Figure 2
PDZK1 is not an immediate early ER-α–dependent gene product but is required for E2-dependent growth of MCF-7 cells. (A) MCF-7 cells were treated with 1 nmol/L E2 for different time intervals or left untreated. Total RNA was subjected to RT-PCR analysis with primers specific to PDZK1 or β-actin (upper two panels). Protein extracts were subjected to immunoblot analysis with antibodies against PDZK1 or GAPDH (lower two panels). (B) MCF-7 cells were treated with 1 nmol/L E2 for 48 h or left untreated. Cells were subjected to immunofluorescence with antibodies against PDZK1. Note the partial nuclear localization of PDZK1 in E2-treated MCF-7 cells (C). (D) MCF-7 cells were treated with 1 nmol/L E2 in the presence or absence of the ER-α antagonist ICI 182,780, the selective estrogen modulator tamoxifen or the EGFR kinase inhibitor AG1478 for 48 h, after which protein extracts were subjected to immunoblot analysis with antibodies against PDZK1 or GAPDH. (E) MCF-7 cells were transiently transfected with siRNA targeting PDZK1 (siPDZK1-1) or control siRNA and treated with 1 nmol/L E2 or left untreated for 48 h. Protein extracts were prepared and subjected to immunoblot analysis with antibodies against PDZK1 or GAPDH (inset). MCF-7 cells were subjected to PDZK1 knockdown with siPDZK1-1 (sequence 1) or PDZK1-2 (sequence 2), after which cell proliferation was assessed by using the MTT assay. *Difference from respective untreated controls; #difference from E2-treated cells; p < 0.05. (F) MCF-7 cells were subjected to PDZK1 knockdown with the siPDZK1, after which cells in the S phase were assessed by fluorescence-activated cell sorter analysis. *Difference from untreated control; #difference from E2-treated cells; p < 0.05. (G) MCF-7 cells were subjected to PDZK1 knockdown with the siPDZK1-1 sequence and treated with 1 nmol/L E2 for 48 h. Protein extracts were subjected to immunoblot analysis with antibodies against c-Myc or GAPDH. (H) MCF-7 cells were subjected to ER-α knockdown with siRNA and treated with 1 nmol/L E2 for 48 h. Protein extracts were subjected to immunoblot analysis with antibodies against c-Myc, PDZK1 or GAPDH. Note that the immunoblot for ER-α was run on a different gel and its GAPDH loading control is depicted in Supplementary Figure S4; the latter figure also depicts immunoblot for ER-α in protein extracts from cells treated with control siRNA.