Fig. 6: NAFLD promotes EGFR palmitoylation via activating de novo palmitate biosynthesis in metastatic CRC cells.

A Representative images of IHC staining for FASN of MC38 allografts in control and NAFLD mice. Scale bars, 50 μm. B The protein level of FASN in HCT 116 and SW 480 cells treated with or without BSA-PA/OA at indicated doses (PA 0 µM, OA 0 µM; PA 50 µM, OA 100 µM; PA 100 µM, OA 200 µM; PA 200 µM, OA 400 µM, respectively) by western blot assay. C Representative fluorescence imaging of BODIPY 493/503 staining (green) in HCT 116 and SW 480 cells treated with BSA-PA/OA (PA 100 µM, OA 200 µM) and/or 10 µM orlistat. Nuclei were stained with DAPI (blue). D Representative quantitative plots of the histogram (left) and MFI (right) of BODIPY 493/503 staining in BSA-PA/OA (PA 100 µM, OA 200 µM) incubated HCT 116 and SW 480 cells treated with or without 10 µM orlistat. E The palmitoylation level of EGFR in HCT 116 and SW480 cells treated with or without BSA-PA/OA (PA 100 µM, OA 200 µM) and 10 µM orlistat by acyl-biotin exchange assay. F The protein level of EGFR in HCT 116 and SW480 cells treated with BSA-PA/OA (PA 100 µM, OA 200 µM) and indicated concentrations of orlistat by western blot assay. G Representative immunofluorescence images for EGFR (red), LAMP1 (green), and DAPI (blue) of BSA-PA/OA (PA 100 µM, OA 200 µM) treated HCT116 cells incubated with or without 10 µM orlistat. Scale bars, 10 μm. Intensity profiles of LAMP1 (green lines) and EGFR (red lines) co-localization signal were shown in plotted lines at three random sites. H Left, the degradation of EGFR treated with BSA-PA/OA (PA 100 µM, OA 200 µM) in HCT 116 cells was evaluated by CHX-chase assay in the presence of orlistat and/or inhibitors for proteasome MG132 and lysosome NH₄Cl. Right, quantification of the intensity determined by the relative level of EGFR remaining (n = 3). I Left, the degradation of EGFR treated with BSA-PA/OA (PA 100 µM, OA 200 µM) in HCT 116 cells was evaluated by CHX-chase assay in the presence of orlistat and/or inhibitors for depalmitoylation ML348, Palm B. Right, quantification of the intensity determined by the relative level of EGFR remaining (n = 3). Data are presented as mean ± SEM. Significance was determined by two-tailed unpaired Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NAFLD non-alcoholic fatty liver disease, BSA bovine serum albumin, PA palmitic acid, OA oleic acid, MFI mean fluorescence intensity, 2-BP 2-bromopalmitate, FASN fatty acid synthases, CHX cycloheximide.