Fig 2: PLCL1 upregulated the expression of the lipid browning related gene UCP1 in ccRCC cells. Transcriptome sequencing was performed for A498 cells overexpressing PLCL1. A) GO enrichment for the indicated cells based on the results from sequencing. B) GSEA assays for the correlation of energy metabolism, fatty acid metabolism, and mRNA levels of PLCL1 according to the TCGA database. FDR < 25%, p < 0.05 was considered statistically significant. C) KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment top 20 for indicated cells based on the results from sequencing. D) The heatmap of cluster analysis based on sequencing results. E) Lipid browning‐related proteins were determined by western blot analysis. GAPDH was used as a loading control. F) GSEA assays for the correlation of PPAR signaling and mRNA level of PLCL1 according to TCGA database. FDR < 25%, p < 0.05 was considered as statistically significant. G) Western blot assay for the protein levels of PLCL1 and UCP1 in the PLCL1‐knockdown cells.

Fig 3: UCP1 repressed ccRCC progression and promoted tumor cell “slimming” through lipid browning. A) The mRNA of UCP1 in two independent gene sets from the Oncomine database (https://www.oncomine.org) in ccRCC. B) The mRNA levels of UCP1 in 30 ccRCC tissues and adjacent nonmalignant tissues. t-test, p = 0.0004. C,D) The protein levels of UCP1 in ccRCC tissues and cell lines including five ccRCC cell lines (786-0, A498, ACHN, CAKI, and OSRC) and normal cell line (293) (Abbreviation: N, Normal tissue; T, Tumor tissue). E) Immunohistochemistry (IHC) staining for PLCL1 in ccRCC tissues and adjacent nonmalignant tissues. F) UCP1-overexpressing cell lines were constructed by plasmid transfection with both A498 and CAKI. Cell growth curves of CCK8 assays for indicated cells. ****, p < 0.0001, **, p < 0.01. G) Migration and invasion assay for indicated ccRCC cells (Magnification: 200×). t-test, **, p < 0.01, *, p < 0.05. H) Photomicrographs of Oil Red O staining of overexpressed UCP1 cell lines and its negative control (Magnification: 400× & 800×). Relative TG (mmol/gprot) tested by triglyceride assay kit. Relative diameters of lipid droplets and relative diameters of cells in the ccRCC cells described above. t-test, ****, p < 0.0001, ***, p < 0.001, *, p < 0.05.

Fig 4: PLCL1 repressed ccRCC progression and promoted tumor cell “slimming” through UCP1-mediated lipid browning. On the basis of PLCL1 overexpression, cells with UCP1 knocked down were constructed by transfecting UCP1 siRNA. We constructed four groups of cell lines for rescue experiments, including cell lines with PLCL1 expression lentivirus control vector and siUCP1 control, cell lines with PLCL1 expression lentivirus and siUCP1 control, cell lines with PLCL1 expression lentivirus control vector and siUCP1, and cell lines with PLCL1 expression lentivirus and siUCP1. A) Western blot assay for the protein levels of PLCL1 and UCP1 in indicated cells. B) Cell growth curves of CCK8 assays for indicated cells. ***, p < 0.001, **, p < 0.01, p = ns (no significance). C) Migration and invasion assay for indicated ccRCC cells (Magnification: 200×). t-test, ***, p < 0.001, **, p < 0.01, *, p < 0.05, p = ns (no significance). D) Photomicrographs of Oil Red O staining of indicated cells (Magnification: 400×). Relative diameters of lipid droplets and the ccRCC cells described above.

Fig 5: PLCL1 regulated the protein stability of UCP1 by affecting UCP1 ubiquitination levels in ccRCC. A) Relative mRNA levels of UCP1 in the cells PLCL1-overexpressing and PLCL1-knockdown cells. p = ns (no significance). B,C) ccRCC cells with stable PLCL1 knockdown and overexpression were treated with cycloheximide (CHX) at the indicated time points. Cells were collected, and UCP1 protein expression was analyzed by western blot. D) Cells with stable PLCL1 knockdown were treated with vehicle (DMSO), MG132 (20 × 10-6 m) or chloroquine (50 × 10-6 m) for 12 h. Western blotting was used to analyze the protein level of UCP1. E) GO enrichment for the indicated cells based on the results from sequencing. F,G) The cells with stable PLCL1 knockdown and overexpression were lysed and subjected to immunoprecipitation with an antibody against UCP1 and analyzed by western blotting with an anti-ubiquitin antibody.