Fig. 4.

Expression of H3.3K36M in Myf5⁺ progenitor cells impairs brown adipose tissue and muscle development. a Schematic of the LSL-K36M transgenic construct. The LSL-K36M transgene consists of the following elements from 5′ to 3′: CAG promoter, four copies of SV40 stop signals (STOP) flanked by two loxP sites, H3.3K36M with FLAG tag, and polyA. b–d LSL-K36M were crossed with Myf5-Cre to generate LSL-K36M;Myf5-Cre mice expressing H3.3K36M in progenitor cells of brown adipose tissue (BAT) and muscle. b Representative morphology of E18.5 embryos. c, d Histological analysis of E18.5 embryos. Sagittal sections of cervical/thoracic area were stained with H&E (c), or with antibodies against the BAT (B) marker Ucp1 (green) and the muscle (M) marker Myosin (red) (d). Scale bar = 300 µm. e–g LSL-K36M were crossed with Cre-ER to generate LSL-K36M;Cre-ER mice. Primary brown preadipocytes were isolated from newborn pups. After SV40T immortalization, cells were treated with 4-hydroxytamoxifen (4OHT) to induce H3.3K36M expression, followed by adipogenesis assay. e Western blot in LSL-K36M;Cre-ER preadipocytes (D0) and adipocytes (D7). f Oil Red O staining at D7 of adipogenesis. Scale bars = 30 μm. g qRT-PCR of Pparg, Cebpa, and Fabp4 expression at D0 and D7 of adipogenesis. h–j Immortalized LSL-K36M;Cre-ER preadipocytes were infected with retroviral vector expressing MyoD. After puromycin selection, cells were treated with 4OHT to induce H3.3K36M expression, followed by myogenesis assay. h Western blot in LSL-K36M;Cre-ER preadipocytes (day 0, D0) and myocytes (day 5, D5). i Cell morphologies were observed under a microscope at D5 of myogenesis. Scale bars = 20 μm. g qRT-PCR analysis of myogenic gene expression at D0 and D5 of myogenesis. All qRT-PCR data are presented as means ± SEM. Three technical replicates from a single experiment were used