TASK-1 knockdown enhances apoptosis in A549 cells.

<p>(A,B) Cells were transfected with TASK-1 siRNA or non-silencing siRNA (control siRNA). 48 hours after transfection, cells were replated, after additional 24 hours cells were treated with different stimuli for 72 hours and apoptosis was assessed by detecting cells with caspase 3 activity by FACS analysis. For apoptosis induction, cells were treated with cisplatin at different concentrations or with DMEM medium containing dialyzed serum and 10 mM or 0 mM D-glucose (balanced with metabolically inert L-glucose). (C) A549 cells were transfected and treated with different concentrations of cisplatin in the same manner as in panel A. Apoptosis was assessed by staining floating and adherent cells with Hoechst dye. Rates of nuclear fragmentation were determined in a blinded manner. (D) A549 cells were transfected in the same manner as in panel A and treated with different concentrations of cisplatin without previous replating. (A,B,D) Apoptosis was assessed by detecting cells with caspase 3 activity by FACS analysis. Results are mean +/- SEM from n = 3 independent experiments. Group comparisons were performed with Two-way ANOVA followed by Bonferroni post-hoc analysis. * <i>P</i><0.05, ** <i>P</i><0.01; *** <i>P</i><0.001. n.s., not significant.</p

Expression of TASK-1 in lung cancer cell lines.

<p>(A) TASK-1 expression in eight different NSCLC cell lines. A representative immunoblot and mean densitometry values are shown. A549 cells, present on all immunoblots, served as a reference for normalization. Bottom: TASK-1 mRNA levels were assessed by quantitative RT-PCR. Beta-actin (ACTB) was used as a reference gene. (B) TASK-3 protein and mRNA levels in eight different NSCLC cell lines. (A,B) Results are mean +/- SEM from three to four independent samples. Group comparisons were calculated with one-group Student′s t-test. (C,D) A549 cells were transfected with non-silencing siRNA (c), TASK-1 siRNA (T) or left untreated (u). (C) Representative immunoblot using a TASK-1 antibody in the presence or absence of a specific blocking peptide is shown. TASK-1 appears at a molecular weight of 52 kDa, the additional faint band at 46 kDa might represent the unglycolsylated form. (D) TASK-1 mRNA levels 48 hours after transfection with TASK-1 siRNA assessed by quantitative RT-PCR. (E) Representative immunoblots of TASK-1 in A549 cells and H358 cells at different time intervals after transfection. Data are shown as mean +/- SEM from n = 3 independent experiments. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

Expression of TASK-1 and of putative downstream effectors of TASK-1, Na⁺-coupled transporters in human NSCLC.

<p>(A) TASK-1 protein was assessed in NSCLC tissue and corresponding non-involved lung from twelve patients using Western blot. Right: Densitometry values for TASK-1 in NSCLC and lungs were normalized to β-actin. (B,C) mRNA levels of members of the <i>SLC5</i> family of Na⁺-coupled transporters and of the Na⁺-driven glutamine transporter <i>SLC38A1</i> were assessed in a publically available GEO dataset (GDS3257) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157453#pone.0157453.ref011" target="_blank">11</a>] published at Gene Expression Omnibus (GEO; <a href="http://www.ncbi.nlm.nih.gov/geo/" target="_blank">http://www.ncbi.nlm.nih.gov/geo/</a>) in lung adenocarcinoma samples (n = 58) and normal lungs (n = 49). The RMA (Robust Multichip Average) expression measure for mRNA abundance is in the log scale. *** <i>P</i><0.001, * <i>P</i><0.05.</p