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. 2023 Jul 10;8(13):e157491. doi: 10.1172/jci.insight.157491

Figure 6. Dia1 and YAP/TAZ are downstream effectors of Syx.

Figure 6

(A) Immunofluorescence images (top) of YAP (red) and TAZ (green) subcellular localization in U251 cells expressing Syx shRNAs. Scale bar: 15 μm. Staggered graphs (bottom) depict percentage of cells with YAP and TAZ in the cytosol (C), nucleus (N), or both (N/C). (B) Immunoblots showing total and phosphorylated YAP1/TAZ, Syx, and GAPDH in U251 cells treated with (bottom) or without (top) MG132. (C) qPCR analysis of relative mRNA levels of CTGF in U251 cells expressing indicated shRNAs. Graph represents the mean ± SEM of 5 biological replicates. (D and E) Graphs show relative luciferase activity of a YAP/TAZ responsive reporter (8xGTIIC) in U251 cells expressing indicated shRNAs (D) or expressing indicated constructs (E). Renilla luciferase activity (Ren-Luc) was used to normalize 8xGTIIC activity. Graphs represent mean ± SEM of 3 biological replicates. (F) Cell viability over indicated time for each cell population as measured by MTT assay (mean ± SD). Shown is representative of 3 biological replicates with 3 technical repeats each. (G and H) qPCR (mean ± SEM of 2–3 biological repeats) and immunoblot analyses of indicated mRNAs and corresponding proteins in U251 cells expressing indicated shRNAs. For Western blots (B and H), different biological samples are separated by dashed lines. The same biological samples run at different times are indicated by larger white space. All other blots were run in parallel. One-way ANOVA with Dunnett’s multiple-comparison test. *P < 0.05, ***P < 0.001.