Fig 1: TRP14 induces autophagy and chemoresistence via the AMPK/mTOR/p70S6K signaling pathway. (A) The phosphorylation levels of AMPK, mTOR and p70S6K were detected by western blot analysis in A2780/DDP and SKOV3/DDP cells. **P<0.01, statistical significance vs. normal A2780 and SKOV3 cells. (B) The phosphorylation levels of p-AMPK, p-mTOR and p70S6K were detected by western blot analysis in SKOV3 and A2780 cells following the overexpression of TRP14. *P<0.05 and **P<0.01, statistical significance vs. normal A2780 and SKOV3 cells. (C) The phosphorylation levels of p-AMPK, p-mTOR and p70S6K were detected by western blot analysis in A2780/DDP and SKOV3/DDP cells following TRP14 knockdown. **P<0.01, statistical significance vs. TRP14-knockdown cells. (D) SKOV3/DDP cells were treated with 4 µM compound C for 12 h. The cells were then treated with the indicated concentrations of cisplatin and cell viability was measured by CCK-8 assay. Values are presented as the means ± SEM (n=3). *P<0.05, statistical significance vs. compound C-treated cells. (E) A2780/DDP cells were treated with 4 µM compound C for 12 h. The cells were then treated with the indicated concentrations of cisplatin and cell viability was measured by CCK-8 assay. Values are presented as the means ± SEM (n=3). *P<0.05, statistical significance vs. compound C-treated cells.
Fig 2: TRP14 knockdown suppresses autophagy-induced cisplatin resistance in SKOV3/DDP and A2780/DDP cells. (A) The levels of TRP14 were detected by western blot analysis in A2780, A2780/DDP, SKOV3 and SKOV3/DDP cells. **P<0.01 indicates statistical significance vs. normal A2780 and SKOV3 cells. (B) A2780/DDP and SKOV3/DDP cells were transfected with TRP14 shRNA. **P<0.01 indicates statistical significance vs. TRP14-knockdown cells. (C) Autophagy-associated genes (LC3, Beclin 1 and ATG5) were examined in cells in which TRP14 was knocked down cells by western blot analysis. **P<0.01 indicates statistical significance vs. TRP14-knockdown cells. (D) Knockdown of TRP14 with shRNA2 in SKOV3/DDP and A2780/DDP cells. The cells were then transfected with GFP-RFP-LC3-plasmid for overnight. Representative images of GFP-RFP-LC3-II-positive puncta were obtained using a confocal fluorescence microscope. *P<0.05, statistical significance vs. TRP14-knockdown cells. (E) Autophagosome and autolysosome vesicles of A2780/DDP and SKOV3/DDP cells were visualized by transmission electron microscopy following transfection with TRP14 shRNA. The white arrows indicate the typical images of autophagosomes and autolysosomes. TRP14 knockdown suppresses autophagy-induced cisplatin resistance in SKOV3/DDP and A2780/DDP cells. (F) MDC staining of A2780/DDP and SKOV3/DDP cells following the knockdown of TRP14. (G) SKOV3/DDP cells were transfected with TRP14 shRNA2. The cells were then treated with or without 200 nM rapamycin (Rapa) for 24 h. After 24 h, the cells were treated with the indicated concentrations of cisplatin (from o to 16 µg/ml) for 24 h and cell viability was measured by CCK-8 assay. The Kruskal-Wallis was used for multiple comparisons and then Mann Whitney U test and Bonferroni's correction were applied. The values are presented as the means ± SEM (n=3). *P<0.05, statistical significance vs. TRP14-knockdown cells. (H) A2780/DDP cells were transfected with TRP14 shRNA2 overnight. The cells were then treated with or without 200 nM Rapa for 24 h. After 24 h, the cells were treated with the indicated concentrations of cisplatin (from 0 to 16 µg/ml) for 24 h and cell viability was measured by CCK-8 assay. The Kruskal-Wallis was used for multiple comparisons and then Mann Whitney U test and Bonferroni's correction were applied. The values are presented as the means ± SEM (n=3). *P<0.05, statistical significance vs. TRP14-knockdown cells.
Fig 3: The positive association of TRP14 and Beclin 1 proteins in ovarian tissues. (A) Human ovarian cancer and marginal tissue specimens (BC11115b) were subjected to immunohistochemistry (IHC) using antibodies against TRP14 and Beclin1. Representative stains from the samples are shown. Scale bar, 100 µm. (B) The levels of TRP14 and Beclin1 were detected by western blot analysis in ovarian cancer tissues. *P<0.05 and **P<0.01, statistical significance vs. low TRP14 expression group. (C) The association between the TRP14 and Beclin1 level was analyzed by the Kruskal-Wallis test. *P<0.05, statistical significance.
Fig 4: TRP14 overexpression promotes autophagy and cisplatin resistance in human ovarian cancer cells. (A) The levels of TRP14 were detected by western blot analysis in A2780 and SKOV3 cells by the overexpression of TRP14. **P<0.01 indicates statistical significance vs. control cells. (B) Autophagy-associated genes (LC3, Beclin1 and ATG5) were examined in A2780 and SKOV3 cells following TRP14 overexpression. *P<0.05 and **P<0.01, statistical significance vs. control cells. (C) Overexpression of TRP14 in A2780 and SKOV3 cells and then cells were transfected with GFP-RFP-LC3-plasmid overnight. Representative images of GFP- RFP-LC3-II-positive puncta were obtained using a confocal fluorescence microscope. *P<0.05, statistical significance vs. control cells. (D) Autophagosome and autolysosome vesicles of A2780 and SKOV3 cells following the overexpression of TRP14 were visualized by transmission electron microscopy. The white arrows indicated typical images of autophagosomes and autolysosomes. (E) TRP14-overexpressing SKOV3/DDP cells were transfected with or without ATG5 shRNA. The cells were then treated with the indicated concentrations of cisplatin (from o to 16 µg/ml) for 24 h and cell viability was measured by CCK-8 assay. The Kruskal-Wallis was used for multiple comparisons and then Mann Whitney U test and Bonferroni's correction were applied. The values are presented as the means ± SEM (n=3). *P<0.05, statistical significance vs. TRP14-overexpressing cells. (F) TRP14-overexpressing A2780 cells were transfected with or without ATG5 shRNA. The cells were then treated with the indicated concentrations of cisplatin (from o to 16 µg/ml) for 24 h and cell viability was measured by CCK-8 assay. The Kruskal-Wallis was used for multiple comparisons and then Mann Whitney U test and Bonferroni's correction were applied. The values are presented as the means ± SEM (n=3). *P<0.05, statistical significance vs. TRP14-overexpressing cells.
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