Fig. 5.

Primary fibroblasts adapt to oncogene-induced replication stress by overexpressing Claspin and Timeless. a Immortalized BJ-hTERT cells expressing an oncogenic version of Ras (Ras^v12) under the control of a doxycyclin-inducible promoter were grown for 60 days in the presence of Dox to induce oncogene-induced senescence (OIS). Thirteen individual clones escaping senescence were isolated. The expression of Claspin, Timeless, CHK1, and ATR mRNAs was determined by qRT-PCR in these clones and in BJ-hTERT cells expressing or not Ras^V12 after normalization of mRNA levels to HPRT and 18S. BJ-hTERT and BJ-Ras^V12 cells were grown in presence of doxycyclin 8 days before analysis. b Western blot analysis of Claspin, Timeless, CHK1, ATR, RAD17 protein levels in BJ-hTERT, BJ-Ras^v12 cells, and in clones #4, #5, and #8. c Western blot analysis of phospho-CHK1 (S345) and γ-H2AX in BJ-hTERT and BJ-Ras^v12 cells and in clones #4, #5, and #8. d DNA fiber spreading analysis of fork progression in these cells after 10 min IdU and 20 min CldU pulses. The median length of CldU tracks (red) was determined for five independent experiments. ****p < 0.0001, ***p < 0.001, ns nonsignificant difference with BJ cells. Mann–Whitney rank sum test. e Growth of BJ-hTERT (black), BJ-Ras^v12 (white) after 8 days in the presence of doxycyclin and clones #4, #5, and #8 (gray). f Hierarchical clustering analysis of BJ-hTERT, BJ-Ras^v12, and clones #4, #5, and #8 based on the expression of DNA repair genes (in two independent experiments: A and B). Blue: low expression, red: high expression