Fig 2: PNS reduced the expression of ATP1A1 and downregulated AKT/ERK signaling pathways. SH: 20 μmoL•L−1; SL: 10 μmoL•L−1. (A) The effects of PNS on regulating the expression of ATP1A1 and AKT/ERK signaling pathways in L02 cells and HepG2 cells. (B) The effects of PNS and sorafenib on regulating the expression and phosphorylation of ATP1A1/AKT/ERK signaling pathways. (C) The image of proteins after PNS treatment in HepG2 cells. (D) The fluorescence image of laser confocal detection of ATP1A1 expression in the HepG2 cell membrane (10 × 60). Scale bar = 50 μm. Data are expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. the L02 control group. ∆ p < 0.05, ∆∆ p < 0.01, and ∆∆∆ p < 0.001 vs. the PNS group.

Fig 3: RB inhibits malignant progression of GBM by targeting ATP1A1 and increasing Na+-K+-ATPase activity in GBM cells. (A) The most likely predicted target of RB. The binding energy of RB with the ATP1A1 protein was −8.7 kcal/mol, proving a good binding interaction. RB interacts with the ATP1A1 protein mainly through the formation of hydrogen bonds as well as hydrophobic interactions. The hydrogen bond length with ASP-121 was 3.1 Å; hydrophobic interactions were formed with Ile-315, Glu-312, Phe-785, Phe-783, Phe-909, Gly-796, and Thr-797. (B) Graphic representation of Na+-K+-ATPase activity. (C) Representative western blot analysis of total ATP1A1 protein expression. (D) Western blotting analysis validated the efficiency of siATP1A1s in GBM cells. (E) GBM cells were transfected with siATP1A1#3 for 48 h and incubated with 4 μM RB for 48 h. Graphic representation of the percentages of G2/M phase cells calculated from the NC, RB, siATP1A1#3 and siATP1A1#3 + RB cells (Supplementary Figure S3D). (F) Western blot analysis of ATP1A1, p21, CDC25C and G2/M-related proteins from NC, RB, siATP1A1#3 and siATP1A1#3 + RB in U251 and A172 cells. (G) Graphic representation of intracellular Ca2+ levels from the NC, RB, siATP1A1#3 and siATP1A1#3 + RB groups in U251 and A172 cells (Supplementary Figure S3E). (H) The numbers of invasive cells as shown in Supplementary Figure S3F. (I) Western blot analysis of the protein expression levels of p-FAK, p-Paxillin and MMP2 in NC, RB, siATP1A1#3 and siATP1A1#3 + RB in U251 and A172 cells. Three independent experiments were performed with data shown as the mean ± SEM by unpaired Student’s t test (B) or one-way ANOVA (E,G,H). *, p < 0.05; **, p < 0.01 and ***, p < 0.001.

Fig 4: RB exerts antitumor efficacy in vivo. (A) Scheme of tumor inoculation and systemic injection. (B) P3#GBM-luciferase cells were orthotopically inoculated into BALB/c nude mice. PerkinElmer IVIS Spectrum monitored tumor growth by detecting bioluminescence. (C) Bioluminescence values on the 7th, 14th, and 21st days after implantation. (D) Kaplan–Meier analysis was performed on the median survival of the tumor-bearing mice, and log-rank analysis was used to measure the statistical significance of the differences. (E) Images of IHC staining for ATP1A1 and Ki-67 in sections from brains of orthotopic P3#GBM tumor-bearing nude mice treated with DMSO or RB. Scale bars, 100 and 20 μm. (F) Immunoreactive scores of Ki-67 and ATP1A1 in sections from the brains of orthotopic P3#GBM tumor-bearing nude mice treated with DMSO or RB in (E). Three independent experiments were performed with data shown as the mean ± SEM by two-way ANOVA (C) or unpaired Student’s t test (F). Survival differences between groups were assessed by the log-rank test (D). *p < 0.05; **p < 0.01 and ***p < 0.001.

Fig 5: PNS and digitonin regulated the expression of proteins and mRNA of ATP1A1/AKT/ERK signaling pathways. Digitonin: 10 nmol/L; PH: 600 mg/L PNS; and PL: 300 mg/L PNS. (A) The representative image of Western blot after treatment with PNS and digitonin and their quantitative analysis are shown. (B) The relative expression of AKT, ERK1/2, and ATP1A1 by Q-PCR and their quantitative analysis are shown. (C) The fluorescence image of laser confocal detection of the expression of ATP1A1 on the HepG2 cell membrane (10 × 60). Scale bar = 50 μm. Data are expressed as mean ± standard deviation. *p < 0.05, **p<0.01, and ***p < 0.001 vs. the control group; Δ p < 0.05, ΔΔ p < 0.01, and ΔΔΔ p < 0.001 vs. the digitonin + PH group; # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. the digitonin + PL group.