Extended Data Fig. 1: AHCY overexpression in different cell types.
Gene expression levels of (A) adenosylhomocysteinase (AHCY) and (B) itchy E3 ubiquitin protein ligase (ITCH) in stromal vascular fraction (SVF) cells before and after adipocyte differentiation and compared to that of (C) agouti signaling protein (ASIP) in differentiated SVF cells are shown from the patient and controls (Ctr) patients 1-4 (n=4; 3 females, 1 male) matched for age (mean age 13.8 years) and body weight state (all presenting with overweight or obesity). Data points of n=3 independent experiments are indicated for each subject. In peripheral blood leukocytes (PBL) AHCY (D) and ITCH (E) expression are shown from the patient and controls. Data points are indicated for each subject. (F) Relative AHCY expression in Simpson–Golabi-Behmel syndrome (SGBS) cells after transfection with pCMV-Entry control vector (Ctr) or pCMV-AHCY (AHCY) in n=3 independent experiments. Transfected SGBS cells were differentiated into adipocytes and the percentage of differentiated cells was determined by cell counting after Hoechst and Nile red staining. Using the same cells adipogenic capacity was compared by Oil red O staining (representative microscopic pictures from one of three independent experiments; scale bar = 500µM) and gene expression of the adipogenic marker gene peroxisome proliferator-activated receptor gamma (PPARG) after differentiation in n=2 independent experiments was determined. Bar plots show the mean ± SEM. (G) AHCY and ITCH expression levels are shown for SVF cell-derived induced pluripotent stem cells (iPSCs) from three clones of the patient and two clones from each of the control (Ctr) children, 1 and 10. Data points for each clone from each subject are indicated. The bar plots A-E and G present the mean values ± SEM as a fold change of the controls. Gene expression data were normalized to the expression of β-actin and TATA-box-binding protein. All bars from controls are shown in blue and from the patient in red. P values derived using two-sided Student’s t-test are given.
