Fig 2: DHX9 inhibitor treatment in MSI-H/dMMR colorectal cancer cells phenocopies siRNA KD. A, Immunofluorescence imaging of LS411N MSI-H/dMMR (top) and NCI-H747 MSS/pMMR (bottom) colorectal cancer cells treated with DMSO or 1 μmol/L ATX968 for 48 hours showing nuclear R-loops (S9.6, green; DAPI, blue; n = 2). B, S9.6 dot blot assay of LS411N MSI-H/dMMR cells treated with 1 μmol/L ATX968 or DMSO for 48 hours ± RNase H1 (n = 2). C, Immunofluorescence imaging of LS411N MSI-H/dMMR (top) and NCI-H747 MSS/pMMR (bottom) colorectal cancer cells treated with DMSO or 1 μmol/L ATX968 for 72 hours showing nuclear G-quadruplexes (BG4, pink; DAPI, blue; n = 2). D, Western blot of γH2AX and pRPA (S8) in LS411N MSI-H/dMMR cells (top) and NCI-H747 MSS/pMMR colorectal cancer cells (bottom) over a 7-day time course after treatment with 1 μmol/L ATX968 (+) or DMSO (−; n = 2). E, Immunofluorescence imaging of LS411N MSI-H colorectal cancer cells treated with DMSO or 1 μmol/L ATX968 for 4 days. DAPI (nuclei), blue; EdU, green; DHX9, pink; n = 2. F, Bar graph of the proportion of cells in phases of the cell cycle (G1, blue; S, red; G2/M, green) after treatment with 1 μmol/L ATX968 in LS411N and NCI-H747 cells for 5 days (n = 2). G, Quantification of Annexin V–positive cells in LS411N MSI-H/dMMR and NCI-H747 MSS/pMMR colorectal cancer cells 1 to 6 days after treatment with 1 μmol/L ATX968 or DMSO (n = 2). Error bars, SD. ***, P < 0.001; ****, P ≤ 0.0001. H, Model schematic depicting the effects of DHX9 inhibition and proposed mechanism of action in MSI-H/dMMR cancer cells. Norm., normal; Pol, polymerase; pRPA, phosphorylated RPA32. (H, Created with BioRender.com.)

Fig 3: wtAAV2-mediated RPA32 exhaustion induces cellular DNA damage.(A) Schematic of the timeline of wtAAV2 infection at different multiplicities in 293T cells for 24 hours before sequential pulsing with IdU and CldU for 20 minutes each prior to DNA fiber analysis. (B,C) DNA fiber length measurements were indicated by each datapoint for (B) IdU-labelled DNA fibers and (C) CldU-labelled DNA fibers. The median lengths of many measurements of IdU and CldU labelled fibers in wtAAV2 infected cells are represented by red bar and green bar respectively. At least 150 individual fibers were measured for each condition for three independent replicates of wtAAV2 infections of 293T cells. Statistical significance was measured by Mann Whitney Wilcoxon test, **** represents P < 0.0001, ns represents differences that are not statistically significant. (D,E) U2OS cells infected with wtAAV2 at an MOI of 10,000 vg/cell were pulsed with EdU (green) for 20 minutes before being processed for gamma H2AX localization (red). The nucleus is shown by DAPI staining (blue) with the nuclear border demarcated by dashed white line. (E) Quantification of the number of gamma H2AX foci per cell in U2OS cells that were EdU-negative (left) and EdU-positive (right) upon being infected with wtAAV2 at different MOI’s for 24 hours. The median number of gamma H2AX foci in at least 30 nuclei is indicated by red bar. (F,G) Immunoblot analysis of AAV2 infection at the indicated MOI’s compared with 2 mM HU for 24 hours in (F) 293T cells and (G) NHDF cells. Cells were harvested as described and analyzed for γH2AX in the nuclear lysates. Tubulin levels were used as loading controls for the immunoblots. (H) Immuno-FISH assays imaging the relative location of the wtAAV2 genome (green) to active forms of the identified cellular DDR factors (red) in iPOND-MS studies described above. U2OS cells were infected with wtAAV2 at an MOI of 10,000 vg/cell for 24 hours before being processed for Immuno-FISH imaging. Nuclei are represented by blue DAPI staining and the nuclear borders are demarcated by dashed white lines. The scale bars represent 10 microns distance.

Fig 4: TRIM21 deficiency amplifies DNA damage responses by preventing PARP1 degradation.a, b Western blot analysis showing the activation of DNA damage repair signal post-DNA damage in SCLC cells upon PARP1 or TRIM21 knockdown. DMS273 and SHP77 cells were treated for 1 h with increasing amounts of camptothecin (CPT) (DMSO, 1, 2, 4 μM) or etoposide (EP) (DMSO, 20, 40, 80 μM), followed by washing and incubation in fresh medium for 4 h. PARP1, TRIM21, CHK1, p-CHK1 (Ser345), RPA32, and p-RPA32 (S4/S8) proteins were analyzed by immunoblotting. c Immunofluorescent staining depicting the change of DNA damage-induced γH2AX foci in SCLC cells upon PARP1 or TRIM21 knockdown. Immunostaining analysis of γH2AX foci formation after 1 h of treatment with 2 μM CPT or 40 μM EP, followed by 4 h of recovery in PARP1 or TRIM21 stably knockdown DMS273 cells. Scale bars, 10 μm. d Quantification of the γH2AX foci in (c). e Comet assays depicting the effect of PARP1 or TRIM21 knockdown on DNA damage in DMS273 cells following the same treatment conditions as described in (c). Scale bars, 100 μm. f Quantification of the tail moment of comet assays in (e). g Colony formation assays measuring the clonogenic capacity of the DMS273 cells treated by CPT with indicated concentrations upon PARP1 or TRIM21 knockdown. h, i Cell viability assays depicting cellular survival following DNA damage upon PARP1 or TRIM21 knockdown. Cells were treated as described in (a, b), and cell viability was assessed using the CellTiter-Glo luminescent assay after 48 h. Data in (d) (n = 80 cells) and (f) (n = 100 cells) are presented as mean ± SD. In (h, i), data from three independent experiments are shown as mean ± SEM. Statistical analysis was performed by two-tailed unpaired t-tests in (d, f, h, i). Source data are provided as a Source Data file.