Figure 4

Role of TM in tumor growth. (a) Effects of the TM silencing vector on the proliferation rates of HT1197 and HT1376 cancer cells. The number of viable cells was counted after incubation for 2, 4, and 6 days. The y-axis represents the viable cell number. (b) Effects of TM inhibition on xenograft tumor growth. Each point represents the mean of three separate experiments ± SD; *, P  <  0.05. Expression of TM was also evaluated by immunohistochemical staining of xenografts. Representative slides are shown at × 400 magnification. (c) Flow cytometry using annexin V-propidium iodide (PI) staining for cell death rates in cells transfected with the TM silencing vector or control vector (CV). (d) Effect of TM silencing vector on apoptosis demonstrated by IF analysis. The results from representative slides are shown (DAPI = blue; cleaved caspase 3 = green). (e) Effect of TM silencing vector on autophagy demonstrated by IF analysis. The results from representative slides are shown (DAPI = blue; LC3 = green). The level of LC3 II was also examined by Western blot analysis in cells transfected with the CV or the TM silencing vector (TM^-). (f) Effects of the TM silencing vector on the expression of apoptosis- and cell aging-related proteins evaluated by Western blot. Data points represent the mean of three separate experiments ± SD. *, P  <  0.05.