Fig 2: Overexpression of S100A11 promoted FOXO1 and autophagy marker expression in vivo and in vitro. (A) Heatmap generated by RNA sequencing data indicated that autophagy was up-regulated in the mice liver treated by S100A11 overexpression and the HFHC diet for 2 weeks. n = 3 individual animals for each group. (B) mRNA expression of FOXO1 and autophagy marker genes in vivo detected by qPCR. ß-actin was used as an internal control. n = 4 individual animals for each group. (C and D) Western blot and quantification of Ac-FOXO1, FOXO1, ATG7, and LC3-II in the liver of the hepatic S100A11 overexpression and HFHC diet–treated mice. The target proteins were normalized to tubulin or ß-actin as indicated. n = 3–4 individual animals. (E) mRNA expression of FOXO1 and autophagy marker genes in vitro detected by qPCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (F) Western blot detection of phosphorylated, acetylated, and unphosphorylated FOXO1, ATG7, and LC3-II in the S100A11 overexpression Hepa 1–6 cells. (G) Quantification of Western blot results from panel F. The target proteins were normalized to GAPDH. (H) Quantification of Western blot results from panel F. p-FOXO1 and Ac-FOXO1 were normalized to FOXO1. (I and J) Protein level and quantification of P62 in the S100A11 overexpression Hepa 1–6 cells detected by Western blot. The target protein was normalized to GAPDH. *P < .05, **P < .01, and ***P < .001. p-FOXO1, phosphorylated forkhead box O1.

Fig 3: Secreted S100A11 exerts its important role in the proliferation of MPM cells via RAGE.a RAGE protein expression in MPM cells. b Inhibition of RAGE signaling through sRAGE significantly suppressed the phosphorylation of STAT3 and MAPK induced by S100A11 recombinant protein. c The effect of sRAGE on the growth of MPM cells examined by MTT assay. Cell proliferation was significantly inhibited by sRAGE

Fig 4: ANXA7 is needed for repair in MCF7-p95ErbB2 cells. (a) Schematic representation of the proteomic setup. MCF7-p95ErbB2 cells were cultured in SILAC medium. Cells were left uninjured (control) or injured by treating with digitonin (20 µg/ml) for 10 min at 37 °C. Cells were disrupted and biotin labelled membrane fragments were affinity purified using streptavidin beads before MS analysis. (b) Plot showing change in cell surface level of proteins presented as the ratio of MCF7-p95ErbB2 cells treated with digitonin over uninjured MCF7-p95ErbB2 as measured by MS analysis (Also see Supplementary Fig. S1A and Table 1). (c) Representative sequential images of HeLa cells showing the translocation of ANXA7-GFP or (d) ANXA7-RFP and ANXA1-GFP (upper panel) or ANXA7-RFP and ANXA5-GFP (lower panel) in MCF7-p95ErbB2 cells in response to focal laser injury (blue arrow indicates injury site). (e) Corresponding translocation kinetic plots. Error bars represent SEM from three independent experiments. (f) Immunoblot showing ANXA7 and S100A11 protein levels in MCF7-p95ErbB2 depleted for ANXA7 or S100A11, as compared to Ctrl siRNA. HSP90 served as loading control (72 h). (g) Plasma membrane repair kinetics upon laser injury measured by membrane impermeable FM1-43 dye influx in MCF7-p95ErbB2 transfected with ANXA7, S100A11 or Ctrl siRNA for 72 h. Error bars represent SEM for at least 10 independent cells per condition. (h) Representative images of MCF7-p95ErbB2 cell transfected with either Ctrl siRNA (upper panel) or ANXA7 siRNA (lower panel) showing the influx of impermeable FM1-43 dye after laser injury (white arrow indicates injury site). The asterisk represent P-values based on Student’s t-test (g): **P = 0.01, as indicated when comparing indicated siRNAs to control siRNA. Full-length blots are presented in Supplementary Fig. S2b.

Fig 5: S100A11 overexpression induced lipid accumulation in Hepa 1–6 and Hep 3B cells. (A and B) qPCR and Western blot detection of the S100A11 expression in Hepa 1–6 stable cell lines by puromycin selection for 2 weeks. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (C) Bodipy staining of the S100A11 overexpression Hepa 1–6 cells treated with or without 0.2 mmol/L OA for 24 hours. Original magnification: 20×. (D) TG measurement in the S100A11 overexpression Hepa 1–6 cells treated with or without 0.2 mmol/L OA for 24 hours. (E) Oil Red O staining of the S100A11 overexpression Hepa 1–6 cells treated with or without 0.2 mmol/L OA for 24 hours. Original magnification: 20×. (F) Quantification of Oil Red O staining from panel E. (G) Oil Red O staining of the S100A11 overexpression Hep 3B cells treated with 0.1 mmol/L OA for 12 hours. Original magnification: 40× (left two panels) and 100× (right two panels). (H) Quantification of Oil Red O staining from panel G. *P < .05, **P < .01, and ***P < .001.