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. 2021 Apr 19;11:578928. doi: 10.3389/fonc.2021.578928

Figure 6.

Figure 6

Transient receptor potential mucolipin 1 (TRPML1) silencing induces cathepsin B activation. (A) Whole cell lysate (WCL), membrane (M), and cytosolic (C) fractions were isolated from siGLO and siTRPML1 T98 cells. (B) WCL, M, and C fractions were isolated from siGLO and siTRPML1 T98 cells. Cathepsin B and LAMP-1 levels were measured in WCL by Western blot and normalized to β-actin. Cathepsin B levels were measured in C fraction by Western blot and normalized to β-actin. Densitometric data shown are expressed as mean ± SE of three separate experiments. *p < 0.05. (C) Cathepsin B activity was measured in siGLO and siTRPML1 T98 and U251 cells. *p < 0.05. (D) siTRPML1 T98 and U251 cells were treated with the inhibitor BAF A1 (25 nM) for 48 h. The Western blot analysis of pro-cathepsin B and mature cathepsin B protein levels was performed. Blots are representative of one of three separate experiments. β-actin was used as loading control. (E) Caspase-3 activity was measured in siGLO, and siTRPML1 T98 cells treated or not with the cathepsin inhibitor Ca074Me. FU, fluorescence units. *p < 0.001vs. untreated siGLO, #p < 0.05 Ca074Me-treated siTRPML1 vs. Ca074Me-treated siGLO cells. (F) Representative flow cytometric profiles of siTRPML1 U251 cells treated or not with Ca074Me and then stained with C12FDG. Gray curve represents unstained cells. MFI, mean fluorescence intensity.