Fig. 1

Hmga2 suppression interferes with embryonic stem cell (ESC) differentiation. a ESCs expressing GFP under the control of Nanog gene promoter were transfected with the control shRNA (sh Ctrl) and two different shRNAs targeting Hmga2, which induced a significant decrease of the Hmga2 protein. Knockdown cells were induced to differentiate into neurons. After 8 days of differentiation, cells were harvested and the number of GFP expressing cells was measured by FACS, subtracting the fluorescent background of parental differentiated E14Tg2a ESCs. The histogram shows the percentage of GFP-positive cells in the indicated samples as mean ± SEM (n = 6). *P <0.05. The efficiency of neuron formation was evaluated at 10 days of differentiation by staining the cells with a neuro-specific antibody (anti-β3-tubulin). Scale bars: 250 μm. b Analysis of the phenotype induced by Hmga2 suppression during neuroectodermal differentiation of ESCs. ESCs were transfected with the indicated siRNA, and induced to differentiate through serum-free embryoid body (SFEB) formation for 2 and 4 days. Sectioned SFEBs were then subjected to immunostaining to analyze the presence of stemness (Oct4, Nanog) and neuroectodermal (Sox1) markers. Scale bars: 100 μm. The histograms represent the percentage of Oct4-, Nanog-, and Sox1-positive cells counted in more than six independent fields. *P <0.05, **P <0.01. The efficiency of Hmga2 silencing by siRNA was measured by western blot. c qPCR analysis of the expression changes of Oct4, Nanog, Rex1, and Klf4 in differentiated ESCs upon suppression of Hmga2. Data represent mean of three independent experiments (n = 3) ± SEM. *P <0.05. **P <0.01. d MPI ESCs were transfected with a control siRNA or a specific Hmga2 siRNA and induced to differentiate into SFEBs. At 4 days of differentiation, SFEBs were collected and the expression of the indicated markers was analyzed by qPCR and western blot. Data represent mean of three independent experiments (n = 3) ± SEM. *P <0.05. e qPCR analysis of the expression of Oct4, Nanog, Rex1, and Klf4 in undifferentiated ESCs upon suppression of Hmga2. Data represent mean of independent experiments (Oct4, Rex1, Klf4, n = 3; Nanog, n = 4) ± SEM. *P <0.05. f Representative images of the double staining of undifferentiated ESCs with Nanog (red) and Hmga2 (green) antibodies. The arrows indicate cells expressing Hmga2, which corresponds to cells expressing the lowest level of Nanog. Scale bars: 20 μm