Figure 4.

The SASP of non-CSLCs is essential for CSLCs. (a) RPMI 8226 cells treated with doxorubicin (2.7, 13.5 and 50 nM) for 2 days were double-stained with CD138 and C₁₂FDG as illustrated in the top panel for 0 and 50 nM doxorubicin. CD138^low and CD138^high senescent cells were quantified by flow cytometry. (b) QRT-PCR analysis of IP-10 and RANTES expressions in CD138^low- and CD138^high-sorted RPMI 8226 cells 2 days after doxorubicin treatment (50 nM); the untreated CD138^low- and CD138^high-sorted RPMI 8226 cells served as reference (P=0.12 and P=0.0031 for IP-10 and RANTES, respectively). (c) CD138^low-sorted RPMI 8226 cells were cultured either in the control medium (medium of untreated cells) or in the conditioned medium (medium of cells treated with 50 nM doxorubicin for 2 days). Cells were stained with CD138 after 1 day of culture and analyzed by flow cytometry (P=0.02). (d) RPMI 8226 cells having migrated overnight towards the control medium or the conditioned medium were analyzed by flow cytometry after CD138 labeling (P=0.0047 and P=0.0045 for CD138^low and CD138^high, respectively). (e) RPMI 8226 cells were infected with lentiviral particles containing shRNA-targeting CHK2 or control shRNA. Whole-cell lysates were prepared and analyzed by western blot with anti-CHK2 antibody to confirm the downregulation of CHK2. Cells were treated (or not for controls) with doxorubicin (50 nM) for 2 days and CD138^low/CD45^high/CD20^high cells were tracked and quantified as described previously (P=0.049). Data are means±S.D. from at least three independent experiments; *P<0.5