Fig 2: Overexpression of PTEN suppresses non-small-cell lung cancer (NSCLC) cell chemoresistance, proliferation and apoptotic resistance. PTEN was knocked down or overexpressed in H1299 and HCC8237 cells. (A and B) PTEN expression in these cells was examined via qPCR and Western blotting. Cellular viability (C) and DDP IC50 values (D) were measured. (E) The proliferation of these cells was monitored via EdU uptake assay in the absence (left) or presence of DDP (2 µg/ml; Right). (F) The proliferation of these cells was monitored via colony formation assay in the absence (left) or presence of DDP (2 µg/ml; Right). (G) The apoptotic ratio values for these cells were measured via Hoechst staining in the absence (left) or presence of DDP (20 µM; Right). (H and I) PI3K-p85, PI3K-p100, PI3K, pAKT and AKT levels were measured via Western blotting the indicated cells in the presence or absence of DDP treatment. GAPDH employed as a normalization control. Outcomes are means ± standard deviation (SD) (*p < .05; **p < .01; ***p < .001)

Fig 3: Depletion of FBXO31 regulates ERK and AKT activity and promotes cell growth in prostate cells(A) Parental and FBXO31 KO RWPE1 cells were treated with vehicle or the DUSP6 inhibitor BCI for the indicated days, and cell viability was determined by ATP-Glo assay. Graph was generated using Prism 8 (mean ± SD; n = 8).(B) VCAP cells stably transduced with a retroviral system expressing doxycycline-inducible FBXO31 or a control vector were treated with doxycycline for the indicated times. WCLs were immunoblotted as indicated.(C) RWPE1 cells were treated with various concentrations of BCI for 6 h and then analyzed by immunoblotting for the indicated proteins.(D) FBXO31 wild-type or KO RWPE1 cells were cultured in medium without EGF for the indicated times (withdrawal [W/D]) and then harvested and analyzed by immunoblotting.(E) FBXO31 wild-type or KO RWPE1 cells were cultured in regular medium or in medium without EGF (W/D). Where indicated, cells were treated with the DUSP6 inhibitor BCI. GTP-bound RAS was quantified using RAF1 Ras-binding domain (RBD) pull-down assay. Western blotting images are representative of 3 independent experiments, and the intensity of bands was quantified by ImageJ. The columns represent the relative levels of RAS-GTP as normalized to the levels of total RAS protein (mean ± SD; n = 3 independent experiments)(F) FBXO31 wild-type or KO RWPE1 cells were cultured in regular medium or medium without EGF. Relative cell viability was measured by ATP-Glo assay. Graph was generated using Prism 8 (mean ± SD; n = 8).

Fig 4: miR-20a promotes in vitro cisplatin resistance and proliferation in non-small-cell lung cancer (NSCLC) cells. (A) Levels of miR-20a expression were assessed in HCC827 and H1299 cells following miR-20a mimic transfection, miR-NC mock transfection or control treatment. The viability (B and C) and DDP IC50 values (D) for NSCLC cell lines treated as in (A) were measured. The proliferation (E and F), colony formation (G and H) and Hoechst staining (I and J) results for NSCLC cell lines treated as in (A). (K and L) Western blotting was used to assess PI3K-p85, PI3K-p100, PI3K, pAKT and AKT expression in the indicated cell lines in the presence or absence of DDP treatment, with GAPDH as a normalization control. Outcomes are means ± standard deviation (SD) (*p < .05, **p < .01, ***p < .001 vs. control; ## p < .01, ### p < .001 vs. mock + DDP)

Fig 5: Exosomes derived from cancer-associated fibroblasts (CAFs) induce tumour growth and chemoresistance, and activate PI3K/AKT pathway in a nude mouse non-small-cell lung cancer (NSCLC) model. HCC827 cells were processed with exosomes derived from normal tissue-associated fibroblasts (NAFs) or CAFs were subcutaneously injected into nude mice, which were treated or untreated with DDP. (A) Subcutaneous tumours in each group were excised and displayed. (B) Tumour volume was measured at the end of 3, 7, 14, 21 and 28 days. (C) qPCR analysis of miR-20a expression in tumours. (D) Western blotting analysis of PTEN, PI3K-p85, PI3K-p100, PI3K, pAKT and AKT. Outcomes are means ± standard deviation (SD) (*p < .05; **p < .01; ***p < .001)