Extended Data Fig. 5: Enhanced ketogenesis during lactation induces transcriptomic and cellular changes in iWAT SVF.

a. Pie chart of differentially expressed genes in 1,3BD SVF vs. Water SVF. Water SVF and 1,3B -SVF were isolated from iWAT of P21 male mice orally administrated water or 1,3BD from P2–P21 (n = 3/group; each sample pools 3-5 animals). b. Pie chart of differentially expressed transcription factor genes in SVF samples in (a). c. Immunofluorescence staining of UCP1 in SVF samples from (a). d. Western blot analysis of PGC1α, PPARγ1/2, CD81, CD137, and β-actin in SVF from (a). β-actin as loading control. e. Densitometry analysis of (d) (n = 3/group). f. Proportion of CD34⁻:CD81⁺ cells in SVF (see Fig. 4i) (n = 3/group). g. Gating strategy for isolating CD81^low and CD81^high cells from SVF of iBAT in P21 CTL, Hmgcs2 KO and 1,3BD-treated mice. h. Quantification of CD81⁺ cell populations from (g) (n = 7 for CTL, 6 for KO, 7 for 1,3BD). i. Mean fluorescence intensity (MFI) of CD81 in cells from (g) (n = 7 for CTL, 6 for KO, 7 for 1,3BD). j. Proportions of CD81^high and CD81^low cells within the CD81⁺ population from (g) (n = 7 for CTL, 6 for KO, 7 for 1,3BD). k. Histogram of BrdU incorporation (left) and quantification of BrdU⁺ cells in CD81^low, CD81^high, CD81^low cells treated with 2 mM βHB for 2 days (CD81^low + βHB cells), and CD81^high cells treated with 2 mM βHB for 2 days (CD81^high + βHB cells) (n = 3/group). l. Oil Red O staining of differentiated CD81^low, CD81^high, CD81^low + βHB, and CD81^high + βHB cells. Cells were isolated from CTL iWAT and cultured under adipogenic conditions for 6 days. Data are expressed as means ± SD. Statistical analyses: two-sided Student’s t-test (e), one-way ANOVA with Tukey’s post hoc test for multiple comparisons (f, h–j) and two-way ANOVA with Tukey’s post hoc test for multiple comparisons (k).

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