Fig 2: Nuclear export of AnxA2, but not of pTyr23AnxA2, is blocked by LmB. PC12 cells were untreated (A,C) or treated for 2 h with LmB (B,D), followed by immunofluorescence staining using monoclonal pTyr23AnxA2 (A,B) or polyclonal AnxA2 (C,D) antibodies. The middle panels show higher magnification images with lines indicating cross-sections of nuclei, with the left-to-right orientation of the lines corresponding to the fluorescence intensity profiles shown to the right. The insets include DAPI staining (blue) to verify the position of the nuclei. Scale bars: 10 µm. (E) A schematic model of the transient Tyr23 AnxA2 phosphorylation events occurring at two distinct sites in response to H2O2. Upon exposure to H2O2, nuclear Tyr23-phosphorylated AnxA2 in PML bodies rapidly dephosphorylates and might become associated with SC-35-positive nuclear speckles. AcD increases the localisation of pTyr23AnxA2 in PML bodies suggesting a role in transcription. Note that the pTyr23AnxA2 form is not exported from the nucleus (via the CRM1-dependent pathway). At the same time cortical AnxA2 is Tyr23 phosphorylated, associates with actin and subsequently with endosomes and multivesicular bodies (MVB) and is exported out of the cell in the lumen of exosomes. pTyr23AnxA2 appears to be ubiquitylated and/or sumoylated.

Fig 3: Incorporation of ubiquitylated pTyr23AnxA2 into extracellular vesicles (exosomes) and decrease in the cortical pool of pTyr23AnxA2 after prolonged treatment of cells with H2O2. (A) PC12 cells were grown in exosome-depleted medium in the presence of H2O2 for 0 min (lanes 1,5), 15 min (lanes 2,6), 1 h (lanes 3,7), or 2 h (lanes 4,8). ECM-bound proteins were released by EGTA (lanes 1–4), whereas extracellular vesicles were isolated from the culture medium by the ExoQuick-TC method (lanes 5–8). 100 µg of protein from the EGTA-released fractions (lanes 1–4) and the control extracellular vesicle fraction (lane 5), or an equal volume of extracellular vesicles from H2O2-treated cells (lanes 6–8) were separated by 10% SDS-PAGE, transferred to nitro-cellulose membranes and probed with antibodies against pTyr23AnxA2, total AnxA2, CD63, TSG-101 and T-cadherin, as indicated. (B) Following 1 h treatment of PC12 cells with 1 mM H2O2, proteins (600 µg) present in purified extracellular vesicles were immunoprecipitated (IP) by monoclonal AnxA2 antibodies (lane 1) after pre-clearance of the samples with normal mouse IgG (lane 2). The proteins were subjected to 10% SDS-PAGE and immunoblot analysis using monoclonal antibodies against pTyr23AnxA2 or ubiquitin by loading half of the immunoprecipitation sample on each gel. The bands representing ubiquitylated AnxA2 (square bracket; Ub-AnxA2) and IgG light chain (LC, arrowhead) are indicated to the right. (A,B) Following incubation with HRP-conjugated secondary antibodies and the ECL-reagent, the reactive protein bands were detected using the ChemiDoc™ XRS+ molecular imager. Note that the secondary antibody used in B only recognises the IgG light chains. Molecular mass standards are indicated to the left. (C) PC12 cells were untreated (C1), or treated for 15 min (C2), 30 min, (C3), 60 min (C4) or 120 min (C5) with 1 mM H2O2. The localisation of pTyr23AnxA2, detected using the monoclonal antibody (green), is shown in the merged images, which also display nuclear staining (DAPI, blue). Scale bars: 10 µm.

Fig 4: Rack1 is required for Anxa2 Tyr23 phosphorylation and enhanced invasiveness of drug-resistant breast cancer cells. a Rack1 knockdown decreased the basal levels of phosphorylated Anxa2 in two drug-resistant cells. Western blotting analysis of the total and phosphorylated Anxa2 expression in MCF-7/ADR and MDA-MB-468/ERP cancer cells transfected with negative control or siRNAs targeting Rack1 for 72 h; ß-actin was used as the loading control. b Rack1 knockdown inhibited EGF-induced Tyr23 phosphorylation of Anxa2. c Knockdown of Rack1 expression in two drug-resistant cells significantly decreased cell migration ability as measured by wound healing assay. Data are shown as mean ± SD; n = 6; ****P < 0.0001 versus control. Statistical analysis was performed by two-way ANOVA. d Knockdown of Rack1 expression attenuated the migration and invasion ability in two drug-resistant cells. For cell migration assay, 1 × 105 cells in 200 µL of serum-free medium were loaded into the upper chamber. For cell invasion assay, 2.5 × 105 cells in 200 µL serum-free medium were loaded into the upper chamber coated with Matrigel. The statistical results are summarized in the right panel. Data as mean ± SD; n = 6; ****P < 0.0001 compared with the control group

Fig 5: Anxa2 regulated the expression of VEGFs. HepG2 cells were treated with three different Anxa2-shRNAs. (A) Protein level and phosphorylation status of STAT3 and Anxa2 were determined by immuno-blotting. (B) The activation of STAT3 was determined via reporter gene assay. (C) The expression of STAT3 target genes was determined by qRT-PCR. (D) The protein level of four VEGFs was determined by immuno-blotting. (E) The concentration of four VEGFs in cell-free supernatant was determined by ELISA. All experiments were performed with triple replicant. Significance analysis was shown as ** (p < 0.01) and *** (p < 0.001).