Fig. 4. TFEB does not induce expression of genes involved in autophagy and lysosomal biogenesis in undifferentiated mESCs.

a mRNA expressions of TFEB and TFEB-lysosomal target genes at respective days of embryonic differentiation (EB) (mESC, D5 EB, and D9 EB). b Cellular localization of TFEB during EB differentiation (D0, D5, and D9) was determined using cellular fractionation. The phosphorylated form of TFEB (pS142 and pS211) was also analyzed. Sox2 and Oct4 were used as positive controls consistent with their roles as regulators for stemness. β-tubulin and Lamin B were used as cytosol and nuclear loading control, respectively. c mESCs and HEK293 cells were transfected with 4XCLEAR reporter for 48 h prior to luciferase analysis. d qPCR analysis to measure the expression of TFEB target genes involved in lysosomal biogenesis and autophagy by GFP-TFEB-AA overexpression in mESC. e GFP-TFEB-AA was overexpressed in mESC for 48 h prior to labeling with Lysotracker Red. Nuclei were stained with Hoechst. Scale bars, 100 μm. f qPCR analysis to measure the expression of TFEB target genes involved in lysosomal biogenesis and autophagy in TFEB KO mESCs. g, h ChIP-qPCR analysis was performed by pulling-down with anti-TFEB antibody, following which qPCR was performed targeting TFEB-lysosomal target gene promoter binding sites at the genomic DNA level in undifferentiated (g) and Day 9 (D9) differentiated (h) mESCs. mRNA was normalized with β-actin. All statistical analyses represent average values of a representative experiment from at least two independent experiments. Error bars represent SD values of triplicate assays. Data are shown as mean ± SD, n = 3. *p < 0.05; **p < 0.01; ***p < 0.001 ****p < 0.0001 compared to the corresponding control group. The student’s t test was used for all statistical analysis.