J Clin Invest.
FGFR3 knockdown in RT112 bladder cancer cells inhibits proliferation and induces G1 cell-cycle arrest in vitro, and it suppresses tumor growth in vivo.
Three different FGFR3 shRNAs were cloned into a Tet-inducible expression vector. RT112 cells stably expressing FGFR3 shRNAs or a control shRNA were established with puromycin selection. (A) Representative blots showing FGFR3 expression in selected clones treated with or without doxycycline (Dox; 0, 0.1, and 1 μg/ml, left to right). (B) [3H]thymidine incorporation by stable RT112 clones. Selected RT112 stable clones, namely clone 4 for FGFR3 shRNA2 (sh2-4), clone 1 for FGFR3 shRNA4 (sh4-1), and clone 16 for FGFR3 shRNA6 (sh6-16), were cultured with or without 1 μg/ml doxycycline for 3 days prior to 16-hour incubation with [3H]thymidine (1 μCi per well). Counts of incorporated [3H]thymidine (in cpm) were normalized to those from cells without doxycycline induction. Error bars represent SEM. (C) DNA fluorescence flow cytometry histograms of RT112 stable cells. RT112 clones expressing control shRNA or FGFR3 shRNA4 were cultured with or without 1 μg/ml doxycycline for 72 hours, and the nuclei were stained with propidium iodide (PI). Similar results were obtained for FGFR3 shRNA2 and -6 (Supplemental Figure 2). (D) The growth of RT112 cells expressing control shRNA (n = 9 per treatment group) or FGFR3 shRNA4 (n = 11 per treatment group) in mice. Mice were given 5% sucrose alone or supplemented with 1 mg/ml doxycycline, and tumor size was measured twice a week. Error bars represent SEM. Similar results were obtained for FGFR3 shRNA2 and -6 (Supplemental Figure 2). P < 0.0001. Lower panel: Expression of FGFR3 protein in tumor lysates extracted from control shRNA or FGFR3 shRNA4 stable cell xenograft tissues.