Fig. 4: SLC7A5 regulates de novo lipid synthesis in an ERBB2-dependent manner.

A GSEA. The analysis shows a significant enrichment with a high NES of 2.0577, a P-value less than 0.001, and an FDR of 0.0119, indicating a strong association between SLC7A5 and this signaling pathway. B Correlation Analysis. TCGA database analysis revealed a positive correlation between ERBB2 and de novo lipid metabolism pathway-associated proteins ACLY, ACACA, and FASN, suggesting ERBB2 is involvement in lipid metabolism remodeling. C Proteasome Inhibition Assay. Knockdown of ERBB2 significantly reduces ACLY levels (line 2), which are restored upon treatment with 50 µM MG-132 for 6 h (line 4). D CHX Half-life Assay. ERBB2 knockdown shortens the half-life of ACLY (Left). Nuclear-cytoplasmic fractionation revealed that ERBB2 signaling selectively regulates ACLY activity/modification in the cytoplasmic compartment (Right). CHX. 10 µg/mL. E Denaturing IP Assay. Overexpression of ERBB2 decreases ACLY ubiquitination. F Co-Immunoprecipitation (Co-IP) Assay. Overexpression of ERBB2 enhances binding between CUL3 and ERBB2, while reducing binding between CUL3 and KLHL25. All in vitro data are derived from at least three independent experiments. G Proteasome Inhibition Assay. SLC7A5 knockdown increases ACLY (line 2), but ERBB2 knockdown further decreases ACLY levels (line 3). This decrease is inhibited by 50 µM MG-132 for 6 h (line 6). H Denaturing IP Assay. ERBB2 knockdown increases ACLY ubiquitination (line 2), which is reduced upon additional knockdown of CUL3(line 3) or KLHL25 (line 4). I Proteasome Inhibition Assay. ERBB2 knockdown decreases ACLY (line 2), but this decrease is reversed by knockdown of CUL3(line 3) or KLHL25 (line 4). MG-132 treatment (50 µM for 6 h) restores ACLY expression (line 7, 8). J Denaturing IP Assay. SLC7A5 overexpression reduces ACLY ubiquitination (line 2), which is increased upon ERBB2 knockdown (line 3). K ELISA. ELISA measurements of Tri-Glycerides, Free Fatty Acids, and Total Cholesterol showed that ERBB2 knockdown reverses the lipid metabolism remodeling caused by SLC7A5 overexpression. L Lipid Droplet Staining. LD540 staining demonstrated a reduction in lipid droplet accumulation induced by SLC7A5 following ERBB2 knockdown. Error bars represent standard deviation. Statistical significance is denoted as follows. * = P < 0.05, ** = P < 0.001, *** = P < 0.0001, ns = non-significant compared to normal or control treatment.