Fig 2: P-glycoprotein silence activates autophagy and inhibits Akt/mTOR activity in endothelial cells following oxygen glucose deprivation/reoxygenation. Endothelial cells (bEnd.3) were transfected with P-glycoprotein (P-gp) or negative (NC) siRNA or un-transfected, and then subjected to either oxygen glucose deprivation/reoxygenation (OGD/R) treatment or normal culture conditions. Twenty-four hours thereafter, cells were harvested for immunofluorescence staining and Western-blotting analyses. (A) Representative Western-blotting images for autophagic proteins, total and phosphorylated Akt and mTOR proteins. (B-D) Quantification of the ratio of LC3-II to LC3-1 (LC3-II/LC3-1, B), Beclin 1 (B) and P62 (C), the ratios of phosphorylated Akt (p-Akt) to total Akt (p-Akt/Akt, D) and phosphorylated mTOR (p-mTOR) to total mTOR (p-mTOR/mTOR, D) (n = 3). (E) Representative images and quantification of LC3 and P62 immunofluorescence staining (n = 3). Scale bars, 40 µm. One-way ANOVA followed by the post hoc Tukey test for B, D, and E. Mann-Whitney test for C. All data are shown as mean ± SD, *P<0.05, **P<0.01 between two groups.

Fig 3: Pi3K pathway plays a major role in mesenchymal phenotype. (A) The correlation of EMT score with protein expression data of TCGA patients was calculated and plotted. The Yellow dots represent the positive correlation (>+0.2), and the green dots represent the negative correlation (<-0.2). (B) The average expression of proteins with a positive correlation with the EMT score was plotted as a heatmap for each EMT class. The proteins are divided into three classes depending upon their role in the PI3K pathway and EMT. (C) A diagram showing the PI3K pathway modulator with a positive correlation with the EMT score. (D) EMT score for each CCLE cell line was calculated, and cells were divided into epithelial, mixed, and mesenchymal groups. The expression of VIM and CDH1 was compared in three groups and plotted. (E) EMT score for each CCLE cell line was calculated, and the EMT score of the top 10 epithelial and mesenchymal cells was plotted. (F) The GSEA analysis was done to show the activation of various pathways in mesenchymal and epithelial cell lines. (G) The differential expression analysis was done between mesenchymal and epithelial cells, and differentially expressed genes were used from the pathway analysis using Metascape. The pathway network was plotted as identified in the analysis. (H) The EMT score was correlated with the IC50 value of drugs as obtained from Genomic drug sensitivity in cancer (GDSC) databases. The drugs were divided into two groups (positive and negative correlation with EMT score). Fisher’s exact test suggests that significantly more drugs showed increased IC50 in high EMT cells. (I) The correlation coefficient of drugs with high correlation (±0.15) with EMT scores were identified and plotted. (J) Three cell lines AGS, MCF (epithelial), and one 143B (mesenchymal) were selected, and levels of mTOR, pAKT, AKT, and VIM were determined with western blot. (K, L) The effect of DNA damaging compounds (I) Etoposide (J) Adriamycin was determined in AGS, MCF7 (epithelial), and 143B (mesenchymal cells) and plotted to show the high resistance of 143B for DNA damaging compounds. (M) The effect of the PI3K inhibitor was measured at two different concentrations to show the high sensitivity of PI3K inhibitors on mesenchymal cell line 143B.

Fig 4: U2 auxiliary factor homology motif kinase 1 (UHMK1) activated the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway in lung adenocarcinoma cells. (a) Volcano plot analysis of our transcriptome data. (b) The heat map of the top 20 significantly upregulated and downregulated genes. (c) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the transcriptome sequencing data. (d) Evaluation of the levels of proteins related to the PI3K/AKT/mTOR signaling pathway by western blotting assay.

Fig 5: Sitravatinib efficiently suppresses the resistance mediated by FGF2 and FL. A-B The IC50 values of sitravatinib (S) and gilteritinib (G) for MV4-11 or MOLM13 cells in the absence/presence of FGF2 or FL. Error bars indicate mean ± standard error, n = 3 independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001. C-F MV4-11 and MOLM13 cells were treated with sitravatinib or gilteritinib at indicated concentrations for 3 h, then FGF2 or FL (10 ng/mL) was added to the culture for incubation of 3 h. Cell lysates were immunoblotted to evaluate p-FLT3, p-STAT5, p-AKT and p-ERK 1/2. The densitometry ratio of p-AKT to AKT and p-ERK to ERK was assessed via ImageJ, and expressed as fold change compared with vehicle control