Figure 7. p16^INK4a downregulates α-klotho expression in humans.

(a) The relative levels of indicated mRNA in hRTECs were examined using RT–qPCR. RNAs were prepared from early-passaged or late-passaged hRTECs (left panels), early-passaged hRTECs transfected with siRNA against p16^INK4a or control (middle panels), or early-passaged hRTECs transfected with or without p16^INK4a expression vector (right panels). (b) Schematic representation of the reporter construct of the human α-klotho gene promoter used in the analysis (left panel). The E2F-binding element is shown as a black rectangle with the sequence and firefly luciferase is shown as Luc. The reporter construct was co-transfected into hRTECs along with Renilla plasmid. Where indicated, cells were also co-transfected with 1.5 μg of expression plasmid encoding E2F1 or E2F3. (c) Early-passage cultured hRTECs were subjected to ChIP analysis using antibodies shown at bottom and PCR primers shown at the top (red arrows). For all graphs, the experiments were performed in triplicate, and representative results from three independent experiments are shown. Data were analysed by Welch's t-test and are displayed as mean±s.d. *P<0.05, **P<0.01.