Figure 4
hPTTG1 overexpression activates CXCR2/p21 signaling through a p53-independent mechanism. (A) MCF-7hPTTG1 cells stably infected with shGFP, shp53-1, or shp53-2 were plated for senescence assays. The percentage of positively stained cells is indicated to the right of the respective images. The results are presented as the mean ± SEM (n = 3). *P < 0.05. (B) Cell lysates were collected from MCF-7hPTTG1 cells and MCF-7hPTTG1 cells stably infected with shGFP, shp53-1, or shp53-2 for immunoblot analysis. (C, G) T47-D, AU 565, HCT116, and p53-null HCT116 cells were transiently transfected with pcDNA3.1 (Mock) or pcDNA3.1-hPTTG1 (hPTTG1), and then these cells were stained with FITC-conjugated anti-CXCR2 antibody for FACS analysis. (D, H) The cell lysates were collected from T47-D, AU 565, HCT116, and p53-null HCT116 cells transiently transfected with pcDNA3.1 (Mock) or pcDNA3.1-hPTTG1 (hPTTG1) for immunoblot assays. (E, F) T47-D and AU 565 cells were transiently transfected with pcDNA3.1 (Mock) or pcDNA3.1-hPTTG1 (hPTTG1), and then these cells were plated for senescence assays. The percentage of positively stained cells is indicated to the right of the respective images. The results are presented as the mean ± SEM (n = 3). *P < 0.05; ***P < 0.0001. (I) Schematic representation of the role of p53 in hPTTG1/CXCR2-mediated senescence. (J) Cell lysates were collected from MCF-7hPTTG1 cells and MCF-7hPTTG1 cells stably infected with shGFP, shCXCR2-1, or shCXCR2-2 for immunoblot analysis. (K) Cell lysates were collected from MCF-7hPTTG1 cells treated with DMSO (vehicle), IgG control, CXCR2 inhibitor (SB225002), anti-CXCR2 antibody, or anti-IL-8 antibody for immunoblot analysis.