Fig. 6: Repression of Ucp1-En4 impairs thermogenic capacity and mitochondrial function in iBAT under cold acclimation conditions.

a Schematic of experimental design of lentiviral injection experiments. b, c qRT-PCR analysis of Ucp1 and lipogenesis-related genes (Acly, Acaca, and Fasn) mRNA expression in iBAT from control and iBAT-En4 mice at 4 °C (n = 5). d Western blot analysis of UCP1 in iBAT from control and iBAT-En4 mice at 4 °C (n = 4). Gray values of protein bands were quantified using Image J software, with Vinculin as a normalizer. e, f Oxygen consumption (VO2) and heat production of control and iBAT-En4 mice in metabolic cages at 30 °C (n = 5). g, i Representative infrared images (left) and quantification of average dorsal skin temperature (right) of control and iBAT-En4 mice at 30 °C or 4 °C (n = 5). h, j Rectal temperature of control and iBAT-En4 mice at 30 °C or 4 °C (n = 5). k, l Representative photographs of iBAT from control and iBAT-En4 mice at 30 °C or 4 °C (left). Representative hematoxylin and eosin staining of iBAT from control and iBAT-En4 mice at 30 °C or 4 °C (right). Scale bar = 50 μm. m, n Immunofluorescence staining of iBAT from control and iBAT-En4 mice at 30 °C or 4 °C for UCP1 (red) and nuclei (blue). Scale bar = 50 μm. o Transmission electron microscopy images of control and iBAT-En4 mice at 4 °C. Scale bar = 1 μm. p Assessment of mitochondrial DNA content by qPCR for iBAT from control and iBAT-En4 mice at 4 °C (n = 5). q Western blot analysis of mitochondrial OXPHOS complex subunits in iBAT from control and iBAT-En4 mice at 4 °C (n = 4). Gray values of protein bands were quantified using Image J software, with Vinculin as a normalizer. Data are presented as mean ± SEM. P values were calculated by two-tailed unpaired Student’s t-tests.