FIGURE 4.

p53 is partially responsible for G₂ arrest in ANXA2-deficient cells. Small interfering RNA (siANXA2) was used to silence ANXA2 in A549, H460, and SiHa cells. A non-targeting siRNA (scramble) was used as a negative control. A and B, Western blotting was used to determine the expression of ANXA2, p53, and p21 at the indicated times. C, the gene expression of A549 cells lacking ANXA2 (siANXA2) or the control (scramble) was analyzed by hierarchical clustering 2 days post-transfection. The data for transcripts with at least a 2-fold difference are shown in a heat map. Selected p53 downstream genes are labeled on the right. D and E, immunostaining followed by fluorescence microscopy was used to determine the expression of ANXA2 (green or red) and p53 (red or green) in scramble-, siANXA2-, and siHtrA2-transfected A549 cells 2 days post-transfection, as well as in shLuc- or shANXA2-transfected A549-derived tumor nodules 30 days postinoculation. DAPI (blue) was used for nuclear staining. Western blot analysis showed the expression of p53 in shLuc- or shANXA2-transfected A549-derived tumor nodules. One representative image obtained from one of three individual experiments is shown. F, p53 was silenced in ANXA2-deficient A549 cells with a lentivirus-based shRNA approach. shLuc was used as a negative control. Western blot analysis was used to detect the expression of ANXA2 and p53 2 days post-transfection. G, the cell cycle was analyzed using PI staining followed by flow cytometry 4 days post-transfection. One representative data set and the percentages of cells in the G₂/M phase are shown as the means ± S.D. from three individual experiments with triplicate cultures. H, ANXA2 was silenced by siANXA2 in A549 cells. Real-time RT-PCR was used to determine the mRNA expression of GADD45A and CDKN1A in ANXA2-deficient A549 cells 2 days post-transfection. A non-targeting siRNA (scramble) was used as a negative control. The data are shown as the mean ± S.D. (error bars) of the -fold change relative to untreated cells in triplicate cultures. I, Western blotting showed the protein expression of ANXA2, CDKN1A (p21), and GADD45A in ANXA2-deficient A549 cells 2 days post-transfection. J, CDKN1A (p21) and GADD45A was silenced in ANXA2-deficient A549 cells with a lentivirus-based shRNA approach. shLuc was used as a negative control. Real-time RT-PCR was performed to determine the mRNA expression of GADD45A and CDKN1A. The data are shown as the means ± S.D. of the -fold change relative to untreated cells from triplicate cultures. K, the cell cycle was analyzed 4 days post-transfection by using PI staining followed by flow cytometry. One representative data set and the percentages of cells in the G₂/M phase are shown as the means ± S.D. from three individual experiments with triplicate cultures. **, p < 0.01; ***, p < 0.001 compared with the scramble or shLuc control group. ###, p < 0.001 compared with the shLuc group. ns, not significant. For Western blot analysis, β-actin was used as an internal control. One representative data set obtained from three individual experiments is shown.