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. Author manuscript; available in PMC: 2021 Jul 18.
Published in final edited form as: Nat Metab. 2021 Jan 18;3(1):59–74. doi: 10.1038/s42255-020-00331-1

Extended Data Fig. 6. Regulation of SR-BI expression by ATF3 or p53.

Extended Data Fig. 6

a. C57BL/6J mice were i.v. injected with Ad-Empty or Ad-hP53 (n=4). After 7 days, hepatic proteins were analyzed by Western blot assays (top panel) and protein levels were quantified (bottom panel). **P=0.0015 versus Empty.

b-d. p53fl/fl mice and hepatocyte-specific p53−/− (L-p53−/−) mice were i.v. injected with AAV8-ALB-Null or AAV8-ALB-hATF3 (n=7 per group). After 30 days, plasma cholesterol lipoprotein profile was analyzed (b). Plasma non-HDL-C (c), ALT and AST (d) levels were quantified. In (c), P=0.232 or 0.687 for AAV-Null versus AAV-ATF3 for p53fl/fl or L-p53−/− mice, respectively. In (d), for ALT, *P=0.048 and **P=0.00025 for AAV-Null versus AAV-ATF3 for p53fl/fl or L-p53−/− mice, respectively; for AST, P=0.547 or 0.97 for AAV-Null versus AAV-ATF3 for p53fl/fl or L-p53−/− mice, respectively.

e and f. p53fl/fl mice and L-p53−/− mice were i.p. injected with either vehicle or hydrocortisone (HC; 2mg/kg) once a day for 7 days (n=7 per group). Plasma cholesterol lipoprotein profile (e) and plasma ALT and AST levels (f) were determined. For ALT, P=0.825 or 0.998 for Vehicle versus HC for p53fl/fl or L-p53−/− mice, respectively. For AST, P=0.936 or 0.941 for Vehicle versus HC for p53fl/fl or L-p53−/− mice, respectively.

g. HepG2 cells were transfected with pGL3-SR-BI-Luc constructs together pCMV-ATF3 or pCMV-Empty (n=8). After 36 h, relative luciferase units (RLU) were determined. **P=2E-6, 0.000017, 1.5E-7, 6.9E-8, 4E-7 or 1E-7 for CMV-Empty versus CMV-ATF3 for −0.29, −1.0, −0.8, −0.6, −0.4 or −0.2 kb SR-BI-Luc, respectively.

h. EMSA was performed to determine the binding site for ATF3 in the proximal SR-BI promoter. Lane 1: free probe. Lane 2: binding assay for testing if ATF3 protein bound to the SR-BI DNA oligonucleotides. Lanes 3-10: competition assays using SR-BI DNA oligonucleotides containing a wild-type or mutant p53 binding site. Lanes 11 and 12: supershift assays in the presence of IgG or an ATF3 antibody. This experiment was repeated once with similar results.

All the data are expressed as mean±SEM. All the data points are biological replicates. A two-tailed Student’s t-test (a, g) or two-way ANOVA with Turkey’s post hoc test for multiple comparisons (c, d, f) was used for statistical analysis.