Skip to main content
. 2016 Aug 17;8(38):62820–62833. doi: 10.18632/oncotarget.11355

Figure 1. Estrogen induces IGF-1R-dependent cell proliferation through non-canonical activation of ERβ2 in AR-null PC-3 cells.

Figure 1

(A) qRT-PCR and Western blot analyses of AR, ER-β and GAPDH in a panel of PC cell lines. The PC cells were maintained for 48 hours in 10% CS-FBS. Then, the target genes and protein levels were measured by qRT-PCR (upper panel), and Western blot analyses (lower three panels), respectively, in PrEC, LNCaP, C4-2b, or PC-3 cells as described in the Methods section. (B) 4-hydroxtamoxifen (4-OHT), a selective estrogen receptor modulator, or ICI182.780 (antiestrogen) inhibited E2- mediated growth stimulatory effects in PC-3 cells. PC-3 cells (5 × 103 cells per well) were incubated with vehicle or 1 uM inhibitor for 48 h. Cell growth was measured by cell counting kit-8 (CCK-8). (C) Time-course analysis of IGF-1R protein expression in E2-stimulated PC-3 cells. (D) PC-3 cells were transiently transfected with IGF-1R siRNA or non-targeting control siRNA, after which cells were treated with vehicle, 10 nM E2 or 50 ng IGF-I for 48 h and cell proliferation was assessed by CCK-8. (E) Selective IGF-1R inhibitor (AG1024; 5 μM) attenuated growth stimulatory effects treated with E2 or E2-BSA-FITC in PC-3 cells for 48 hours. (F) PC-3 cells cultured in chamber slides were stimulated with E2-BSA-FITC and then fixed under non-permeabilization conditions, and subjected to immunofluorescence staining with antibodies against ERβ2 and IGF-1R. The E2-BSA-FITC (green) stimulation triggered co-localization of IGF-IR (red) and ERβ2 (blue) on the plasma membrane of PC-3 cells. The upper right panel shows that permeabilization is required to detect ERβ in the nucleus. The lower panel shows that under permeabilized conditions, IGF-1R (green) co-localizes with ERβ2 (red) in PC-3 cells. DAPI is indicated in blue. The images were captured using Leica fluorescence microscope. Bar graphs represent mean ± SEM values in triplicates. * and ** denotes significance at P < 0.05 and P < 0.01, respectively, compared to vehicle-treated cells (n = 3).