Figure 8
From: NOTUM promotes thermogenic capacity and protects against diet-induced obesity in male mice
NOTUM abolishes WNT3A’s inhibitory effect on brown adipocyte differentiation in vitro. Confluent pre-BAT cells were treated for 7 days with media promoting brown adipocyte differentiation that contain BSA (1 μg/ml, control), 10 ng/ml human WNT3A, 300 ng/ml human NOTUM, or 10 ng/ml human WNT3A and human 300 ng/ml NOTUM for 7 days before gene expression analysis and immunoblotting. The mRNA (a) (n = 4–6/group) and protein (b) (n = 3/group) levels of genes involved in brown adipocyte differentiation and Wnt signaling are shown. For (b) one sample from baseline (Day 0, D0) was included as reference. For (b), The images shown in the top 4 panels were obtained from the same blot: C/EBPα, PPARγ, and β-ACTIN images were obtained from the the top part of the same blot after stripping and re-probing, whereas image of UCP1 was obtained from the bottom part of the same blot. The images shown in the middle 2 panels of (b), b-CATENIN and β-ACTIN, were obtained from top and bottom portions of the same blot, respectively. The images shown in the bottom 3 panels of (b), ATGL, HSL, and β-ACTIN, were obtained from the same blot. HSL was obtained from the top portion of the blot. ATGL and β-ACTIN were obtained from the bottom portion of the blot after stripping and re-probing. Individual proteins to β-ACTIN ratios are shown in the right panel. (c) The mRNA levels of genes involved in TGF-β signaling and collagen deposition are shown (n = 4–5/group). (d) Activation of TGF-β signaling in pre-BAT at Day 7 as determined by p-SMAD2 to SMAD2 ratio is shown (n = 3/group). One sample at day 0 (D0) was shown as reference. The same blot was first probed with the anti p-SMAD2 antibodies to obtain the p-SMAD2 image, the blot was then stripped and re-probed with the anti SMAD2 antibody to obtain the SMAD2 image. The ratios of p-SMAD2 to SMAD2 are shown in the right panel. Symbols: *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, as compared to control group. #: p < 0.05, ##: p < 0.01, ###: p < 0.001, ####: p < 0.0001, as compared to WNT3A group. The Holm–Sidak post hoc analysis was used for between-group comparisons after statistical significance was established by ANOVA (see “Material and methods” section).
