Fig. 2: RSR is functional and efficient in CRC-SCs.

a–c Evaluation of RSR proficiency in neoR/SENS-CRC-SCs subjected to exogenous replication stress (RS) as illustrated in the scheme in a. Specifically, cells were left untreated or either sequentially exposed to 1 mM hydroxyurea (HU) and, after drug washout, 1 µM nocodazole (N) (a, b; the so-called HU + N assay, see Materials and Methods), or treated for 24 h with 100 nM gemcitabine (GEM) or prexasertib (CHK1i) (a, c). Flow-cytometric cell cycle profiles upon staining with a DNA intercalant (DAPI) alone (c) or together with an anti-pH3 antibody (b) and quantitative data (c; means ± SEM from 6 independent experiments) are reported. In b, mitotic (pH3⁺) cells are in red. See also Supplementary Fig. S3a–c. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (one-way ANOVA and Dunnett T3 post-hoc test) compared to untreated conditions. d Immunofluorescence analysis in neoR-CRC-SCs and SENS-CRC-SCs left untreated or exposed for 24 h to 100 nM CHK1i and stained with an anti-pRPA32 antibody (top) or incubated with 10 μM IdU prior to anti-BrdU staining (bottom). Representative images and quantification of pRPA32⁺ cells and cells with parental ssDNA are reported. Results are expressed as means ± SEM and individual data points. Numbers refer to the number of independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (unpaired t-test with or without Welch’s correction) as indicated. In the bottom part, statistical analysis was performed only in the histogram on the right. e Western-blot analysis in #1SENS-CRC-SCs and #1neoR-CRC-SCs treated or not with 100 nM CHK1i using the depicted antibodies (nucleolin as loading control). #1neoR^a and #1neoR^b correspond to cells collected after 12 and 17 weeks during CHK1i resistance generation. See also Materials and Methods and Supplementary Fig. S3d. All significant P values are shown in Supplementary Table S4. Supplementary figures associated: Supplementary Fig. S3.