Fig 2: S100A11 activates NF-?B pathway through ANXA2, and p65 regulates S100A11 at transcriptional level. A, Western blot analysis p-IKKa, IKKa, I?Ba, p65 and p-p65 in indicated GBM cells. GAPDH was used as the loading control. B and C, Western blot analysis of p65, GAPDH in cytoplasmic extracts (Cyto) and p65, Histone3 in nuclear extracts (NE) in indicated GBM cells. D, Immunofluorescence assay was conducted the distribution of p65 in indicated GBM cells. Scale bar, 20 µm. E, Exogenous expression of ANXA2 rescued the NF-?B pathway inhibited by knockdown of S100A11. F, P65 was knockdown in U87 and U251 cells. S100A11 levels in U87 and U251 cells were measured by qRT-PCR. G, A diagram shows a potential p65-binding site in the promoter region of S100A11. H, Chromatin immunoprecipitation assays detected the binding of p65 with the S100A11 promoter in U87 and U251 cells. Normal rabbit IgG was used as a control. The resultant DNA was analysed with semiquantitative PCR using site-specific primers

Fig 3: ANXA2 could regulate the nuclear translocation of p50 following treatment with TNF-a. a~d. The change of subunits (p50, p65) of NF-?B family in SK-N-BE(2) cells treated without or with TNF-a (100 ng/ml) for 1 h. Cytosol and nuclear fractions were prepared special extraction assay. Densitometric analysis showed subunit p50 was involved in NF-?B activation. e~g. Under the same stimulation of TNF-a, densitometric analysis clearly showed the subunit p50 was downregulated in the nucleus of ANXA2 knockdown NB cells compared with control cells following treatment with TNF-a *: p < 0.05, **: p < 0.01, ***: p < 0.001, The plotted error bars represent mean ± SEM from 3 independent experiments

Fig 4: ANXA2 knockdown induced more apoptosis with chemotherapeutic agents and activated the apoptotic pathway. a, b. After treated with doxorubicin (0.5 µM) or etoposide (10 µM) for 48 h, Hoechest 33342 staining showed significantly increased numbers of apoptotic cells in ANXA2 knockdown group compared with the control group. c, d. AnnexinV/PI flow cytometric analysis showed significantly increased apoptosis in the ANXA2 knockdown group compared with control group following etoposide treatment. Each quantitative bar chart represents the sum of quadrant 2 and 4 (n = 3 independent experiments). e~g. After treatment with doxorubicin (0.5 µM) and etoposide (10 µM) for 48 h, pro-apoptotic proteins AIF and Cleaved-PARP were increased, particularly in ANXA2 knockdown groups. The plotted error bars represent mean ± SEM from 3 independent experiments. *: p < 0.05, **: p < 0.01

Fig 5: Annexin A2 interacts with both Prx2 and STAT3.a Co-precipitation: Both Prx2 (upper panels) and STAT3 (lower panels) co-precipitate AnxA2. The blots are representative of n = 3 independent experiments. PD pulldown; IB immunoblot. Source data are provided as a Source data file. b Proximity ligation: AnxA2 interacts with Prx2 and STAT3 (upper panels). S100A10 interacts with AnxA2 (positive control), but not with Prx2 or STAT3 (middle panels). Single antibody experiments are included as negative controls. Representative images show PLA foci in red and DAPI in blue. Scale bars: 10 µm. The bar graphs (lower panels) quantify the number of PLA foci per cell (n = 50–60 cells in n = 2 technical replicates). The results are representative of n = 3 independent experiments. c Depletion of AnxA2 by siRNA diminishes the association between Prx2 and STAT3. Co-precipitation of Prx2 by SBP-STAT3 (left panels) and co-precipitation of STAT3 by Prx2-SBP (right panels). The blots are representative of n = 3 independent experiments. PD pulldown; IB immunoblot. Source data are provided as a Source data file. d Deletion of AnxA2 compromises the Prx2-STAT3 interaction as detected by PLA. Representative images show PLA foci in red and DAPI in blue (left panels). Scale bar: 10 µm. The bar graphs (right panels) show the frequency of PLA foci in AnxA2 KO cells relative to wild type cells (n = 40–50 cells in n = 3 technical replicates), using two independent clones of AnxA2 KO cells. The interaction of AnxA2 with Prx2 or STAT3 in AnxA2 KO cells represents the background signal. The results are representative of n = 3 independent experiments. All bar charts in this figure represent the mean ± SD. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant; based on a two-tailed unpaired t-test.