Fig 2: Foxk2 induced adipogenic differentiation in primary BMSCs. The overexpression (A, B) or knockdown (H, I) of Foxk2 was verified using RT‐qPCR and Western blotting in primary BMSCs after infection of Ad‐Foxk2 or transfection of Foxk2 siRNAs. The infected (C–G) or transfection (J–N) BMSCs were then cultured in AIM, and stained with Oil Red O after 5 days (C, J, scale bars represent 100 μm). The extent of Oil Red O staining was quantified by measuring absorbance at OD520 after extraction with isopropanol (D, K). After 3 days, RNA (E, L) and protein (F, M) levels of the adipocyte‐specific genes were assessed by RT‐qPCR and western blot analysis, respectively. Western blot bands were further quantified with greyscale analysis (G, N). β‐actin was used as an internal control. Data are presented as mean ± S.D., with statistical significance determined by a two‐tailed Student's t‐test: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the control group.

Fig 3: Foxk2 was enhanced in iWAT and eWAT of obese mice. (A) RT‐qPCR analysis was conducted to quantify Foxk2 expression across various tissues in C57BL/6J mice (n = 4). The expression level of Foxk2 in the liver was normalised to 1. (B‐D) RT‐qPCR was used to assess the relative expression of Foxk2, PPARγ, Srebp1c, Fasn, Fabp4, and Lpl in specific adipose tissues. Data are presented as mean ± S.D. (n = 8). Statistical significance was determined using a two‐tailed Student's t‐test with significance levels indicated as p < 0.05 (*), p < 0.001 (***), and p < 0.0001 (****). Values are presented relative to non‐obese db/m mice.

Fig 4: Adipogenic treatment facilitated Foxk2 nuclear translocation. (A) Western blotting was conducted to evaluate Foxk2 expression in C3H/10 T1/2 cultured by basal medium or AIM after 3 days. (B) The above cells immunostained for Foxk2 (AF594, red) and DAPI (blue). DAPI was used to label the nuclei. Scale bars represent 50 μm. (C) Frequency of Foxk2+ (AF594+) cells among DAPI+ cells. (D) Nuclear and cytoplasmic fractions were extracted from the Ad‐Foxk2‐infected cells, and the target genes in each fraction were assessed by immunoblotting. β‐actin and histone H3 were used as markers for the cytosolic and nuclear fractions, respectively. (E) Quantification of Foxk2 expression in the nuclear fraction was performed using greyscale analysis. (F) C3H/10 T1/2 cells infected with Ad‐Foxk2 and cultured in basal medium or AIM for 3 days in the presence or absence of LY294002 (10 μM), MK‐2206 (5 μM) or rapamycin (100 nM). Scale bars represent 50 μm. (G) Frequency of Foxk2+ (AF488+) cells among DAPI+ cells. (H) The target genes in nuclear and cytoplasmic fractions were assessed by immunoblotting. (I) The cells in (H) were also stained with Oil Red O, and representative images are shown. Scale bars represent 100 μm. (J) Oil Red O extracted with isopropanol was measured at OD520. (K, L) Protein levels of Foxk2 and the adipocyte‐specific genes were assessed by western blot analysis. The data are presented as mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to the control group. A two‐tailed Student's t‐test was applied for comparisons between two groups, and one‐way ANOVA followed by Tukey's test was applied for comparisons among more than two groups.

Fig 5: Adipogenic induction stimulates Foxk2 expression in progenitor cells. (A, C, E) RT‐qPCR was used to measure the mRNA expression levels of PPARγ and Foxk2 in primary BMSCs (A), C3H/10 T1/2 cells (C), and ST2 cells (E) at specified time points during adipogenic differentiation. (B, D, F) Western blotting analysis was performed to assess the protein levels of Foxk2, C/EBPα, PPARγ, and FABP4 in primary BMSCs (B), C3H/10 T1/2 cells (D), and ST2 cells (F) at the same time points. Data are presented as means ± S.D. Statistical significance was determined using ANOVA followed by Tukey's test, with significance indicated as *p < 0.05, compared to the control group.