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. Author manuscript; available in PMC: 2016 Mar 14.
Published in final edited form as: Nat Genet. 2015 Oct 19;47(12):1482–1488. doi: 10.1038/ng.3423

Figure 2.

Figure 2

PARN deficiency results in decreased TERC levels, telomerase activity and telomere length. (a) qPCR of TERC transcripts in patient-derived and normal (wild-type) fibroblasts (n = 2 biological replicates). (b) qPCR of PARN transcript levels in iPSCs (n = 2 biological replicates). (c) Left, representative immunoblot of PARN, dyskerin and actin protein levels in iPSCs. Right, PARN and dyskerin protein levels normalized to actin levels (n = 4 biological replicates). (d) RNA blot of TERC following denaturing agarose gel electrophoresis of RNA from iPSCs. Ethidium bromide staining of 18S rRNA was used as a loading control. TERC levels normalized to those in WT1 cells are indicated. (e) Telomere repeat amplification protocol (TRAP) assay for telomerase activity in iPSCs, using fivefold dilutions of input cell extract. The internal control (IC) amplification standard is indicated. (f) Southern blot of telomere length by terminal restriction fragment length analysis in normal and patient-derived (clones C1 and C2) iPSCs, as well as control TERC-haploinsufficient (TR+/−) iPSCs. Passage numbers are indicated. MW, molecular weight marker. (g) qPCR of PARN, TERC and DKC1 transcripts from HEK293 cells transduced with lentivirus encoding shRNA directed against PARN versus luciferase (control) (n = 4 biological replicates). (h) Left, representative immunoblot of PARN, dyskerin and actin protein levels in HEK293 cells (from g) transduced with lentivirus encoding shRNA directed against PARN versus luciferase. Right, PARN and dyskerin protein levels normalized to actin levels (n = 4 biological replicates). For all panels, error bars represent s.d. Significance is indicated: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; NS, not significant.