Figure 2: Klotho decreased GLUT4 plasma membrane translocation and prevented suppressory effects of insulin on mRNA expression of GLUT4 in differentiated 3T3-L1 cells. Differentiated 3T3-L1 cells were pretreated with different concentrations of klotho (100-1000 pM) for 1 h and then treated with insulin (20 nM) and anti-GLUT4 Alexa flour 488 antibody. Then, cells were fixed and GLUT4 positive cells were counted using a flow cytometer. (a) Flow cytometer charts indicate cell distribution and (b) percent of GLUT4 positive cells in comparison with control. Negative control just treated with anti-GLUT4 Alexa flour 488 antibody, but positive control also received insulin (20 nM). For gene expression assays, cells were treated with klotho (800 pM) and insulin (20 nM) for 12 h. Then total RNA was extracted and GLUT4 mRNA expression was evaluated using qRT-PCR. (c) Results were presented as fold of change of GLUT4 expression in comparison with untreated cells. GLUT4 translocation assay was repeated three times (n = 3), independently. GLUT4 mRNA expression analysis was performed three times in duplicated forms (n = 6).* P < 0.05 and ** P < 0.01 indicate significant differences compared to the positive control and control groups in parts b and c, respectively. GLUT4, glucose transporter type 4; qRT-PCR; quantitative real-time polymerase chain reaction.