Fig 1: Lipid droplets enhance MDM2‐mediated p53 degradation via the Cyb5r3‐Myh9 interaction. A) Fluorescence imaging showing that anti‐p53 immunofluorescence (green) colocalized with lipid droplets (LDs) stained with Nile red (red). B) Immunoprecipitation and Western blot analysis of MDM2‐mediated p53 ubiquitination (Ub) on LDs. LDs were purified from equal numbers of control, OA‐treated, and OA+AMG‐232‐treated tumor cells. AMG‐232 inhibits MDM2 activity. ADRP and GAPDH label LDs and the cytosolic fraction, respectively. Fetal bovine serum was used as a control. C) Immunoprecipitation and Western blot analysis demonstrated the interaction between p53 and Cyb5r3 on purified LDs from tumor cells. D,E) Western blot analysis of p53 levels in tumor cells with Cyb5r3 knockdown (shCyb5r3‐1 and shCyb5r3‐2) cultured with mouse or human serum. D) Representative images and E) quantification. F) Immunoprecipitation and Western blot analysis of the interaction between p53 and Myh9 on purified LDs from tumor cells. G) Fluorescence imaging reveals Myh9 localization on LDs, visualized by Nile red staining. H,I) Western blot analysis of p53 levels in tumor cells with Myh9 knockdown (shMyh9‐1 and shMyh9‐2) cultured with mouse or human serum. H) Representative images and I) quantification. J) Fluorescence imaging shows the effect of Myh9 and Cyb5r3 knockdown on p53 localization to LDs in OA‐treated tumor cells. K) Immunofluorescence analysis of breast and pancreatic cancer tissues derived from obese patients (BMI ≥ 30 to validate the relationship between Myh9/Cyb5r3 and p53 expression. Experimental conditions: human ND serum from healthy BMI patients and human HFD serum from overweight patients; mouse ND serum from normal diet groups and mouse HFD serum from high‐fat diet groups; and 100 µm OA or 4 µm AMG‐232. β‐actin was used as a control for Western blotting. Scale bars: 5 µm (A,G,J) and 100 µm (K). Statistical analysis: One‐way ANOVA with post hoc test for E and I. Significance: ***, p < 0.001. Control: fetal bovine serum; ND: normal diet; HFD, high‐fat diet; OA: oleic acid.
Fig 2: The lipid droplet‐p53 regulatory circuit enhances tumor growth. A) A CCK‐8 assay was used to measure the proliferation of Myh9‐knockdown (shMyh9‐1 and shMyh9‐2) tumor cells in response to 100 µm OA stimulation; B) A CCK‐8 assay was used to quantify the proliferation of Cyb5r3‐knockdown (shCyb5r3‐1 and shCyb5r3‐2) tumor cells upon 100 µm OA stimulation; C) A CCK‐8 assay was used to evaluate the proliferation of RPS3A‐knockdown (shRPS3A‐1 and shRPS3A‐2) tumor cells following 100 µm OA stimulation. Overexpression (OE) was used for the shRNA rescue experiments. The control conditions utilized fetal bovine serum, and OA denotes oleic acid treatment. Error bars denote the s.d. in A‐C (n = 3 per group). P‐values were calculated using one‐way ANOVA with a post hoc test in A, B, and C. ***, p < 0.001; ns, p > 0.05.
Supplier Page from Sino Biological, Inc. for Mouse CYB5R3 Gene ORF cDNA clone expression plasmid, N-Myc tag