Extended Data Fig. 2: CKB depletion does not affect adipogenesis.

a. Representative immunofluorescence microphotographs of adipocyte progenitors stained with Hoechst and antibodies targeting DPP4. Scale bar=200 μm. b-e. In vitro differentiated human adipocytes were transfected with scrambled non-silencing (siC) or CKB-targeting (siCKB) (three-four replicates/condition). Effects on gene expression of adipogenic/thermogenic markers (experiment repeated twice) (B), adiponectin secretion (five replicates/condition) (C), cellular triglyceride levels (four replicates/condition) (D) and lipid droplet morphology (from experiments in panel D) (E) were determined. Scale bar in panel E = 10 μm. Data in bar charts are shown as mean ± SEM. f. PCr and Cr levels in in vitro differentiated human adipocytes transfected with scrambled non-silencing (siC) or CKMT2-targeting (siCKMT2) oligonucleotides (n = 5 replicates/group). Data are shown as mean ± SEM. *p = 0.024 for PCr and 0.045 for PCr/Cr, respectively by Student’s two-sided t-test. g. Gene set enrichment analysis (GSEA) of genes correlating with CKB expression in human WAT and regulated by siCKB transfection in human in vitro differentiated adipocytes. Hallmark gene sets from MsigDB were used to calculate the enrichment. Details on the analyses are found in the Methods section. The data are presented as dot plots. The color of the dot indicates the normalized enrichment score (NES) for each pathway. The size of the dot represents the negative log₁₀ adjusted p-value for the enrichment of each pathway. h. CKB and CCL2 mRNA levels in in vitro differentiated human adipocytes, derived from a female donor, transfected with siC) or siCKB (four replicates/condition). Data are shown as mean ± SEM. *p = 0.024, ****p < 0.0001 by Student’s two-sided t-test. i. Same experiment as in panel H but displaying CCL2 secretion (four replicates/condition). Data are shown as mean ± SEM.****p < 0.0001 by Student’s two-sided t-test. Abbreviations: Adj. p-val.=adjusted p-value, Cr=creatine, NES = normalized enrichment score, PCr=phosphocreatine, WAT = white adipose tissue.