Fig 2: Anxa2 tyrosine phosphorylation is required for the invasiveness of drug-resistant breast cancer cells. a The expression of Anxa2 and its mutants, Anxa2Y23A and Anxa2Y23D, were effectively rescued in Anxa2-silenced MCF-7/ADR cells as verified by Western blot analysis using anti-Anxa2- or anti-GFP-specific antibodies. ß-actin was used as a loading control. b The re-expression of Anxa2WT and the phospho-mimicking Anxa2Y23D mutant, not the phospho-deficient Anxa2 Y23A mutant, in Anxa2-silenced cells rescued the migration and invasion ability. The cell migration and invasion assays were performed by transwell assay. Data are shown as mean ± SD; n = 6; ****P < 0.0001 and nsP > 0.05 indicate no statistical significance. The statistical results are summarized in the following panel

Fig 3: Localization of CTSB and members of the proteolytic cascade in the caveolae. Representative WB showing expression of (A) CTSB (pro-CTSB, sc-CTSB, dc-CTSB) and CAV1, or (B) ANXA2, pro-uPA and uPAR in caveolae-enriched fractions (2–5, 15 µL) and cell lysates (CL) from hTM cells. l.e.: lower exposure; h.e.: higher exposure. (C) Representative WB showing expression of pro-CTSB, pro-uPA, and uPA in conditioned media (15 µL) from hTM cells.

Fig 4: Ahnak interacts with and regulates cell surface expression of a1 subunits of L-type VGCCs. a GST or GST-p11/Anxa2 peptide hybrid was incubated with detergent extract of mouse hippocampus. After precipitation of GST proteins by glutathione-agarose, proteins in brain extract (input) and co-precipitated proteins were analyzed for subunits of L-type VGCCs by immunoblotting. Precipitated GST proteins were stained with ponceau S (bottom). b Illustration of full length of Ahnak, N-terminal fragment (N), repetitive elements in the central region (R), and C-terminal fragment (C). Estimated molecular mass of the fragments in SDS-PAGE is shown. c COS-7 cells were co-transfected with a plasmid expressing HA-tagged Cav1.2 or V5-tagged ß4b subunit together with a plasmid expressing Strep-tagged Ahnak fragment of N, R, or C. After pulldown of Strep-tagged Ahnak fragment with Strep-Tactin beads, co-precipitated HA-Cav1.2 or ß4b-V5 subunit and endogenous p11 and Anxa2 were detected by immunoblotting as indicated. Immunoblotting (IB) images of HA-Cav1.2 or ß4b-V5 subunit are from HA-Cav1.2- or ß4b-V5 subunit-transfected cells, respectively. IB of endogenous p11 and Anxa2 and Coomassie brilliant blue (CBB) stain of Ahnak fragments are representative images. d-g Voltage-induced L-type calcium current and surface expression of Cav1.2 and Cav1.3 are significantly reduced in primary cultured Ahnak KO neurons. Primary cortical neurons were prepared from WT or Ahnak KO embryos. Whole-cell patch-clamp recordings of somatic L-type Ca2+ currents were performed on DIV 11–12 before (black) and after (red) the bath addition of nimodipine (10 µM) (d, e). Representative traces (d). Histograms showing the current density of the somatic Ca2+ currents (n = 5 cells per group) (e). f, g Cell surface proteins were biotinylated and precipitated with streptavidin-coupled beads. Total Cav1.2 (f), Cav1.3 (g), and actin in lysates and the precipitated surface Cav1.2 (f) and Cav1.3 (g) were detected by immunoblotting. Quantification of surface level (the surface level normalized to total level) of Cav1.2 or Cav1.3 and total level (total Cav1.2 or Cav1.3 level normalized to total actin) (n = 5 per group). Bar graphs are means ± SEM. *p < 0.05, **p < 0.01, t test

Fig 5: ATR may link to ANXA2 in MMc-induced COL6 secretion. (a) HRGECs were cultured with DMSO (control) or ATRi for 1 h, then MMc (12 µg/mL) was added in the cultures. Two h after MMc addition, the cells were washed and cultured with or without ATRi for the indicated number of hours. The cells were immunostained with anti-COL6 and anti-ANXA2 antibodies. Scale bar: 50 µm. (b) The ANXA2 signal ratio (nucleus/cytoplasm) were measured for more than 25 cells using the samples of a. (c) HRGECs transfected with control or ANXA2 siRNA were cultured with DMSO (control) or ATRi for 1 h, and then MMc (12 µg/mL) was added in the cultures. Two h after MMc addition, cells were washed, and cultured with or without ATRi for the indicated number of hours. The cells were immunostained with anti-COL6 antibody. The percentage of cells with cytoplasmic COL6 (COL6-positive cells) was measured by fluorescent images. A total of more than 100 cells of four independent fields per experiment was analyzed. (b, c) Statistical significance was determined by the Mann–Whitney U test. **p < 0.01.