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. 2019 Apr 5;15(10):1738–1756. doi: 10.1080/15548627.2019.1596475

Figure 6.

Figure 6.

Activation of TFEB induces autophagy upregulation in UNC13D-deficient cells. (a) qPCR array was performed using the RT2 Profiler™ PCR Array System and 84 autophagy and lysosomal genes were analyzed in wild-type (WT) and unc13d-null (Jinx) MEFs. The amount of mRNA in the samples, standardized to 5 housekeeping genes, was expressed as ΔΔCT as described in Material and Methods. See Table S1 for gene information. (b) The upregulation of autophagic and lysosomal genes in UNC13D-deficient cells was validated by quantitative RT-PCR. The target gene expression level was calculated using the 2-ΔΔCT method, which was normalized to actin. The expression levels were relative to the fold change of the corresponding control (WT), which were defined as 1. (c) Atg9b mRNA level was analyzed by qPCR in mock and Tfeb-shRNA (shTfeb) treated WT and Jinx MEFs. (d) WT and Jinx MEFs were infected with lentiviral mouse shRNA against Tfeb (TRCN0000085548, Dharmacon) for 96 h. Expression levels of TFEB were analyzed by Western blot. (e) Quantitative analysis of (d), presented as mean ± SEM from 3 independent experiments. *p < 0.05, ***p < 0.001, Student’s t-test. (f) The indicated macroautophagy markers in mock transfected and Tfeb-knockdown cells were analyzed by Western blot and quantified using ImageJ. (g) Quantitative analysis of LC3B-II expression. Results are represented as mean ± SEM from 3 independent experiments. *p < 0.05, **p < 0.01, Student’s t-test. (h) Quantitative analysis of SQSTM1 protein expression. The results are presented as mean ± SEM from 3 independent experiments. (i) SQSTM1 and LC3B-II protein expression levels were analyzed by Western blot in mock and Tfeb-shRNA transfected WT and Jinx cells under fed, serum starvation (starv) and serum starvation with BafA conditions. (j) Quantitative results of (i) are represented as mean ± SEM from 3 independent experiments. *p < 0.05, Student’s t-test. (k) Phospho-RPS6KB, total RPS6KB, SQSTM1 and LC3B levels in wild-type (WT) and unc13d-null (Jinx) MEFs were analyzed by western blot under fed and serum starvation (Starv.) conditions in the presence or absence of 1 µM rapamycin.