Figure 2
From: Annexin A7 is required for ESCRT III-mediated plasma membrane repair
ANXA7 is needed for positioning ALG-2 at the repair site. (a) Representative sequential images of MCF7-p95ErbB2 cell expressing ALG-2-RFP and ANXA7-GFP exposed to laser injury (blue arrow indicates injury site) and (b) corresponding kinetic blot from three independent experiments. Error bars represent SEM. Also, see Supplementary Movie 1. (c) Representative immunoprecipitation experiment using RFP, GFP, ALG-2 or control IgG antibodies from lysates of MCF7-p95ErbB2 cells overexpressing ANXA7-mRFP or ANXA7-tGFP and exposed to scrape injury. Following IP, immunoblot analysis was carried out using ANXA7 and ALG-2 antibodies. (d) Representative sequential images of the translocation of ALG-2-GFP in response to laser injury (blue arrow indicates injury site) in MCF7-p95ErbB2 cells transfected with control siRNA (upper panel) or ANXA7 siRNA (lower panel) (72 h). (e) Time for peak of ALG-2 accumulation at the repair site following laser injury of MCF7-p95ErbB2 transfected with indicated siRNAs (72 h). Error bars represent SD for 10 independent cells per condition. The asterisk represent P-values based on Student’s t-test: **P ≤ 0.01. (f) Immunoblot showing ANXA7 and p95ErbB2 protein levels in MCF7-p95ErbB2 Ctrl-CRISPR cells as compared to A7-CRISPR cells. Tet– refers to cells washed to remove tetracycline to induce p95ErbB2 expression. HSP90 and CDK7 served as controls for equal loading. (g) Cell membrane repair kinetics upon laser injury measured by membrane impermeable FM1-43 dye influx in MCF7-p95ErbB2 Ctrl-CRISPR cells as compared to A7-CRISPR cells (A7-CRISPR cells show compromised repair). Error bars represent SD for at least 7 independent cells per condition. (h) Representative images of translocation behavior of ALG-2-GFP upon focal laser injury (blue arrow indicates injury site) in MCF7-p95ErbB2 A7-CRISPR cells expressing Ctrl-RFP plasmid (left panel) or wildtype ANXA7-RFP (right panel). (i) Time for ALG-2 to reach injury site in MCF7-p95ErbB2 A7-CRISPR cells expressing Ctrl-RFP plasmid or wildtype ANXA7-RFP. (j) ALG-2-GFP distribution (µm2) or (k) ALIX-GFP distribution (µm2) around the injured membrane in A7-CRISPR cells expressing either Ctrl-RFP or ANXA7-RFP measured by Volocity software. (l) Representative sequential images of ALIX-GFP translocation upon laser injury (blue arrow indicates injury site) in MCF7-p95ErbB2 A7-CRISPR cells expressing Ctrl-RFP plasmid (left panel) or wildtype ANXA7-FP (right panel). Note, ANXA7-RFP tend to form unspecific aggregates when overexpressed (red puncta around the nucleus). The aggregates are inert and do not respond to membrane injury. White line indicates region of interest (ROI). Error bars represent SD for at least 5–6 independent experiments. P-values based on Student’s t-test: ***P ≤ 0.001. Full-length blots for 2c and 2f are presented in Supplementary Figs S2c and S3 respectively.
