Fig 2: ASA inhibits the stemness of ESCC cells.a Immunoblot analysis for stem cell markers in KYSE180 and KYSE410 cells treated with the indicated drug combinations for 72 h. b Immunoblot analysis for stem cell markers in KYSE180 and KYSE410 cells treated with ASA at the indicated concentrations for 48 h. c Representative images from colony formation assay and the colony count of KYSE180 and KYSE410 cells pretreated with 2 µg/mL DDP, 5 mM ASA or the combination for 48 h (n = 3). d Representative images from spheroid formation assay and the spheroid count/diameters of KYSE180 and KYSE410 cells pretreated with 2 µg/mL DDP, 5 mM ASA or the combination for 48 h (n = 3). e Representative flow cytometry analysis of mitoxantrone efflux in untreated and ASA-treated cells. Mitoxantrone was examined at 670 nm, with blank and DMSO as a negative control. f Statistic analysis of Geo Mean of the fluorescence collected from three parallel experiments. g Inhibition of phosphorylation of Akt determined by immunoblot analysis. In all the panels, error bars indicate means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s. not significant. Student’s t test.

Fig 3: Effect of higher Hsp90 levels following ASA treatment on the mitochondrial proteins and the influence of intracellular Hsp70 on Hsp90. (A) Representative western blotting results of mitochondrial HSP90, Akt, p-Akt and PKM2 are shown, and the ratios of phosphorylated/total protein abundance are presented. (B) Representative western blotting results of mitochondrial Bcl-2 and p-Bcl-2, and the ratios of phosphorylated/total protein abundance are shown. (C) Representative western blotting results of cellular Hsp70 and HSF-1 are shown. (D) Representative immunohistofluorescence staining images showing the co-localization between Hsp90 and Hsp70 (Hsp90, green fluorescence; Hsp70, red fluorescence; nucleus, blue fluorescence; the merged image of Hsp90 and Hsp70 in the cytoplasm, yellow fluorescence; the merged image of Hsp90 and Hsp70 in the nucleus, white fluorescence) in myocardial tissues. Scale bar=100 µm. (E) Representative western blotting results of mitochondrial cyt c are shown. (F) Representative western blotting results of cytosolic cyt c and caspase-3 are shown. The results are expressed as the mean ± SD; n=5. *P<0.05 and **P<0.01 vs. Con, ##P<0.01 vs. ASA + HS, and the comparison between ASA and HS is indicated by &&P<0.01.

Fig 4: Evaluation of the expression levels of vasodilation-associated proteins. (a) Representative Western blot analysis. (b,c) Quantification of phosphorylated (p)-PI3K/PI3K (b), p-Akt/Akt (c), and p-eNOS/eNOS (d) in EA.hy926 cells treated with different concentrations of DPHC. The protein bands were ultimately developed and photographed with the FUSION Solo Vilber Lourmat system. Quantitative data were analyzed using Image J 1.50i software (NIH, USA). Results are expressed as the mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05, ** p < 0.01. *** p < 0.001, significant difference compared to the control group. DPHC: diphlorethohydroxycarmalol; PI3K: phosphoinositide 3-kinase; Akt: protein kinase B; eNOS: endothelial nitric oxide synthase.

Fig 5: MT1G deficiency induces PI3K/AKT signaling in gastric cancer cell. (A-B) Gastric cancer cells (NCI-N87 and HGC-27) were divided into four groups based on its different treatment with plasmids transfection, including MT1G overexpression plasmids and related control plasmids, MT1G knockdown plasmids-shMT1G and matched negative control plasmids-shNC. The expression of AKT, p-AKT, PI3K, p-PI3K in MT1G overexpression or knockdown NCI-N87 (A) and HGC-27 (B) cells was measured by western blotting. Error bars represent data from three independent experiments (mean±SD). **P<0.01. ##P<0.01.