Fig. 2: CKB silencing induces a pro-inflammatory profile in human white adipocytes.
From: Impaired phosphocreatine metabolism in white adipocytes promotes inflammation
a, Expression of indicated genes in cells of human subcutaneous WAT. Results are displayed as z-scores. b, Expression of indicated genes during adipogenesis. c, CKB mRNA levels in human adipocytes (six replicates per condition, repeated three times) transfected with non-silencing (siC) or CKB-targeting (siCKB) oligonucleotides. ****P < 0.0001. d, Western blot displaying CK-B and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in human adipocytes (three replicates per condition, repeated three times) transfected with siC or siCKB. ***P = 0.0002. e, Creatine kinase activity measured in lysates of human adipocytes (three to four replicates per condition, repeated three times) transfected with siC or siCKB. ***P = 0.0003. f, Metabolite levels in human adipocytes (ten replicates per condition, repeated twice) transfected with siC or siCKB. **P = 0.0093 for phosphocreatine and 0.002 for phosphocreatine/creatine ratio. g, Western blot showing the protein levels in mitochondrial and cytoplasmic fractions of human adipocytes transfected with siC, siCKB or siCKMT2 (repeated three times). Creatine kinase activity was determined in paired lysates as indicated in the right panel from one experiment. h, Principal component analysis based on microarray data from human adipocytes (three replicates per condition) transfected with siC or siCKB. Ellipses indicate 95% confidence intervals. i, log2(fold-change) of genes regulated by siCKB in human adipocytes (upper half, from microarray data presented in h). For each gene, the association (Spearman’s rank correlation coefficient ρ value) with CKB expression in WAT transcriptomic data from cohort 2 is shown (lower half). Leading edge genes in the GSEA ‘HALLMARK_INFLAMMATORY _RESPONSE’ pathway identified in Extended Data Fig. 2g are shown. j, CCL2 mRNA expression in human adipocytes (six replicates per condition, repeated three times) transfected with siC or siCKB. ***P = 0.0002. k, CCL2 secretion measured by ELISA from human adipocytes (three replicates per condition, repeated three times) transfected with siC or siCKB. **P = 0.0059. l, Correlation between CCL2 and CKB mRNA levels in human WAT from cohort 2. m, Correlation between CCL2 WAT secretion and CKB mRNA levels in human WAT from cohort 2. In c–f, j and k, Student’s two-sided t-test was used. In l and m, standardized β and P values are shown for multiple regression analysis after BMI correction. Data in c–f, j and k are shown as mean ± s.e.m. APC, adipocyte progenitor cells; ATM, adipose tissue macrophages; cyto, cytoplasm; Cr, creatine; M1, M1-macrophages; M2, M2-macrophages; mito, mitochondria, PC, principal component; PCr, phosphocreatine.
