Figure 4

CLIC4 regulates lipid raft expression, but not cell surface expression, of MMP14 in ARPE19 cells. (A) Representative immunoblots of ARPE19 lysates stably expressing inducible CLIC4-sh/turboRFP, treated with (+) or without (−) Dox for three days. Dox treatment drastically reduced the expression level of CLIC4 but not MMP14 except the 53-kDa species (asterisk), which became less detectable. (B) A representative image of a mixture of ARPE19 cells stably expressing inducible CLIC4-sh/turboRFP, which were treated with Dox not, were stained with an antibody recognizing the extracellular domain of MMP14 under non-cell permeabilized conditions from three independent experiments. Scale bar = 10 µm. Asterisks marked the transfected cells that displayed the red fluorescence. The histogram shows the MMP14 signal density (intensity/surface area) of 37 cells for “WT” and 25 cells for CLIC4-KD. (C) Surface biotinylation assays of inducible CLIC4-sh1 expressing ARPE19 cells, treated with or without Dox. Total cell lysates (input) and MMP14 immunoprecipitates were immunoblotted with the indicated antibodies or streptavidin for biotin detection. A histogram shows the ratios of surface expressed MMP14 relative to the total immunoprecipitated MMP14. (D) Immunoblots of 5%-20% sucrose fractionations of TritonX-100 lysates of naïve ARPE19 cells probed with indicated antibodies. The light, detergent-insoluble fractions (#10, #11) are considered to contain lipid rafts. (E) (Left) ARPE19 cells stably expressing inducible CLIC4-sh/turboRFP, Dox treated or not, were subjected to detergent-resistant membrane partition analysis. The detergent-soluble (S) and detergent-resistant (R) fractions were immunoblotted. Arrows point to the smaller molecular mass, cleaved species of Flotillin-1 and MMP14. (Right) A histogram shows the relative fraction of cleaved forms of Flotillin-1 (49-kDa) and MMP14 (~53-kDa) into the detergent-resistant membranes (DRM). Y-axis shows the ratio of [the signal intensity of lower molecular mass species in the R fraction]/[the signal intensity of both low and high molecular mass species in both R and S fraction]. KD of CLIC4 by Dox (+) significantly reduced the expression of the proteolytic form of MMP14, but not Flotillin-1, in the detergent-resistant membrane fractions. Bars show means ± S.D. from three independent experiments. p value, t-test. n.s.: not significant.