Fig 1: Proteins/lipids related to ERRa were enriched in mitochondrial function in EC (A) The relationship between TFEB-ERRa and acc, fasn, acadm are determined by RT-qPCR in KLE and ECC-1 cells. (B) The association of oxygen consumption rates (OCR), basal, maximal respiration, spare respiratory capacity, and ATP production regulated by TFEB-ERRa are shown. (C) The effect of ERRa regulation on TFEB, LPCAT1, LPCAT3, MMP2, and Cortactin expression in EC cells is analyzed using western blot (WB). (D) The effect of XCT790 treatment on LPCAT1, LPCAT3, MMP2, and Cortactin proteins for 24 h were evaluated between control and TFEB-overexpressing by WB. (E) Representative Scanning electron microscope (SEM) micrographs of KLE and ECC-1: TFEB-ERRa axis and XCT790 treatment for 24 h regulated pseudopod, in comparison to the controlled group. Micrographs are screened at scale of 10–30 µM. *, p < 0.05. Statistical tests: Student’ s t-test or ANOVA.
Fig 2: Accumulation of UFA-containing GPs induced by ERRa is required for EC progression (A) The baseline characteristic including BMI, menopause, FIGO stage, histologic type, MI, LNM and ERRa expression of patients (EC = 35 vs controls = 19) tissues for lipidomic. (B) Different classes of lipids were tested in 35 EC tissues and 19 control tissues. (C) Venn diagram showing the distribution of TFEB-ERRa axis related lipids and ERRa-dependent lipids in clinical samples. (D)Systematic lipidomic changes between EC and control tissues assessed by OPLS-DA. (E) Systematic lipidomic changes between LNM and non-LNM tissues assessed by OPLS-DA. (F) Significant lipid species in EC and normal control tissues and its PC/SM evaluation. (G) Analysis of TFEB-ERRa associated lipids, ERRa-dependent lipids and EC as well as LNM. (H) The univariate binary logistic regression analyses of Age, BMI, CA125, PC (18:1/18:2) + HCOO and PC (32:2) + H. (I) The correlation analysis of PC (18:1/18:2) + HCOO and ERRa. *, p < 0.05. Statistical tests: Student’ s t-test(E–F), Logistic regression(G), Pearson’s rank correlation analysis(H). BMI: Body Mass Index.
Fig 3: TFEB promoted M. pneumoniae-induced autophagy. (a) The expression of LC3 II detected by immunofluorescence. 3-MA suppressed the upregulation of LC3 puncta induced by M. pneumoniae. (b) Quantification of A. (c) The protein expression detected by western blot. Downregulation of TFEB reversed the increase of LC3 II induced by M. pneumoniae. (d) Quantification of C. (e) The expression of LC3 II detected by immunofluorescence. TFEB knockdown suppressed the upregulation of LC3 II induced by M. pneumoniae. **P < 0.01, ##P < 0.01.
Fig 4: Bioinformatics analysis revealed that TFEB promotes ERRa transcription to participate in EC progression TCGA database (Sample size: Normal = 23; EC = 543) results are shown. (A) The expression of TFEB varies at different FIGO stages (B) and at different pathological grades. (C) The association of TFEB with OS in the patient/specimen quartiles is shown (Low: 1st quartile distribution; Median: 2nd-3rd quartile distribution; High: 4th quartile distribution). (D) The expression of ERRa varies at different FIGO stages (E) and at different pathological grades. (F) The association of ERRa with OS is shown. (G) The correlation between the expression levels of TFEB and ERRa in EC tissue. (H) ChIP analysis of the ERRa promoter occupancy in KLE cells is performed as described in the Materials and Methods section. TFEB is immunoprecipitated using an anti-Flag antibody, and DNA enrichment is performed using qPCR. The ATP6V1H promoter is used as a positive control and the GLA promoter is used as a negative control. (I) KLE cells are co-transfected with Flag-TFEB, ERRa promoter labeled with luciferase reporter, and Renilla luciferase control. 48 h after transfection, the cells are analyzed and the relative luciferase activity is measured and normalized to the Renilla luciferase control. (J) The putative ERRa-binding sites (ERREs), as predicted by the online program JASPAR (https://jaspar.genereg.net/analysis), are located in the TFEB (P1-P7) gene promoter regulatory regions. (K) KEGG pathway analysis (Ordinate: the KEGG signal path; abscissa: enrichment score). Results of GSEA in fatty acid metabolism and adipogenesis pathways. (L) The association of fatty acid metabolism and adipogenesis with tumor invasion is shown (Low: 1st- 2nd quartile distribution; High: 3rd-4th quartile distribution). Statistical tests: ANOVA (A-B, D-E), Kaplan–Meier estimator (C, F), Pearson correlation analysis (G), and Student’ s t-test (L). p < 0.05 suggests significantly different. TCGA: The Cancer Genome Atlas; FIGO: Federation International of Gynecology and Obstetrics; OS: Overall Survival; ChIP: Chromatin Immunoprecipitation; KEGG: Kyoto Encyclopaedia of Genes and Genomes; GSEA: Gene Set Enrichment Analysis.
Fig 5: Torin 1 enhanced autophagic flux via activating TFEB resulting in the degradation of mut-TGFBI. (A) Torin 1 enhanced autophagic flux marked by decreased SQSTM1 levels and increased LC3-II and CTSD levels in both WT and MT. (B–D) show normalized data in (A) * p < 0.05, ** p < 0.01, n = 3 for each group; (E) The SQSTM1 and mut-TGFBI were degraded gradually over time in MT, and data analysis is shown in (F) * p < 0.05, ** p < 0.01, n = 3 for each group. Error bars indicate SD.
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