TASK-3 Gene Knockdown Dampens Invasion and Migration and Promotes Apoptosis in KATO III and MKN-45 Human Gastric Adenocarcinoma Cell Lines

Figures

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  Figure 1. mRNA expression of TASK channels in KATO III and MKN-45 cell lines. (A,B) expression of TASK-3 (KCNK9) and TASK-1 (KCNK3) in wild-type (WT) cells or cells transduced with shRNAs directed against GFP (shGFP) or TASK-3 (shBP9). Gene expression was normalized against the expression of the gene encoding for the ribosomal protein L19 (RPL19) using the ΔΔCt method. Error bars correspond to mean ± SEM (n = 3). *** p < 0.001, **** p < 0.0001, compared with WT, based on ANOVA followed by Dunnett’s test.

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  Figure 2. Protein levels of TASK-3 and TASK-1 in KATO III and MKN-45 cell lines. (A,B) Representative immunoblots for TASK-3, TASK-1, and GAPDH are shown for wild-type (WT) cells as well as cells transduced with shRNAs against GFP (shGFP) or TASK-3 (shBP9). (C,D) Relative abundance of TASK-3 and TASK-1 protein based on densitometric analyses. Data are expressed as mean ± SEM of three independent experiments. **** p < 0.0001, compared with WT, based on ANOVA followed by Dunnett’s test.

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  Figure 3. Effect of knocking down TASK-3 on cell proliferation and viability. (A,C) Proliferation curves of wild-type (WT) KATO III and MKN-45 cells, or the same cells transduced with shRNAs against GFP (shGFP) or TASK-3 (shBP9) after 0, 24, 48, 72, and 96 h of incubation. (B,D) Cell viability curves as a percentage of viability of KATO III and MKN-45 cells under the indicated conditions following an incubation of 0, 24, 48, 72, and 96 h. Error bars represent the mean ± SEM of three independent experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with WT based on ANOVA followed by Tukey’s post-test.

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  Figure 4. Effects of knocking down TASK-3 on cell cycle regulators and apoptosis. (A,B) The expression of cell cycle regulators in wild-type (WT) KATO III and MKN-45 cells, or the same cells transduced with an shRNA against GFP (shGFP) or TASK-3 (shBP9). (C) Cytometry analyses of apoptosis using annexin V and propidium iodide (PI). (D–G) Percentage of cells in early or late apoptosis based on percentages obtained in C. Error bars correspond to the mean ± SEM of three independent experiments. Cell cycle markers were evaluated by a two-way ANOVA test with a p ≤ 0.05. Early and late apoptosis were evaluated by ANOVA followed by Dunnett’s test. *** p <0.001, compared to WT. ns = no significant difference.

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  Figure 5. Effect of knocking down TASK-3 on cell migration and invasion in KATO III and MKN-45 cells. (A,B) Wound healing assays: images obtained at 0, 3, 6, 12, 24, and 48 h after scratch formation. (C,D) Percentage of wound closure for both cell lines in each condition. Scale bar 40 µm. (E,F) Transwell invasion assays: representative images obtained for KATO III and MKN-45 wild-type (WT), or cells transduced with shGFP (control) and shBP9 (experimental) cells. Scale bar 40 µm. (G−H) The number of invasive cells for both cell lines in each condition. Data are shown as mean ± SEM of three independent experiments. Wound closure percentage was assessed by ANOVA followed by Tukey’s post-test. ** p < 0.01, **** p < 0.0001. Cell invasion was evaluated by ANOVA followed by Dunnett’s test. *** p < 0.001, compared to WT.