“…127 To scrutinize whether the induced nuclear localization of TFEB by CCT020312 in HeLa cells is consistent with its transcriptional activation, we conducted RNA-Seq analysis of the same batch of cells treated with CCT020312 (2.5µM) for 20 h. RNA-Seq analysis revealed that CCT020312 treatment (2.5µM) significantly (adjusted p-value < 0.05) upregulated 1039 genes and downregulated 1177 genes (Supplemental Table 1), including 152 associated with the autophagy-lysosomal pathway (Figure 3C). 128 To discern which genes are specifically modulated by the TFEB transcriptional program, we cross-referenced our findings with TFEB transcriptional program gene sets from previous studies, 125–127,129 identifying 172 genes significantly altered by CCT020312 treatment (Supplemental Table 1, Figures 3C, 3D, S3L). These genes, including lysosomal acid hydrolases and selective autophagy receptors exhibited significant upregulation relative to vehicle-treated samples, establishing a link between CCT020312-induced gene expression changes and autophagy-lysosomal pathway regulation (NEU1: 2.1x DMSO, CTSL: 1.5x DMSO, ASAH1: 1.4x DMSO, LIPA: 1.4x DMSO, CTSD: 1.3x DMSO, CTSF: 1.3x DMSO, SQSTM1: 1.6x DMSO, CALCOCO2: 1.4x DMSO).…”