Fig 2: 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 3: The knockdown of circ-APBB1IP affected the levels of key proteins involved in cell apoptosis, migration, invasion, and the ERK1/2 signaling pathway. (A, B) Expression levels of apoptosis-related protein (Bax, Bad, Bak, Bcl-2, cleaved caspase 3, cleaved caspase 7, cleaved caspase 8, cleaved caspase 9), (C, D) migration-related protein (MMP-2, MMP-9) and invasion-related protein (E-cadherin, N-cadherin) were measured by Western blot assay. (E, F) The ERK1/2 signaling pathway markers were determined by western blot. The data are presented as the mean ± SD for three independent experiments. T-test, ns p > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: The combination of BFOV and metformin improved hepatic inflammatory state and energy metabolism via downregulating inflammatory signaling pathways and activating phosphorylation of AMPK and AKT in the liver of HFC57 mice. (A) Hepatic NO content. (B) Hepatic iNOS activity. (C‐E) Representative bands and quantitative analysis of main factors involving in the inflammatory pathway in the liver by Western blot, including (C) NFκB‐p65 (65KD), (D) iNOS (130KD) and (E) MMP‐2 (72KD). (F, G) Representative bands and quantitative analysis of ratio of phosphorylation AMPKα (62KD) and phosphorylation AKT (60KD) expression in liver by Western blot, each phosphorylation was normalized by the total amount of (F) AMPK (62KD), (G) AKT (60KD). β‐Actin served as a loading control. Data represented the mean of at least three independent experiments ± SEM. One‐way ANOVA: * P <.05, ** P <.01, *** P <.001 vs. Con. # P <.05, ## P <.01, ### P <.001 vs. BFOV + Met

Fig 5: Flubendazole-induced mitophagy enhances anti-migration potential in MDA-MB-231 cells.MDA-MB-231 cells were transfected with negative-control or DRP1 shRNA for 24 h and treatment with or without flubendazole (0.5 μM) for 24 h. A, B The scratch assay was used to measure the migration capabilities of the cells. Representative images and statistics were shown. Scale bar, 100 µm. C, D Transwell assay was used to measure the number of migrated cells. Representative images and statistics were shown Scale bar, 50 µm. E Immunoblotting of E-cadherin and MMP-2 expression. β-actin was measured as the loading control. F–I The expression of MMP-2 and E-cadherin were analyzed by immunofluorescence. Scale bar, 20 µm. Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance compared with respective control groups (all P-values were obtained by one-way ANOVA).