Figure 1.
Figure 1. Modulation of EXOSC3 and PAPD5 rescue telomere integrity in DKC1_A353V hESCs. (A) Schematic depicting shRNA cassette insertion into the AAVS1 locus of hESCs. shRNA sequences used for each cassette are described in supplemental Methods and supplemental Table 1. HA, homology arm; RES, resistance cassette. (B) Quantification of EXOSC3 (left) and PAPD5 (right) levels in WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 by quantitative reverse transcription polymerase chain reaction. (C) Western blot for EXOSC3 and PAPD5 in WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 hESCs. LE, long exposure. β-Actin is shown as loading control. Quantification of band intensities is shown (relative to β-actin). (D) Quantification of TERC in WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 by quantitative reverse transcription polymerase chain reaction. (E) Relative abundance of oligoadenylated reads at mature 3′ end of TERC in WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 hESC. (F) Telomerase activity by telomere repeat amplification protocol in WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 hESCs. Range of concentrations represents fourfold serial dilutions. L.C., loading control. (G) Telomere length analysis by telomere restriction fragment (TRF) of WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 hESCs at different passages. Passage numbers are described for each lane. For shEXOSC3 and shPAPD5 transfected cells, passage numbers reflect passage at transfection (35), plus number of passages since transduction. Quantification of mean telomere length is shown. (H) Quantification of interphase quantitative fluorescence in situ hybridization analysis, cells at same passage number as panel G. At least 40 nuclei were analyzed in each cell line. (I) Representative immunoblot analysis of γH2AX in WT, DKC1_A353V (passage 57), DKC1_A353V_shEXOSC3 (passage 35+28), and DKC1_A353V_shPAPD5 (passage 35+28) hESCs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a loading control. Numbers indicate band intensity relative to GAPDH. All experiments were conducted using n = 3, mean ± standard error of the mean, *P ≤ .05, unless otherwise indicated. Statistical analysis was performed using 1-way analysis of variance followed by Tukey’s post hoc test or by Bonferroni posttest in panel H. In panel H, ***P ≤ .0001. n.s., not significant; ZFN, zinc finger nucleases.

Modulation of EXOSC3 and PAPD5 rescue telomere integrity in DKC1_A353V hESCs. (A) Schematic depicting shRNA cassette insertion into the AAVS1 locus of hESCs. shRNA sequences used for each cassette are described in supplemental Methods and supplemental Table 1. HA, homology arm; RES, resistance cassette. (B) Quantification of EXOSC3 (left) and PAPD5 (right) levels in WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 by quantitative reverse transcription polymerase chain reaction. (C) Western blot for EXOSC3 and PAPD5 in WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 hESCs. LE, long exposure. β-Actin is shown as loading control. Quantification of band intensities is shown (relative to β-actin). (D) Quantification of TERC in WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 by quantitative reverse transcription polymerase chain reaction. (E) Relative abundance of oligoadenylated reads at mature 3′ end of TERC in WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 hESC. (F) Telomerase activity by telomere repeat amplification protocol in WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 hESCs. Range of concentrations represents fourfold serial dilutions. L.C., loading control. (G) Telomere length analysis by telomere restriction fragment (TRF) of WT, DKC1_A353V, DKC1_A353V_shEXOSC3, and DKC1_A353V_shPAPD5 hESCs at different passages. Passage numbers are described for each lane. For shEXOSC3 and shPAPD5 transfected cells, passage numbers reflect passage at transfection (35), plus number of passages since transduction. Quantification of mean telomere length is shown. (H) Quantification of interphase quantitative fluorescence in situ hybridization analysis, cells at same passage number as panel G. At least 40 nuclei were analyzed in each cell line. (I) Representative immunoblot analysis of γH2AX in WT, DKC1_A353V (passage 57), DKC1_A353V_shEXOSC3 (passage 35+28), and DKC1_A353V_shPAPD5 (passage 35+28) hESCs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a loading control. Numbers indicate band intensity relative to GAPDH. All experiments were conducted using n = 3, mean ± standard error of the mean, *P ≤ .05, unless otherwise indicated. Statistical analysis was performed using 1-way analysis of variance followed by Tukey’s post hoc test or by Bonferroni posttest in panel H. In panel H, ***P ≤ .0001. n.s., not significant; ZFN, zinc finger nucleases.

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