Fig. 2: Primary mouse NRIP1KO adipocytes from both male and female mice display increased thermogenic gene expression and uncoupled oxygen consumption.
From: CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease
a, b Ucp1, Cidea, Fabp3 and thermogenic gene expression, fold change over NTC. In (a, b) male = blue (n = 3), female = peach (n = 3). Error bars denote mean ± SEM. c UCP1 (33 kDa) protein expression in primary adipocytes, 6 days post differentiation with various RNP concentrations. Lanes 1 to 6 contain 20 μg of protein lysate and lane 8 contains 2.5 μg of protein lysate from mouse BAT. Tubulin, as loading control. d Oxidative phosphorylation protein expression in primary male adipocytes on day 7 post differentiation targeted with NTC or NRIP1 sgRNA-M1-M7. Lanes 1 to 8 contain 20 μg protein while lane 9 contains 2.5 μg protein (mouse BAT). GAPDH, loading control. e Oxygen consumption rates (OCR) in mature primary male adipocytes targeted with NTC or NRIP1 sgRNA-M6 without norepinephrine (NE) stimulation and (f) with NE stimulation. Statistical comparison for OCR in technical replicates was done for (e, f) using one-way ANOVA with Sidak’s multiple comparison test. ****p < 0.0001 (g). Summary of OCR in panels (e, f) at 40 min in primary male adipocytes with and without NE stimulation. Statistical comparison using two – way ANOVA with Sidak’s multiple comparison test. ****p < 0.0001, ***p = 0.0003. h OCR in mature primary female adipocytes targeted with NTC or NRIP1 sgRNA-M6 without NE stimulation and (i) with NE stimulation. Statistical comparison for OCR technical replicates for (h, i) was using one-way ANOVA with Sidak’s multiple comparison test. ****p < 0.0001 (j). Summary of OCR at 40 min in primary female adipocytes. Statistical comparison using two – way ANOVA with Sidak’s multiple comparison test. NE **p = 0.0065, NE + Oligo *p = 0.0112, NE + FCCP ***p = 0.0005. Biological replicates confirmed the increased OCR by NE treated NripKO cells compared to NE treated NTC cells (p = 0.02, n = 4) as well as increased OCR by NE treated NripKO cells plus oligomycin compared to NE plus oligomycin in NTC cells (p = 0.03, n = 4) (Supplementary Fig. 3). Molecular weight markers in (c and d) are shown in the source data. Error bars in (e–j) denote mean ± SEM.
