Figure 6. Asprosin activates Ptprd in a cell autonomous manner.

(a) phosphorylated-Stat3 (p-Stat3) levels measured using ELISA in hypothalamic neural lysate of WT normal chow fed lean mice, 15 days post tail-vein transduction with Ad-empty or Ad-hFBN1 (3.6 x 10⁹ pfu/mouse; three technical replicates of n= 7 or 8 biological replicates per group) viruses.

(b) p-Stat levels measured using ELISA in hypothalamic neural lysate of normal chow fed lean Ptprd^+/+ and Ptprd^−/− male mice (three technical replicates of n= 7 or 8 biological replicates per).

(c-d) A representative western blot and relative quantification of β-actin, p-Stat3 and Stat3 in hypothalamic neural lysate of normal chow fed Ptprd^+/+ and Ptprd^−/− male mice (three technical replicates of n = 6 biological replicates per group).

(e) PTPRD mRNA levels measured in HEK293T cells 24h post transfection with scrambled (control) and Ptprd siRNA (25nM; three technical replicates of n = 3 biological replicates per treatment).

(f-g) A representative western blot and relative quantification of beta actin, p-Stat3 and Stat3 in HEK293T cell lysate 48h post transfection with control and PTPRD siRNA (25nM; three technical replicates of n = 4 biological replicates per treatment).

(h) Stat3-response element driven luciferase activity measured in HEK293T cells transduced with 2μg 4xM67 pTATA-TK-Luc plasmid with control or PTPRD siRNA treatment (25nM; three technical replicates of n = 11 biological replicates per treatment).

(i,j) Validation of asprosin overexpression in HEK293T cells using IL2-his-asprosin expressing mammalian expression plasmid (empty plasmid as control;i) and Ad5-IL2-his-Asprosin (Ad5-empty as control; j) under Ptprd knockdown condition (non-targeted scrambled siRNA as control; four technical replicates of n =4 biological replicates per group).

(k) Percent change in Stat3-response element driven luciferase activity measured in HEK293T cells 72 hours post co-transfection of 2μg 4xM67 pTATA-TK-Luc plasmid with serial dilution of empty or IL2-his-asprosin expressing mammalian expression plasmid (0.25, 0.5, 1 and 2 μg plasmid; two technical replicates of n = 5 or 6 biological replicates per treatment).

(l) Stat3-response element driven luciferase activity measured in HEK293T cells 72 hours post co-transfection of 2μg 4xM67 pTATA-TK-Luc plasmid with 1 pg empty or asprosin expressing plasmid under conditions of control or PTPRD knockdown (25nM; three technical replicates of n = 9 or 10 biological replicates pertreatment).

(m,n) A representative western blot and relative quantification of β-actin, p-Stat3 and Stat3 of HEK293T cell lysate, 72 h post co-treatment of control and PTPRD siRNA (25 nM) with control or asprosin expressing Ad5 vectors (100vp/cell; three technical replicates of n = 4 biological replicates per treatment).

*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; by two-tailed Student’s t-test. Data are represented as mean ± SEM. See also Figure S9.