Figure 4.

CHK1 inhibition leads to increased replication stress only in MMC-resistant TNBC. (A) Examples and frequency distribution of DNA fiber lengths (CldU) in untreated and MMC treated cells. Exponentially growing cells were sequentially labeled with CldU and IdU in the absence or presence of MMC (0.1 µM). DNA was spread and incorporated CldU and IdU was detected with appropriate antibodies. Shown are means ± SEM of DNA fiber length (CldU) frequency distributions of three independent experiments. Asterisks represent significant differences (** p < 0.01; *** p < 0.0001, Student’s t-test). (B) Immunodetection of activated intra-S phase checkpoint proteins. Cells were treated with 1.5 µg/mL MMC for 1 h and proteins were extracted 24 h later. Proteins were separated and transferred by Western blotting. Detection of proteins was performed with appropriate antibodies. HSC70 served as the loading control. Phosphorylation of the untreated control was used for standardization and ratios of phosphorylated to non-phosphorylated protein are shown. Data from three independent experiments were used for quantification. Errors are mean values + SEM. (C) DNA fiber lengths of CldU labeled tracts after treatment with MMC in the presence of the CHK1 inhibitor MK8776 (1 µM). Exponentially growing cells were incubated for 2 h with MK8776 and sequentially labeled with CldU and IdU (plus 0.1 µM MMC), DNA was spread and incorporated nucleotides were detected with the appropriate antibodies. The frequency distribution of DNA fiber lengths in the first label (CldU) of three independent experiments is shown. Asterisks represent significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n.s. not significant; Student’s t-test).