Fig. 2: Loss of Asc-1 function induces beiging in white adipocytes.

a Immunofluorescence staining of ASC-1 (green), F-Actin (gray), and lipids (red) in SCF of 2 week old Asc-1 wt and ko mice (n = 1). Size bar 100 µm. b Correlation between Asc-1 and Ppary expression in 52 immortalized preadipocyte clones derived from adult subcutaneous SVF. c Relative Ucp1 expression before (day 0) or after 8 days of differentiation of MACS-sorted ASC-1⁺ and ASC-1⁻ preadipocytes with either insulin alone, the normal differentiation protocol (Adipo) or the differentiation protocol plus 1 µM rosiglitazone (n = 4). Asc-1 knockdown (shAsc-1) and control (shScr) immortalized subcutaneous preadipocytes were grown to 100% confluence, and RNA or protein was taken from the day of differentiation start (preadipocyte) and after 8 days of differentiation with (Rosi) or without rosiglitazone (adipocyte). d Relative gene expression of Asc-1 (n = 6, only shAsc-1 preadipocyte n = 5), Fabp4 (n = 6, only shScr adipocyte n = 5), Ucp1 (n = 10, only shScr preadipocyte and shAsc-1 Rosi n = 9), and Tfam (n = 6). e Western blot of UCP1 (33 kDa), PPARy (54 kDa), and β-actin (42 kDa). f Basal respiration (n = 20 shScr and 24 shAsc-1 technical replicates) and proton leakage (n = 19 shScr and 24 shAsc-1 technical replicates) calculated from the OCR measured by a Seahorse flux analyzer after differentiation with rosiglitazone, normalized to DNA content (n = 3 biologicalreplicates). g Balb/c nude mice were injected with shAsc-1 and shScr preadipocytes over the sternum. Histology (n = 1; size bar 20 µm) and h–i immunofluorescence staining of engrafted tissue 6 weeks post transplantation. Green: UCP1 (n = 2), red: lipids, blue: DAPI, and gray: PERILIPIN-1 (n = 1 shScr-2 shAsc-1). Size bar 100 µm. Statistics were calculated using ordinary two-way ANOVA with Tukey’s multiple comparison post hoc test or two-tailed unpaired Students t test (f). Data are shown as mean ± SEM.