Fig. 1: Endogenous MYC prevents transcription-replication conflicts in PDAC cells.

a Immunoblot showing MYC expression in KPC cells. Where indicated, doxycycline (1 µg/ml) was added for 48 h. Vinculin was loading control (n = 3; n is number of independent experiments unless otherwise stated). b Quantitative image-based cytometry showing DNA content (Hoechst) on the x-axis and EdU incorporation on the y-axis of cells carrying doxycycline-inducible shRNA targeting MYC. “-MYC” indicates samples in which MYC depletion was induced by doxycycline addition (1 µg/ml) for 48 h and “+MYC” ethanol-treated control samples. EdU was added 30 min before fixation. For each independent replicate, 1000 cells were randomly selected and shown in this plot (n = 3). The table on the right shows mean values, standard deviations, and P values (unpaired two-sided t-test). c Bar plot showing the percentage of γ-H2AX positive cells in EdU⁻ or EdU⁺ cells, derived from (b). Data are presented as mean ± s.d. (n = 3; unpaired two-sided t-test). d Representative immunofluorescence images used for the analysis shown in (b). The merged image includes all stains including Hoechst to mark nuclei. Scale bar: 10 µm. e Single-cell quantification of nuclear PLA foci between either PCNA or RAD9 and total RNAPII. The following total number of cells was examined over three independent experiments: +MYC: PCNA-RNAPII n = 7715; −MYC: PCNA-RNAPII n = 2794; +MYC: RAD9-RNAPII n = 4934; −MYC: RAD9-RNAPII n = 3768; two-tailed Mann–Whitney U-test). Single antibody controls are shown for the +MYC condition (number of cells analyzed in one experiment: RNAPII: n = 2451; PCNA: n = 3232; RAD9: n = 2767). In the box plot, the central line shows the median and the borders of the boxes extend from the 25th to the 75th percentile, and the whiskers were plotted using the Tukey method and outliers are shown as black dots. f Immunoblot of cells harboring doxycycline-inducible shRNA targeting MYC. MYC depletion was induced as in (a) and AZD6738 (0.3 µM) was added for 48 h. Beta-actin was loading control (n = 3). g Bar plot showing the relative number of AsiSI-normalized reads, quantifying double-strand breaks as determined by BLISS sequencing. Doxycycline was added for 48 h to deplete MYC and AZD6738 (2 µM) for 2 h was added where indicated. Data are presented as mean ± s.d. (n = 3; unpaired two-sided t-test). Source data are provided as a Source Data file.