Fig. 4: Dio3os suppression reduces T3 availability and PRDM16 activity, impairing brown adipogenesis in mouse embryonic fibroblasts and ex vivo organoids.

a Diagram shows the Dlk1-Dio3 imprinting locus. Dio3os is a maternally imprinted long-coding RNA (LncRNA), located ~1.6 kb upstream of the paternal Dio3 gene. IG-DMR: imprinting control region with different DNA methylation. b mRNA expression of Dio3os in BAT of female fetuses and neonates at E18 and P0 (n = 6). Expression was normalized to 18 S rRNA. c–n Pluripotent mouse embryonic fibroblasts (MEFs) were transfected with Cas9 nuclease, CRISPR-tracrRNA, scramble, or Dio3os crRNA (Dio3os-cas9). MEFs were induced brown adipocyte differentiation for 6 days. MEFs in Dio3os-cas9 were also treated with 50 nM T3 (Dio3os-cas9 + T3) during brown adipocyte induction. c Following 72 h transfection, mRNA expression of Dio3os was measured in MEFs. Expression was normalized to 18 S rRNA (n = 4). d, e mRNA expression (d) and protein content of Dio3 (e) after 2 days of brown adipogenic commitment (n = 4). f T3 concentration in differentiated brown adipocytes (2 days) (n = 5). g mRNA expression of thyroid hormone receptors in differentiated brown adipocytes (2 days) (n = 4). h After brown adipogenic commitment for 2 days, immunoblotting measurement of PRDM16 protein content. β-actin was used as a loading control. i Population of committed brown preadipocytes (Lin: EBF2+/PDGFRa+) measured by flow cytometry sorting (FACS) (n = 4). j Immunostaining of mitochondria (mito-tracker) and UCP-1 in differentiated brown adipocytes (6 days) (n = 3). Scale bar 100 µm. k After brown adipogenic commitment for 2 days, PRDM16 was immunoprecipitated and protein lysates were further separated by SDS-PAGE. PRDM16, PGC-1a, and CtBP1 were measured by immunoblotting (n = 4). IgG was used as a negative control. l Quantification of PGC-1a and CtBP1 protein immunoprecipitated with PRDM16 in isolated brown preadipocytes of fetal BAT (E18.5) (n = 6). PRDM16, PGC-1a, and CtBP1 were detected by immunoblotting. IgG was used as a negative control. m Mouse BAT-stromal vascular fractions (SVFs) were induced to form vascular spheroids (5 days), and the presence of endothelial cells was visualized by CD31+ immunostaining. Spheroids were transfected with Cas9 nuclease, CRISPR-tracrRNA, scramble, or Dio3os crRNA (Dio3os-cas9) following brown adipocyte differentiation for 25 days. During brown adipogenic induction, Dio3os-cas9 spheroids were treated with low dose T3 (15 nM; Dio3os-cas9+ LT3) or high dose T3 (50 nM; Dio3os-cas9+ T3) (n = 4). n Heatmap displaying mRNA expression of mitochondrial biogenic and oxidative phosphorylation genes. mRNA expression was normalized to 18 S rRNA (n = 4). o Immunostaining staining of lipids using BioDIPY (red color) and nucleus using DIPY (blue color) (n = 4). Scale bar was 50 µm. Number and size of lipid droplets were quantified using image-J 3D project. Data are presented as mean ± s.e.m. Unpaired Student’s t-test with two-tailed distribution and one-way ANOVA with adjusted multiple-comparison were used for data analyses.