Figure 2. Pin1 inhibition by juglone increases PTH mRNA in vivo posttranscriptionally.
(A and B) Juglone i.p. (A) PPIase activity assays of parathyroid cytoplasmic lysates from rats injected i.p. with juglone or vehicle. (B) Serum PTH in rats as in A. (C–F) Juglone applied topically. (C) PPIase activity of parathyroid cytoplasmic lysates from rats treated with juglone or vehicle applied topically; data in A and C are presented as mean ± SEM (n = 3) from 1 of 3 repeat experiments; P < 0.05 at 5–30 seconds. (D) Immunoblots of the extracts in C for Pin1 and GAPDH. (E) Northern blots of PTH mRNA levels after juglone, in rats as in C. The lanes were run on the same gel but were noncontiguous (white line). (F) qRT-PCR analysis for PTH mRNA in rat parathyroids as in C; *P < 0.05. (G) Nuclear run-ons of PTH transcription in isolated nuclei of parathyroids from 5 rats in each group after topical juglone (J) or vehicle (C). (H) Quantification of PTH mRNA transcription in G, showing no difference in PTH transcription. The same result was obtained in a repeat experiment. (I) RNase protection analysis (RPA) of total parathyroid RNA from rats after topical juglone or vehicle. Left panels: RPA using an antisense probe for the PTH mRNA 3′ UTR or for control GAPDH mRNA. Right panel: RPA using an antisense probe for the first intron (newly transcribed pre–PTH mRNA). The intact probes before and after RNase treatment (arrows to the left and right of the gels, respectively) and hybridization with tRNA as a nonspecific control are shown. Juglone increased PTH mRNA but not the pre–PTH mRNA or GAPDH mRNA.