CLIC3 promotes invasion. (A) MDA-MB-231 cells were nucleofected with non-targeting siRNA (si-con) or siRNAs targeting CLIC3 (si-CLIC3 #3 or #4), then allowed to invade into plugs of fibronectin-supplemented Matrigel. Invasion is quantified as the proportion of cells migrating further than 45 µm. The experiment was repeated using pools of MDA-MB-231 cells with stable expression of empty vector (MDA-MB-231-pcDNA3) or Flag-tagged siRNA-resistant CLIC3 (MDA-MB-231-Flag-CLIC3). Data are mean±s.e.m. from three independent experiments, performed in duplicate; P-values were calculated with a Mann–Whitney U test. (B) MDA-MB-231 cells were plated onto a collagen plug that had been pre-conditioned by primary human fibroblasts, allowed to invade for 6 days, then stained for cytokeratin. Scale bar: 100 µm. At least four individual plugs per siRNA duplex were generated in two independent experiments. Values are mean±s.e.m. Each data point represents an individual x10 magnification field. P-values were calculated with a Mann–Whitney U test. (C) MCF10DCIS.com cells were plated on a thin layer of Matrigel and phase-contrast micrographs captured after 6 days (upper panels). Individual acini were outlined and circularity determined using ImageJ with a value of 1 representing perfect circularity. Each data point represents a single acinus with data generated in three independent experiments (si-con, n = 121; si-CLIC3 #3, n = 242; si-CLIC3 #4, n = 218); the bar represents the median. P-values were calculated with a Mann–Whitney U test. To visualize the basement membrane acini were stained for α6 integrin or laminin 5 (middle panels). The actin cytoskeleton and nuclei were visualized with phalloidin–Alexa-Fluor-546 and DAPI staining respectively (lower panels). Scale bars: 20 µm.

CLIC3 is localized to the late endosome/lysosome and regulates MT1-MMP recycling. (A) MDA-MB-231 cells expressing fluorescently tagged proteins or exposed to Lysotracker Green were imaged by live-cell confocal microscopy. The zoom panels show a magnified view of the area outline by the dashed square. Right, colocalization is expressed as a percentage of yellow versus red pixels for ≥30 cells from three or more experiments. Box, median and interquartile range; whiskers, minimum to maximum. Scale bar: 10 µm. Single-channel images are presented in supplementary material Fig. S3A. (B) Colocalization of endogenous CLIC3 and MT1-MMP in fixed paraffin-embedded MDA-MB-231 cells. Examples of two separate cells, with differing focal planes are shown in the upper and lower panels respectively. Scale bars: 5 µm. (C) MDA-MB-231 cells expressing GFP–MT1-MMP and Cherry–CLIC3 were imaged by live-cell confocal microscopy. Movies were acquired and stills are shown from the time points indicated. Scale bar: 10 µm. Single-channel images are presented in supplementary material Fig. S3B. (D) MDA-MB-231 or MCF10DCIS.com cells were nucleofected with non-targeting siRNA (si-con) or siRNA targeting CLIC3 (si-CLIC3 #4). Recycling of MT1-MMP was determined as described in Roberts et al. (Roberts et al., 2001). Values are mean±s.e.m. from three independent experiments. (E) MT1-MMP recycling was determined following CLIC3 siRNA nucleofection in MDA-MB-231 cells with stable expression of empty vector (pcDNA3) or Flag-tagged siRNA-resistant CLIC3 (Flag-CLIC3). (F) MDA-MB-231 cells were nucleofected with non-targeting siRNA or siRNAs targeting Rab27A and Rab27B (si-Rab27A+B) and recycling of MT1-MMP determined.