Figure 3
CLIC4 regulates matrix digestion at focal adhesions in ARPE19 cells. (A) Immunoblotting of ARPE19 lines stably expressing Dox-regulated control-sh, CLIC4-sh1, or CLIC4-sh2. Cells were treated with (+) or without (−) Dox for 3 days before the cell lysates were harvested for immunoblotting using anti-CLIC4 and anti-actin antibodies. (B) ARPE19 cells stably expressing inducible CLIC4-sh1/turboRFP, either treated with or without Dox, were mixed and plated on the same fluorescein-conjugated gelatin coverslips for 5 hours. Representative single-channel view of gelatin (in black and white) and merged color image are shown. The “red” CLIC4-KD cells clearly had reduced gelatin degradation activity. (C) Quantification of gelatin degradation activity of Dox treated (+) or untreated (−) ARPE19 cells stably transfecting control-sh, CLIC4-sh1, or CLIC4-sh2 as well as the naïve (“WT”) ARPE-19 cells. N > 400 for each sample, 3 repeats. Error bars, standard deviation. p, t-test; n.s., not significant. (D) A mixture of ARPE19 with (i.e., KD) or without (i.e., WT) CLIC4 suppressed as described in (B) were plated together on the fluorescein-conjugated gelatin coverslip for 5 hours, and immunostained with vinculin. The bottom two panels show enlarged views of the boxed areas in the top panels. (E) Histograms show four different parameters of evaluating the effect of FA assembly by CLIC4 KD using vinculin immunolabeling described in D. n = 435 foci from 8 “red” CLIC4-KD cells. n = 343 foci from 6 control “WT” cells. The graphs in (C,E) represent means ± S.D. of three independent tests. p value, student’s t-test. Scale bars (in B,D) = 10 µm.
