Fig. 1: Loss of α4 in brown preadipocytes impaired insulin signaling and adipogenesis. | Nature Communications

Fig. 1: Loss of α4 in brown preadipocytes impaired insulin signaling and adipogenesis.

From: Phosphatase protector alpha4 (α4) is involved in adipocyte maintenance and mitochondrial homeostasis through regulation of insulin signaling

Fig. 1

a Abundance of α4 mRNA in mouse brown preadipocyte cells during the differentiation process before or on days 2, 4, and 6 after the induction of differentiation. Box plots are defined in terms of minima and maxima by whiskers, and the center and bounds of box by quartiles (one-way ANOVA post hoc Bonferroni test, n = 6 technical replicates per group, *p = 0.01, ***p < 0.0001). b Immunoblotting analysis of α4 in lysates from brown preadipocyte cells during the differentiation process before or 2 and 6 days after induction of the differentiation. (n = 4). c Immunoblotting for α4 and Flag in lysates from Control mouse brown preadipocytes or cells overexpressed 3XFlag-α4 (α4-OE). Densitometric analysis of α4 and Flag in mouse brown preadipocytes. Data are mean ± SEM of n = 5 (Two-tailed Student’s t-test, ***p < 0.0001). d Adipocyte markers in differentiated brown adipocytes from Control (n = 8) and 3XFlag-α4 transfected (α4-OE) cells (n = 8). Data are mean ± SEM (Two-tailed Student’s t-test, Leptin: *p = 0.02, UCP1: ***p = 0.0004). e Mitochondrial oxidative phosphorylation activity in mouse differentiated brown adipocytes from Control (n = 9) and α4-OE (n = 10) cells. Data are mean ± SEM (Two-tailed Student’s t-test, *p < 0.05). f Quantitation of basal respiration (*p = 0.02), maximal respiration capacity, ATP production and Spare Respiratory Capacity (*p = 0.01). Data are presented as mean ± SEM (Two-tailed Student’s t-test, Control (n = 9), α4-OE (n = 10)). g Levels of adipocyte markers in differentiated brown adipocytes from Control cells and cells subjected to knockdown of α4 by shRNA (α4 KD) (n = 8 biologically independent cell clones per group). Data are mean ± SEM (Two-tailed Student’s t-test, Adiponectin: **p = 0.003, PPARγ: *p = 0.02, Glut4: *p = 0.003, Adrb3: **p = 0.07, AP2: ***p = 0.0009, UCP1: **p = 0.006). h Immunoblotting analysis results showing insulin-dependent signaling molecules for IR, Akt, ERK, and S6 phosphorylation in lysates from α4 KD-treated or Control adipocytes after 100 nM insulin stimulation for the indicated durations. i–l Relative changes in protein phosphorylation based on the densitometric immunoblotting analysis (Supplementary Fig. 1h) of cell lysates of adipocytes from α4KD and Control for 5 min using 100 nM insulin. Data are presented as mean ± SEM (One-way ANOVA post hoc Bonferroni test; three clones were used in three independent experiments, p-IR: ***p = 0.0007, p-Akt (S473): **p = 0.001, p-S6 (S235/S236): ***p = 0.0001). Source data are provided as a Source Data file.

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