Figure 4.

Loss of suppressor of Ty 16 (Spt16) decreases the levels of MCM7 and Rb and activates DNA damage responses (DDR). A, A549, NCI‐H1299, and NCI‐H460 human lung cancer cells were transfected with a control siRNA or two separate Spt16‐specific siRNA for 48 h. Cells were then harvested and western blot analyses were performed. This experiment was repeated three times independently. B, A549 and NCI‐H1299 cells were transfected with a control shRNA or an Spt16‐specific siRNA for 48 h. Quantitative RT‐PCR analyses were then performed. C, A549 and NCI‐H1299 human lung cancer cells were transfected as in (A). Cell extracts were then prepared and western blot analyses were performed. This experiment was repeated three times independently. D, A549 and NCI‐H1299 cells were transfected as in (A). Immunofluorescence (IF) analyses were then performed with phosphorylated BRCA1 (p‐BRCA1) antibodies. This experiment were was repeated three times independently. E, NCI‐H1299 cells were transfected with indicated constructs for 48 h. Western blot (left) and quantitative RT‐PCR (right) analyses were performed. F, Immunohistochemistry (IHC) in xenograft tumors generated as in Figure 2D. The levels of MCM7 and Rb were examined and quantified. Bars and error bars are mean ± SD; n = 3 independent repeats. A two‐tailed unpaired Student’s t test was performed. *P < 0.05, **P < 0.01, ***P < 0.001