Extended Data Fig. 7: Estrogen blocks p53 transcriptional activation of ghrelin upon UVB, Related to Fig. 6.
From: Food-seeking behavior is triggered by skin ultraviolet exposure in males
a, ER-α protein levels in human adipose tissue. β-actin used as the loading control. Quantification of the protein amount normalized to β-actin (Q) is indicated. b, Left panel: Experimental design. Right panel: Immunofluorescence of cells stained for ghrelin (red), perilipin 1 (Plin1, an adipocyte marker, green) and nuclei (with DAPI, blue) (n = 10 random fields from 3 biological replicates). Relative ghrelin intensity normalized to DAPI. c, p53, p21 and ghrelin relative mRNA levels in 3T3-L1 differentiated adipocytes treated with a vehicle, 100 nM DHT or 100 nM β-E2 and with UVB (50 mJ/cm2) or control irradiation after 24 h (n = 3 biological replicates). Data normalized to 18S. d, Upper panel: Luciferase activity upstream of the human ghrelin promoter (−3,000 bp upstream) in the presence of ER-α or p53, or an empty vector (control) in HeLa cells with or without 100 nM β-E2 treatment for 24 h. Lower panel: Luciferase activity similar as in upper panel in H1299 cells upon ER-α or an empty vector (control) expression. Firefly luciferase activity normalized to Renilla luciferase activity (n = 3 biological replicates). e–g, ChIP levels (fold) normalized to input of p53 and IgG occupancy over ghrelin promotor (e); p53 (f) and NCOR1 (g) occupancy over p21 promotor in LiSa-2 adipocytes treated with 100 nM β-E2 or vehicle and irradiated with UVB (50 mJ/cm2) or mock-UVB (control) and harvested after 24 h (n = 3 biological replicates). h, NCOR1 protein level in LiSa-2 adipocytes treated as in (e). β-actin was used as the loading control. Quantification of the protein amount normalized to β-actin (Q) is indicated. i, 17-β estradiol plasma protein levels in OVX or sham female mice after 5 weeks of either daily UVB (50 mJ/cm2) or mock-UVB (control) exposures (n = 7 biologically independent mice per condition). j, Weekly mean body weight (grams) of the OVX/sham female mice after daily UVB (50 mJ/cm2) or mock-UVB (control) irradiation before and 3 weeks after OVX/sham surgery (n = 29 sham before/after and n = 25 OVX before/after biologically independent female mice). k, Elevated-plus maze (EPM) responses in female OVX/sham mice after 4 weeks of either daily UVB (50 mJ/cm2) or mock-UVB (control) exposures. Right panel: Representative heat maps from each condition (n = 10 biologically independent mice per condition). l, ghrelin plasma protein levels of in OVX or sham female mice after 5 weeks of daily UVB (50 mJ/cm2) or mock-UVB (control) exposures (n = 10 biologically independent mice per condition). m, p53 relative mRNA levels from the skin of OVX or sham female mice treated as in (l) (n = 5 biologically independent mice per condition). Data normalized to 36b4. n, ER-α relative mRNA levels from the skin of OVX or sham female mice treated as in (l) (n = 5 mice per condition). Data normalized to 36b4. o, Aromatase relative mRNA levels in human female skin adipose tissue 26 h after treatment with estrogen inhibitor (leterozole (5 µM)) or vehicle (DMSO). Data normalized to 36b4. For all relevant panels: Data are presented as mean ± SEM; an unpaired t-test p-values are shown, or statistical details for OVX or UVB factors in the ANOVAs (F-values, degrees of freedom, p-value) with interaction appears in Supplementary Table 10, or two-way ANOVA analysis with multiple correction test appears in Supplementary Table 11.
