FIGURE 2.
A, AKR1C3 re-expression in Siah2 KD cells. Siah2 KD Rv1 cells were transduced with lentivirus harboring AKR1C3. Cells (control pLKO.1, shSiah2, or shSiah2 + AKR1C3) were analyzed by Western blotting with AKR1C3 and tubulin antibodies. B and C, effect of AKR1C3 re-expression on proliferation of Siah2 KD cells. Rv1 cells in A were maintained in media containing either FBS (B) or CS-FBS (C) and assayed for proliferation at indicated time points. Siah2 KD inhibited the growth of Rv1 cells in FBS media (pLKO.1 versus shSiah2: p < 5 × 10−4 at 24 h, p < 10−5 at 48 h, and p < 5 × 10−6 at 72 or 96 h) or CS-FBS media (pLKO.1 versus shSiah2: p < 5 × 10−6 at 24 or 96 h, p < 10−3 at 48 h, and p < 10−4 at 72 h). AKR1C3 re-expression partly promoted growth of Siah2 KD Rv1 cells in FBS media (shSiah2 versus shSiah2 + AKR1C3: p < 5 × 10−4 at 24 or 72 h, p < 5 × 10−3 at 48 h, and p < 5 × 10−6 at 96 h) or CS-FBS media (shSiah2 versus shSiah2 +AKR1C3: p < 5 × 10−5 at 24 or 96 h, p < 0.05 at 48 h, and p < 0.001 at 72 h). D, effect of AKR1C3 re-expression on colony formation by Siah2 KD cells. Rv1 cells in A were maintained in soft agar for 3 weeks, and colony number per-field was determined. The colony formation was reduced upon Siah2 KD (pLKO.1 versus shSiah2: p < 5 × 10−6) but was partly increased upon re-expression of AKR1C3 (shSiah2 versus shSiah2 + AKR1C3: p < 5 × 10−5). E, effect of AKR1C3 re-expression on orthotopic prostate tumor formation by Siah2 KD cells. Rv1 cells in A were injected into dorsal prostates of nude mice. Three weeks later, tumors were monitored and weighed (n = 6 for each group). The tumor weight was decreased upon Siah2 KD (pLKO.1 versus shSiah2: p < 5 × 10−4) but was partly increased upon re-expression of AKR1C3 (shSiah2 versus shSiah2 + AKR1C3: p < 0.05). F, effect of AKR1C3 re-expression on proliferation or apoptosis of Siah2 KD Rv1 cells in orthotopic prostate tumors. Paraffin sections derived from indicated tumors were analyzed by staining with Ki67 (a proliferation marker) and active caspase-3 (an apoptosis marker). Staining was visualized by DAB (brown) plus a hematoxylin counterstain (blue). G, quantification of Ki67 and active caspase-3 staining shown in F. The number of positively stained nuclei and total nuclei was determined in five random high power fields. The percentage of positively stained cells for Ki67 was reduced upon Siah2 KD (pLKO.1 versus shSiah2: p < 0.005) but was partly increased upon re-expression of AKR1C3 (shSiah2 versus shSiah2 + AKR1C3: p < 0.05). The percentage of positively stained cells for active caspase-3 was increased upon Siah2 KD (pLKO.1 versus shSiah2: p < 0.001) but was partly decreased upon re-expression of AKR1C3 (shSiah2 versus shSiah2 + AKR1C3: p < 0.05).