Fig 1: 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.
Fig 2: Expression level of miR-124 in human ovarian tissues and PDCD6 is a target of miR-124. (A) Representative hematoxylin and eosin staining of ovarian tumor tissue specimens (magnification, ×200). (B) Representative hematoxylin and eosin staining of non-neoplastic ovarian tissue specimens (magnification, ×200). (C) Expression of miR-124 was detected in 30 human ovarian cancer tissues by polymerase chain reaction. Data are presented as log2 values of the change in ovarian tumor tissues relative to non-neoplastic ovarian tissues. (D) The expression of miR-124 in ovarian cancer tissues was decreased compared with in non-neoplastic ovarian tissues (***P<0.001). (E) The putative association between PDCD6 and miR-124 was revealed by TargetScan. (F) The dual-luciferase reporter assay revealed regulation of PDCD6 by miR-124, including wild-type PDCD6 or mutant PDCD6 3′-UTR transfected with or without miR-124 mimics or scramble. (G) Diagram of the PDCD6 3′-UTR with potential binding-sites for miR-124. The data from three in vitro independent experiments are presented (**P<0.01). NC, negative control; miR, microRNA; UTR, untranslated region; mut, mutant; PDCD6, programmed cell death 6.
Fig 3: ANXA7 is required for ALG-2 binding to supported membranes. (a) Schematic representation of supported membrane model composed of primary and secondary membranes. Non-vesicular membrane patches with open edges were used in the subsequent experiments (POPC/POPS, 9:1 molar ratio). (b) Representative sequential images before and after addition of recombinant ANXA7-GFP protein to a membrane patch stained with DiD (DiD channel: upper panel. Lower panel: GFP channel) in the presence of Ca2+ (Also see Supplementary Movie S2). Note, ANXA7-GFP induces a pearl-like membrane conformation. (c) Similar experiment as in b but in the absence of Ca2+ (top panel) or POPS (lower panel). (d) Sequential images before and after addition of recombinant ALG-2-GFP protein to a membrane patch stained with DiD in the presence of Ca2+. (e) Addition of ANXA7 (without GFP) and ALG-2-GFP proteins trigger ALG-2-GFP membrane binding. Accumulation of ALG-2-GFP into pearl-like peripheral structures induced by ANXA7 are indicated by yellow arrows (upper panel, DiD. Lower panel: GFP channel).
Fig 4: Expression of PDCD6 reversed the miR-124-induced cellular apoptosis. (A) The expression level of miR-124 was determined in SKOV3 and OCVAR3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector. The expression level of miR-124 markedly increased in cells transfected with miR-124 mimics. Transfection with pcDNA3.1/PDCD6 restored the expression of PDCD6 in SKOV3 and OCVAR3 cells even with co-transfection of miR-124 mimics. (B) The expression levels of PDCD6 were determined in SKOV3 and OCVAR3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector. The expression level of PDCD6 was markedly suppressed in cells transfected with miR-124 mimics. Transfection of pcDNA3.1/PDCD6 restored the PDCD6 expression level in SKOV3 and OCVAR3 cells even with co-transfection of miR-124 mimics. (C) The expression level of PDCD6 protein was determined in SKOV3 and OCVAR3 following co-transfection with NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector. The expression level of PDCD6 protein was suppressed in cells transfected with miR-124. Transfection with pcDNA3.1/PDCD6 restored the expression level of PDCD6 protein in SKOV3 and OCVAR3 cells even with co-transfection of miR-124 mimics. (D) Transfection of miR-124 mimics alone or with pcDNA3.1/vector induced apoptosis of SKOV3 and OCVAR3 cells. (E) The cell cycle status of SKOV3 and OCVAR3 cells following co-transfection of NC or miR-124 mimics and either the pcDNA3.1/PDCD6 or pcDNA3.1/vector was detected by flow cytometry analysis. NC, negative control; miR, microRNA; PDCD6, programmed cell death 6.
Fig 5: Proposed model for plasma membrane repair mediated by ANXA7. 1. In resting cells ANXA7, ALG-2 and ALIX are predominantly cytosolic. 2. Following plasma membrane injury and influx of Ca2+ into the cytoplasm ANXA7, ALG-2 and ALIX forms a complex, which is anchored to the injured membrane through the core domain of ANXA7. 3. Here, ALG-2 and ALIX initiates the sequential assembly of ESCRT III components by recruiting Chmp4 proteins including Chmp4b, which assemble into membrane bound spring-shaped filaments. 4. Membrane excision occur upon contraction of the spiral structure leading to shedding of the damaged membrane. Other annexin family members including ANXA4, ANXA5 and ANXA6 participate in the repair network and are needed for wound healing (not shown here).
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