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. 2021 Aug 13;10(8):2080. doi: 10.3390/cells10082080

Figure 4.

Figure 4

α7-nAChR-Akt signaling increased YAP activation, DNA binding, and transactivation abilities of the YAP-TEAD complex upon nicotine exposure. OE and SAS cells transfected with either non-targeting siRNA or α7-nAChR siRNA were treated with 1 μM nicotine for 48 h. (A) The expressions of α7-nAChR, phospho-YAP (Ser127), YAP, and BiP were assessed by Western blot analysis. (B) The graphs show the quantification of Western blots. Band intensities were quantified using ImageJ software. The relative expressions of α7-nAChR and BiP were normalized to GAPDH expression. The relative expression of phospho-YAP (Ser127) was normalized to total YAP expression. (C) Subcellular localization of YAP and TEAD were detected by investigating the expression levels of these molecules in the cytoplasmic and nuclear fractions. The cytoplasmic and nuclear extracts were obtained by NE-PER nuclear and cytoplasmic extraction reagents and investigated for YAP and TEAD expressions by Western blot analysis. β-actin and Lamin B were used as loading controls for the cytoplasmic and nuclear extracts, respectively. (D) The interaction of YAP with TEAD was evaluated by co-immunoprecipitation analysis. Western blot analysis of YAP and TEAD was performed after immunoprecipitation with anti-YAP, anti-TEAD, or anti-rabbit IgG antibodies. (E) Occupancy of TEAD on the HSPA5 promoter was analyzed by chromatin immunoprecipitation assay. Sheared chromatin was immunoprecipitated with anti-TEAD or anti-rabbit IgG antibodies followed by capture with protein agarose beads. The eluted chromatin was subjected to PCR amplification to detect DNA fragments of the HSPA5 promoter region containing the TEAD binding site. (F) The promoter activity of HSPA5 was examined by luciferase reporter assay. α7-nAChR-silenced OE and SAS cells were co-transfected with Cypridina luciferase reporter plasmids constructed with the HSPA5 promoter containing the TEAD binding site and red firefly luciferase plasmids, followed by treatment with 1 μM nicotine for 48 h. Luciferase activity was detected using a luciferase dual assay system. Firefly luciferase activity was used to normalize Cypridina luciferase activity. SC, non-targeting siRNA-transfected cells. α7-KD, α7-nAChR siRNA-transfected cells. GAPDH and β-actin served as the loading controls. Rabbit IgG was used as a negative control. * p < 0.05 by one-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as the mean ± SEM of three independent experiments. SEM, error bars.