Research Paper Volume 9, Issue 12 pp 2647—2665

Figure 4. The loss of PDIA3 shows in Clock^∆19 mice. (A) Relative expression assessed by qPCR of the PDI family genes (Pdi, Pdia3, and Pdia6). Note that the PDI family genes, especially Pdia3, were down-regulated in Clock^Δ19 mice. RNA was collected from WT and Clock^Δ19 mice at ZT0 and ZT12 (n=4). Data were normalized to Gapdh expression. **, P < 0.01 and *, P < 0.05 versus control. (B-C) Immunoblots of PDI family proteins (PDI, PDIA3, and PDIA6) at ZT0 and ZT12 from WT and Clock^∆19 mice. Quantification of the immunoblots show significantly reduced expression of PDIA3. **, P < 0.01 and *, P < 0.05 versus control. n=4 mice per group. (D) The dual luciferase method shows that BMAL1:CLOCK can regulate Pdia3 at the transcriptional level. Per1, which is activated by BMAL1:CLOCK, was used as a positive control. HEK293T cells were co-transfected with Pdia3-luc and Bmal1:Clock or Bmal1:Clock^∆19, and the results are expressed as the firefly luciferase activity normalized to renilla luciferase activity. Note that overexpression of BMAL1:CLOCK (dark gray bar) transactivated the Per1 and Pdia3 reporter genes more than in the control transfections (light gray bar). Activation of the Pdia3 reporter was significantly decreased by 30% when Clock^Δ19 was overexpressed with BMAL1 (black bar). The results are expressed as the mean ± S.E.M (n=4). **, P < 0.01 and *, P < 0.05. (E-F) ChIP assay showing the binding of CLOCK to the E-box (CACGTG) of Pdia3 in liver and hypothalamus tissue. Error bars show the S.E.M of the ChIP PCR reactions performed in triplicate (n=4). **, P<0.01; *, P<0.05.