Fig 1.
Generation and verification of the TERT CKO mice.
(A) Schematic gene-targeting map of TERT gene. The construction of TERT floxed and TERT CKO alleles are shown before and after tamoxifen treatment. TERT gene exon 1–2 was floxed after recombination between WT and targeted alleles. Primers pairs P1/P2 and P3/P5 were used to detect floxed TERT alleles. Primer pair P1/P5 was used to detected the TERT gene excision by Cre. (B) Representative Southern blot analysis. The ES clone with homologous recombination was digested with the restriction enzyme Bsr GI followed by Southern blotting with Dig-labeled 5’ probe as shown in (A). The detected WT and targeted alleles are 14.58 and 7.3 kb, respectively. (C) PCR genotyping using genomic DNA from mouse tails. The PCR fragments for WT was 154bp, for TERT fl/+ they were 154 and 343 bp, for TERT fl/fl it was 343 bp, and for TERT CKO it was 215bp after tamoxifen-induced Cre excision. “Ladder” referred to 100 bp DNA ladder.
Fig 2.
TERT and telomerase in floxed TERT vs WT mice.
Total RNA or protein lysates were prepared from the lung tissues and MLF from BLM or PBS-injected TERT fl/fl or WT mice. TERT gene expression was analyzed by qRT-PCR and expressed as 2-ΔΔCT (n = 3) in (A), and the telomerase activities were detected by TRAP-ELISA kit and expressed as fold change over their PBS control, respectively (B). n = 3. *, P < 0.05.
Fig 3.
The in vitro excision of MLF TERT by Cre activation.
The MLF were isolated from TERT fl/fl mice, and then transduced with 100 MOI of AdCre vector or AdGFP control vector. Six days after transduction, TERT mRNA (A) and telomerase (B) were analyzed, respectively, as described in the legend of Fig 2. n = 3. (C) The MLF from TERT fl/fl or TERT fl/fl/,Cre+/- mice were treated with 5 μM of 4-OHT in vitro at the same time for 6 days, and TERT mRNA was analyzed. n = 3. *, P < 0.05.
Fig 4.
Selective knockout of TERT gene in TERT CKO mice.
(A) MLF, AEC II, thymic T, and splenic B cells were isolated from TERT CKO and WT mice. TERT mRNA expression was examined in these cells. n = 3 for each. (B) Telomerase activity was measured in the MLF from TERT CKO and control mice. n = 3. (C) The TERT mRNA expressions in TERT CKO and control lung tissues are shown. n = 3. *, P < 0.05.
Fig 5.
The impairment of pulmonary fibrosis in TERT CKO mice.
(A) The MLF were isolated from TERT CKO or WT mice at 21 days after BLM injection, and analyzed for TERT mRNA by qRT-PCR. The expression was expressed as the fold change of the level in PBS-treated WT MLF. n = 3–5 mice per group. (B) The lungs from TERT CKO and control mice were homogenized at day 21 after BLM treatment, and measured for whole lung collagen content by HYP assay. n = 3–5 mice per group. (C) Lung tissue RNA extracted from the indicated murine strains was also analyzed for type I collagen mRNA by qRT-PCR. n = 3–5 mice per group. (D) Lung tissue lysates were prepared by RIPA buffer, and analyzed for α-SMA protein in the indicated murine strain by Western blotting (top panel). Quantitative data was normalized by the internal control GAPDH, and shown as the percentage of the GAPDH signals (bottom panel). n = 3–4 mice per group. (E) Representative H & E stained lung tissue sections at day 21after BLM treatment are shown. Original magnification × 20. *, P < 0.05.
Fig 6.
Effects of TERT CKO on MLF proliferation, apoptosis and differentiation.
(A) The MLF were isolated from TERT CKO and control mice, and analyzed for proliferation by counting the cell numbers at the indicated time points. n = 5. (B) MLF proliferation by WST-1 assay at 48 hours after PDGF treatment. n = 6. (C) Apoptosis was also detected in MLF by annexin V-FITC/PI staining and flow cytometry without and with TNF-α/CHX treatment. The FITC+/PI- cells were referred to as the apoptotic cells. n = 3. (D) MLF isolated from TERT CKO or control mice were cultured and analyzed for TERT and a-SMA mRNA expression after treatment with buffer only or with TGF-β1 for 48 hours. The mRNA expression levels were expressed as the fold changes over the WT MLF for each gene. n = 3. *, P < 0.05.
Fig 7.
Effect of TERT overexpression on α-SMA expression.
BJ and BJ 5ta fibroblasts were plated in 6-well plates. The cells were starved with DMEM supplemented with 0.5% FBS for 4 hours before TGF-β1 treatment for an additional 24 for mRNA or 48 hours for protein analysis. (A) BJ and BJ5ta cells were analyzed for TERT mRNA by qRT-PCR. n = 3. *, P < 0.05. (B) The cell lysates were harvested in RIPA buffer, and analyzed for α-SMA protein expression by Western blotting. A representative blot was shown. The GAPDH was used as internal control.