Fig. 1.

DIRAS3 expression is up-regulated upon long-term WL in human ASCs. (A) DIRAS3 expression relative to actin was analyzed by q-RT-PCR in early passage ASCs derived from WLDs (n = 4), NWDs (n = 3) and ODs (n = 3). (B) (left panel) Tissue lysates from different donors were examined for DIRAS3 expression by Western blotting. U-2OS cells overexpressing DIRAS3 served as input control (OV). Equal protein quantities were loaded on SDS PAGE based on BCA quantification. Staining of total protein served as control for equal loading. (Right panel) Densitometric band intensities of the DIRAS3 Western blot were quantified using ImageJ. (C) ASCs were cultured for 48 h in 10% FCS or in the absence of serum (Wo FCS) and relative expression of DIRAS3 mRNA was quantified by q-RT-PCR normalized to actin. (D) Detection of DIRAS3 protein in lysates from ASCs cultured in medium supplemented with 2.5% FCS and for four days without serum (starvation) by Western blotting. ASCs infected with a DIRAS3 overexpressing lentivirus (DIRAS3 OV) served as control. All error bars represent the means ± SEM. p values * = p < 0.05, ** = p < 0.001 and *** = p < .0001. The significance of difference between means was assessed by analysis of variance (ANOVA) (A and B) and Student's t test (C).