Fig. 4: MAD2L2 acts at stalled replication forks in a shieldin-independent manner.

a Schematic and representative images of ssDNA-positive cells in control (sgCTRL) and SHLD1-, SHLD2- or SHLD3-depleted HeLa cells. Following initial labeling with CldU (red) for 16 h, cells were either left untreated or harvested 3 h after irradiation (IR) with 25 Gy. Scale bar, 50 μΜ. Quantification of three independent experiments is shown in (c). b Schematic of the native CldU assay to detect ssDNA. Created with BioRender.com. c Quantification of ssDNA-positive cells. Bars represent the mean ± SD. Each dot represents one of three independent experiments. Statistical analysis was performed according to one-way ANOVA with Dunnett’s multiple comparisons test. d Schematic and quantification of fork degradation assays in control and SHLD1-, SHLD2- or SHLD3-depleted HeLa cells. Cells were treated with 4 mM HU for 4 h before being harvested. A representative fiber experiment from three independent biological replicates is shown. e Schematic and quantification of fork degradation assays in control and MAD2L2-depleted HeLa cells transduced with the indicated shRNAs. Experimental conditions were similar as in (d). Open circles, control shRNA; closed circles, 53BP1 or RIF1 shRNA. A representative fiber experiment from three independent biological replicates is shown. f Control and MAD2L2-depleted HeLa cells were transfected with siRNA control luciferase or siRNA targeting DNA2. Cells were treated and labeled as indicated above. A representative fiber experiment from two independent biological replicates is shown. Statistical analysis for the fiber assays in (d–f) was performed according to two-tailed Mann-Whitney test. Pink bars represent the mean. Additional replicates and combined fiber plots are provided in the Supplementary Information.