Extended Data Fig. 2: Induction of pS6 and Mc4r in the VMHvl depends on oestrogen signalling through ERα.
From: Oestrogen engages brain MC4R signalling to drive physical activity in female mice
a, Immunofluorescence microscopy images of ERα and pS6(S244/S247) in the VMHvl (left panels) and ARC (right panels) of Esr1fl/fl and OVX female mice 4 h post oestradiol benzoate treatment. b, Immunofluorescence microscopy images of ERα (red) and pS6(S244/S247) (green) in the VMHvl in conditional knockout (Esr1Nkx2-1Cre) 4OVX female mice following 4 h after oestradiol benzoate treatment. c, Immunofluorescence microscopy images of ERα (red) and pS6(S244/S247) (green) in the VMHvl of intact adult male mice following 4 h post vehicle or oestradiol benzoate treatment. Bar graph to the right shows increased number of pS6(S244/S247)-positive neurons in the VMHvl of male mice after oestradiol benzoate treatment as done for OVX female mice (unpaired two-tailed t-test, t6 = 8.569, P = 0.0001). d, Mammalian Phenotype Ontology terms most significantly enriched and top five significantly enriched Reactome Pathways among DEGs in the VMHvl (vehicle versus oestradiol benzoate). e, qPCR analysis of the indicated target genes in VMHvl from oestrus female mice, proestrus female mice, and male mice; data points represent values from individual mice (one-way ANOVA Nmur2: F2,15 = 8.469, P = 0.0035, post hoc: oestrus (E) versus proestrus (P) P = 0.0454, proestrus versus male P = 0.0030, and oestrus versus male P = 0.1438; Esr1: F2,11 = 10.18, P = 0.0031, post hoc: oestrus versus proestrus P = 0.1650, proestrus versus male P = 0.0374, and oestrus versus male P = 0.0033). Holm–Šidák multiple comparisons test. f, Mc4r expression levels in VMHvl from OVX oestradiol-benzoate-treated (n = 6) female mice normalized to vehicle treatment (n = 3) (unpaired two-tailed Student's t-test t6 = 6.519, P = 0.0006). g, ISH showing Mc4r expression (red arrows) in the VMHvl of an oestrus female, a proestrus female, and an intact male. h, Representative ISH (Mc4r, red arrows) and immunofluorescent (pS6, yellow arrows) staining in the VMHvl (dashed circle) from OVX female mice treated with vehicle for 4 h, oestradiol benzoate for 2 h, or oestradiol benzoate for 4 h. i, Full size images showing bilateral expression of Mc4r and Rprm in intact female mice staged for oestrus and proestrus. Rprm expression is unchanged in both oestrous stages. Data presented as box plots (see Fig. 1c legend for description). Micrographs are representative of data from 5 mice.
