/X3/X2-depleted cells. Enrichment relative to No Ab control is plotted as means ± SD (n = 3, two-tailed unpaired t test). (I) Relative S9.6 nuclear intensity in EdU-positive and negative cells after C/X3 depletion. Data are represented as a box plot (n = 4, two-tailed unpaired t test with Welch’s correction on means of biological replicates, ≥132 cells were analyzed per condition). P values are indicated. ns, nonsignificant.
Fig 2: CX3 promotes the recruitment of FANCM to R-loop sites.(A) ChIP-qPCR of RAD51C/XRCC3/XRCC2 with or without RNH1-Flag overexpression. Enrichment relative to No Ab control is plotted as means ± SD (n = 3, two-tailed unpaired t test). (B) Representative images (left) and quantification (right) of PLA foci of indicated proteins with S9.6. Data are represented as a scatter plot. Black lines indicate mean values (n = 3, two-tailed unpaired t test on means of biological replicates, ≥329 cells were analyzed per condition). (C) Co-IP of RAD51C with or without HU (1 mM, 4 hours) treatment. Endogenous protein levels were shown in the input. (D) Co-IP of XRCC3 with or without HU (1 mM, 4 hours) treatment. Endogenous protein levels were shown in the input. (E) Co-IP of FANCM with or without HU (1 mM, 4 hours) treatment. Endogenous protein levels were shown in the input. (F) The front and back views of the FANCM-RAD51C-XRCC3 complex. All monomers are shown as surface representation (FANCM: slate; RAD51C: light pink; and XRCC3: light orange). The binding interfaces of FANCM, RAD51C, and XRCC3 are encircled in black and are marked by pink, orange, and dark blue, respectively. (G) Zoom-in view of the two FANCM domains: N-terminal translocase (dark blue) and C-terminal ERCC4 region (smudge green), which exhibit interactions with different regions of RAD51C (pink) and XRCC3 (orange). The interacting residues are represented as sticks. (H) Representative images (left) and quantification (right) of S9.6 + FANCM PLA in control, C/X3/X2-depleted 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, ≥322 cells were analyzed per condition). Scale bar, 5 μm. (I) FANCM ChIP-qPCR in control, C/X3/X2-depleted cells. Enrichment relative to No Ab control is plotted as means ± SD (n = 3, two-tailed unpaired t test). P values are indicated.
Fig 3: CX3 complex protects cells from replication stress–induced R-loops.(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.
Supplier Page from Abcam for Anti-XRCC3 antibody