Journal: Nature Communications
Article Title: YAP1 mediates survival of ALK-rearranged lung cancer cells treated with alectinib via pro-apoptotic protein regulation
Figure Lengend Snippet: Increases in Mcl-1 and Bcl-xL expression were mediated by YAP1. a Relative gene expression of MCL1 and BCLXL detected by qRT-PCR in 4 ALK-rearranged lung cancer cell lines ( n = 4 independent experiments). Significance was evaluated using a one-way ANOVA followed by Sidak’s test. b Evaluation of Mcl-1 expression, Bcl-xL expression, and YAP phosphorylation (pS127). Cell lysates were blotted using an immunoblotting assay with the indicated antibody. c Initial survival rate (top) and apoptosis activity (bottom) of KTOR1 (left) and KTOR2 (right) when cells were exposed to ALC or vehicle in combination with the knockdown of MCL1 and BCLXL . Summarized results are shown in the panels. The number of independent experiments is shown in each panel. Complete results are presented in Supplementary Fig. 6c, d . Significance was evaluated using a one-way ANOVA followed by Sidak’s test. d – e Increases in Mcl-1 and Bcl-xL expression were canceled by the inhibition of YAP1. YAP1 activity was inhibited using siRNA ( d ) or verteporfin ( e ). Traced experiments using H2228 are shown in Supplementary Fig. 6f, g . f Sequence analysis of putative YAP1/TEAD binding sites (labeled as A, B) in the upstream regions of MCL1 and BCLXL . The ChIP-PCR primer pairs designed were shown as arrows. g ChIP-qPCR on KTOR1 and KTOR2, which confirmed the presence of YAP1/TEAD binding sites in the MCL1 and BCLXL upstream regions and increased binding induced by the ALC treatment. BCLXL , a known YAP target, was also used as a positive control. Results are expressed as Percent Input (Two biologically independent cells, two-independent primers, two-independent experiment in each cell lines and two-independent qRT-PCR quantification in each experiment). The titration of DNA digestion, electrophoresis of input samples, and specificity of ChIP-qRT-PCR primers were shown in Supplementary Fig. 8 . Significance was evaluated using a one-way ANOVA followed by Sidak’s multiple comparison test. h Schematic explanation of Fig. 6. The inhibition of YAP1 down-regulated Mcl-1 and Bcl-xL expression. The inhibition of both Mcl-1 and Bcl-xL induced apoptosis and inhibited initial survival. ALC alectinib, DMSO dimethyl sulfoxide, VER verteporfin, siSC si scramble control. Error bars indicate ± S.D. * P
Article Snippet: ALC, crizotinib, ceritinib, and VER were dissolved in dimethyl sulfoxide (DMSO) (Nacalai Tesque) at a concentration of 5 mmol/L.
Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Inhibition, Sequencing, Binding Assay, Labeling, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Positive Control, Titration, Electrophoresis