CCT7 and TRIP6 are identified as novel Mode II TFEB regulators. (A) Volcano plot of the proteins enriched in WT HeLa cells treated with LLOMe for 3 h relative to ATG7 KO HeLa cells treated with LLOMe for 3 h (n = 3 biologically independent samples). The red square indicates that the log₂ ratio is less than −0.5. 93 proteins are potential Mode II regulators. (B) Quantification of TFEB::mNG nuclear-translocated cells in ATG7 KO HeLa cells stably expressing TFEB::mNG. Cells were transfected with scrambled siRNA (si Con) or the indicated siRNA and were treated with 1 mM LLOMe for 3 h (n = 3 biologically independent samples). (C) Strategy for interactome analysis of TFEB::mNG with or without MK6-83 treatment. WT or ATG7 KO HeLa cells stably expressing TFEB::mNG were collected at the indicated time points after MK6-83 treatment (0 h: untreated control), and subjected to IP/MS. (D) Volcano plot of the proteins enriched in WT HeLa cells treated with MK6-83 for 3 h relative to ATG7 KO HeLa cells treated with MK6-83 for 3 h (n = 3 biologically independent samples). (E) Representative immunoblots of CCT7, TRIP6, and GAPDH in WT HeLa cells, transfected with siLuc, siCCT7, or siTRIP6 to confirm knockdown efficiency. (F) Representative immunoblots of FLAG and TFEB in WT or ATG7 KO HeLa cells with or without stable TFEB::mNG expression. 3×FLAG::CNBP was transiently transfected, and cells were either untreated (control) or treated with 1 mM LLOMe for 3 h, followed by immunoprecipitation with an antibody against mNG. (G) Representative fluorescence images of antibody staining for CCT7 or TRIP6 (green) in WT or ATG7 KO HeLa cells under either untreated condition (control), starvation, or LLOMe for 3 h. The arrowheads show CCT7/TRIP6 dots. Scale bars, 50 µm. (H) Quantification of image data shown in G (n = 3 biologically independent samples). (B and H) P values were determined by ANOVA with Tukey’s multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData FS5.