Fig 2: Generation of spheroids by a conventional massive floating culture lowers the efficiency of the differentiation under a cytokine-free condition. Single cell-dissociated hESCs were cultured in a 96-well flat bottom low attachment plate. Observations under phase contrast microscopy (a) and histogram analyses of the spheroid size (b) indicated that spheroids of diverse sizes with irregular shape were created. Staining with a live cell probe and a dead cell probe (c) showed that there were abundant dead cells which were not incorporated into spheroids. Examinations on gene expression kinetics indicated that fold inductions of the genes for BA makers (PPARG, PRDM16, UCP1), a myoblast marker (MYF5), and a BA inducer (BMP7) (d) were reduced compared to Figure 5.

Fig 3: Effect of HBO on the expression of BAT markers. (A) Expression of a key set of brown fat marker genes (UCP1 and PGC-1a) was confirmed by immunoblot. (B) Quantification of protein levels. Actin was used as a loading control for protein expression. Values are shown as ± S.E.M * p < 0.05, *** p < 0.001 compared to control (n = 3 per each condition).

Fig 4: Effect of ADAMTS5 genotype on AT during cold exposure for 72 h.(A, B) Gene expression levels of Ucp-1 (A) and Cidea (B) in sWAT of WT (+/+) and homozygous deficient (-/-) mice from the ADAMTS5-P (+/+: n = 10; -/-: n = 8) and ADAMTS5-J strains (+/+: n = 11; -/-: n = 7). Gene expression levels are normalized to the housekeeping gene Tbp and shown relative to WT mice at +4°C. (C,D) UCP-1 protein levels in sWAT. The Western blots (C) are quantitated by densitometry, normalized to ß-actin and expressed relative to WT (D). (E-G) Relative ADAMTS5 gene expression in sWAT (E), GON AT (F) and BAT (G) of WT mice kept at 24°C or at 4°C. Gene expression levels are normalized to the housekeeping gene Tbp and shown relative to WT mice at 24°C. Data are means ± SEM of 4–8 and 5–11 determinations for panel D and panel E-G, respectively. ** p<0.01, *** p<0.001 versus 24°C according to the non-parametric Mann-Whitney U test. ADAMTS5, a disintegrin and metalloproteinase with thrombospondin type 1 motifs member 5; WAT, white adipose tissue; UCP-1, uncoupling protein-1; s, subcutaneous; WT, wild-type; GON, gonadal; BAT, brown adipose tissue and Tbp, TATA-box binding protein.

Fig 5: Representative picture of immunoblot (A) and protein levels of UCP-1 and tyrosine hydroxylase (TH) (B) in 3 fat depots (EP, RP+PR and IG indicates epididymal, retroperitoneal combined perirenal, and inguinal fat, respectively) of offspring at P84.Data are mean±SEM, n = 22 (from 5 litters)/group. The comparison was based on the protein levels relative to C (taking as 1) within the same fat depot. *P<0.05. In EP fat, UCP-1 is barely detectable.