Fig 1. Confirmation of astrocytic IR knockout model (IRKO^GFAP).

(A) Experimental study of cross-section of hypothalamus at Bregma −1.82 mm and Bregma −2.30 mm for IR^loxp and IRKO^GFAP (n = 3–4 per group) using TIRF and confocal microscopy (B) IF cross-sections (50 nm) of hypothalamus for IRKO^GFAP stained with GFAP and NeuN antibodies (n = 3–4 per group). (C) FACS dot plot showing the sorting gates for tdTomato^low/APC^low, tdTomato^low/APC^high, tdTomato^high/APC^low and tdTomato^high/APC^high from mice brain hypothalamic cells (n = 2 per group). RTPCR of hypothalamic gene expression levels of isolated astrocytes from FACS were reported as relative quantification (RQ = ^2-ΔΔCt) for IR^loxp, IRKO^GFAP and IRKO^GFAP nonastrocyte. IR^loxp (black bar), IRKO^GFAP (white bar), and IRKO^GFAP nonastrocyte (dashed white bar). Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus IR^loxp group. (D) Western blotting of protein expression from brain tissues were imaged and quantified for IR^loxp and IRKO^GFAP mice. IR^loxp (black bar) and IRKO^GFAP (white bar), (n = 4 per group, two brains pooled per lane). Values are expressed as means ± SEM. *P < 0.05 IRKO^GFAP versus IR^loxp group. The underlying data can be found in S1 Data. APC, allopycocyanin; FACS, fluorescence-activated cell sorting; GFAP, glial fibrillary acidic protein; IR; IRKO^GFAP, astrocyte-specific insulin receptor deletion; RQ, relative quantification.