Fig. 5: Trametinib regulates the expression of TFEB-regulated autophagy-lysosome genes.

A Representative western blot analysis of Human brain lysates for indicated proteins. B Bars correspond to densitometric analysis of level of pERK/ERK. Data were presented as the mean ±  S.E.M. Student’s t-test (p = 0.0349; n = 4–5). C Bars correspond to densitometric analysis of level of pTFEB/TFEB. Data were presented as the mean ± S.E.M. Student’s t-test (p = 0.0036; n = 4–5). D Representative western blot analysis of 5XFAD mice brain cortex lysates for indicated proteins. E Bars correspond to densitometric analysis of pTFEB/TFEB. Data were presented as the mean ± S.E.M. One-way ANOVA followed by Dunnett’s post hoc analysis (F_{(2, 6)}  = 10.57, p = 0.0108; n = 3). F Representative western blot analysis of pTFEB(S142) and TFEB from cell lysates of primary cortical neuron. GAPDH was used as loading control. G Bars correspond to densitometric analysis of level of pTFEB(S142). Data were presented as the mean ± S.E.M. Two-way ANOVA followed by Dunnett’s post hoc analysis (F_{(3, 12)} = 15.49, p = 0.0002; n = 5). H Representative western blot analysis of TFEB in the cytosolic and nuclear fractions of primary cortical neuron. GAPDH and lamin B1 were used as cytosolic and nuclear fractions marker, respectively. I Bars correspond to densitometric analysis of level of TFEB in the cytosolic (F_{(3, 12)} = 7.834, p = 0.0037; n = 5) and nuclear fractions (F_{(3, 12)} = 13.12, p = 0.0004; n = 5). Data were presented as the mean ± S.E.M. Two-way ANOVA followed by Dunnett’s post hoc analysis. J qPCR of TFEB target genes in primary cortical neuron (F_{(3, 24)} = 1.234, p = 0.3191, Ctsf; F_{(3, 15)} = 2.644, p = 0.0871, Ctsb; F_{(3, 15)} = 12.61, p = 0.0002, Atp6v1d; F_{(3, 15)} = 6.137, p = 0.0062, Atp6v1h; F_{(3, 24)} = 1.107, p = 0.365, Vps8; F_{(3, 24)} = 10.33, p = 0.0001, Sqstm1; F_{(3, 24)} = 10.90, p = 0.0001, Maplc3; F_{(3, 24)} = 3.08, p = 0.0466, Wipi1; F_{(3, 22)} = 9.804, p = 0.0003, Becn1; F_{(3, 24)} = 3.536, p = 0.0291, Uvrag; n = 6–9). Data were presented as the mean ± S.E.M. Two-way ANOVA followed by Dunnett’s post hoc analysis. K Expression analysis of the TFEB target genes by qPCR in the cortex of 5XFAD (F_{(2, 6)} = 4.678, p = 0.0596, Ctsf; F_{(2, 6)} = 13.98, p = 0.0056, Ctsb; F_{(2, 6)} = 0.6408, p = 0.5595, Atp6v1d; F_{(2, 6)} = 0.6372, p = 0.5611, Atp6v1h; F_{(2, 6)} = 13, p = 0.0066, Vps8; F_{(2, 6)} = 6.976, p = 0.0272, Sqstm1; F_{(2, 6)} = 1.042, p = 0.4089, Maplc3; F_{(2, 6)} = 9.417, p = 0.0141, Wipi1; F_{(2, 6)} = 3.874, p = 0.0831, Becn1; F_{(2, 6)} = 6.938, p = 0.0275, Uvrag; n = 3). Data were presented as the mean ± S.E.M. One-way ANOVA followed by Dunnett’s post hoc analysis. *p < 0.05; **p < 0.01; ***p < 0.001.