Figure 2
Chemotherapy-induced senescence of human tumor cells restricts VSV infection.
(A) Microscopy images of human tumor A549 cells showing morphology (left panels) and SA-beta-gal staining (right panels) of untreated (A549-NT, upper panels) and bleomycin-induced senescent (A549-B, bottom panels) A549 cells. Quantification of the SA-beta-gal positive cells is shown below (at least 200 cells were counted per condition). (B) Western-blot analysis of senescence markers p53 and p21 in untreated A549 cells (A549-NT) or after bleomycin treatment of A549 cells (A549-B). GAPDH is shown as loading control. (C) Expression levels of CDKN1A (coding for p21) mRNA relative to GAPDH (x10−3) as determined by qRT-PCR in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells. (D) Viral titers (PFU/mL) determined in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells after the indicated periods of infection at a MOI of 0.5 PFU/cell. (E) Western-blot analysis of VSV protein synthesis in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells after the indicated periods of infection at MOIs of 0.05 PFU/cell (upper panel) or 10 PFU/cell (lower panel). Actin is shown as loading control. (F) Microscopy images of MEFs showing morphology (left panels) and SA-beta-gal staining (right panels) of untreated (MEFs-NT, upper panels) and bleomycin-induced senescent (MEFs-B, bottom panels) MEFs. Quantification of the SA-beta-gal positive cells is shown below (at least 200 cells were counted per condition). (G) Viral titers (PFU/mL) determined in untreated (MEFs-NT) or bleomycin-treated (MEFs-B) MEFs after the indicated periods of infection at MOIs of 0.05 PFU/cell (left panel) or 10 PFU/cell (right panel). (G) Percentage of apoptotic cells measured after mock or VSV infection at MOI of 10 PFU/cell, in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells. Data are mean values +/− SE from at least three different experiments. *p < 0.05, **p < 0.01, ***p < 0.001 Student’s t test.
