Generation of FIZZ1 KO mice.

<p>(A) Gene targeting strategy and restriction map of FIZZ1 gene. The diagram showed the wild type FIZZ1 gene allele and the gene targeted allele. The black boxes E1–E4 represent the four exons of FIZZ1 gene. All four exons of the FIZZ1 gene were replaced by a Neo gene cassette. (B) Southern blotting analysis of an ES cell clone with homologous recombination. A 3′ probe (shown in A) was used to detect 26 kb WT and 10 kb KO alleles. (C) PCR genotype using mouse tail DNA from wild type (“WT”), FIZZ1 heterozygote (“Het”) and homozygous knockout (“KO”) mice. The PCR fragment for WT was 525 bp, and KO was 351 bp. “Lad” referred to 100 bp ladder.</p

AdFIZZ1 effects on lung fibrosis.

<p>The lung FIZZ1 mRNA (A) at the indicated time points and protein at day 21 (B) after AdFIZZ1 endotracheal administration alone, or with BLM injection (C) were analyzed by qPCR. The results were shown as mean ± SE of triplicate samples or animals. Type I collagen production in the lungs was analyzed at day 7 after indicated treatments with 200 ng lung tissue lysates by ELISA (D). Data were shown as mean ± SE with 5 samples in each group. Lung α-SMA mRNA at day 7 (E) and protein (F) after AdFIZZ1 injection with BLM or PBS were analyzed by qPCR or Western blotting, respectively. A representative blot was shown in (F).*P<0.05 compared to their respective controls. *P<0.05 or †P<0.001 compared to control.</p

Cytokine expression in BMDC. GM-CSF-induced BMDC were separated by MACS with anti-CD11c microbeads.

<p>IFNγ, FIZZ1 and MCP-1 were detected in two distinct cell populations. *P<0.05 vs. respective controls.</p

Effects of FIZZ1 deficiency on BLM-induced pulmonary fibrosis.

<p>WT control or FIZZ1 KO mice were treated with PBS (CON) or BLM as indicated. The lungs were harvested at day 21, and analyzed for lung HYP content (A). The values were expressed as percentages of their respective PBS control. Data were shown as mean ± SE with 5 mice in each group. *P<0.05 or **P<0.001 compared to control. Type I collagen, α-SMA and mRNA and protein in the lungs were also analyzed by qPCR (B) and Western blotting (C), respectively. A typical blot from 5 mice in each group was shown. The lung RNA from day 7 after PBS (CON) or BLM treatment was also analyzed for cytokines mRNA expression by qPCR (D). Results were expressed as 2^−ΔΔCT. The values were expressed as percentages of their respective PBS control. The numbers of total BAL cell (E) and BAL macrophage/monocyte (F) were counted at day 7 of BLM or PBS treated WT or FIZZ1 KO mice. Data were shown as mean ± SE (n = 7 mice for total BAL cell, 4 mice for macrophage/monocyte).</p

FIZZ1 effects on BM cell recruitment.

<p>(A) Fresh isolated whole BM cells from day 7 BLM (“BLM-BM”) or PBS (“CON-BM”) treated mice were preloaded with fluorescent dye, and then plated into 5 µm-inserts in 24-well transwell plates. After 2 hours of incubation with the indicated doses of FIZZ1 in the lower chambers, the cells that have migrated to the lower chambers were quantitated by measuring the fluorescence with an excitation and emission wavelengths of 494 and 517 nm, respectively. The results were normalized to the controls and expressed as percentages of controls, and shown as mean ± SE (n = 3). In (B) purified BM-derived DC from day 7 BLM (“BLM-BMDC”) or PBS (“CON-BMDC”) treated mice were similarly analyzed as in (A) for migration to media only (“None”) or to 50 ng/ml FIZZ1 in the lower chambers. The results were expressed as in (A). (C) BM from GFP transgenic mice were transplanted into WT or FIZZ1 KO mice to create GFP BM chimera mice of the respective recipient strain. After stable engraftment the mice were treated with BLM and 7 days later were analyzed for GFP expression in the lung cell population by flow cytometry. Three populations were discerned corresponding to the cells with undetectable (R1), low (R2) or high (R3) GFP fluorescence. One representative data was shown from two individual experiments, and the combined lung cells from two mice were used for flow cytometry in each group. Circulating FIZZ1 protein was measured in the sera (1∶50 dilution) by ELISA assay 21 days after BLM or PBS treatment (D). Results were shown as mean ± SE with 5–6 mice in each group. (E) Effect of FIZZ1 on migration of primary cultured mouse lung fibroblasts was also analyzed as described in legend for (A). *P<0.05 compared to control.</p

Generation and verification of the TERT CKO mice.

<p>(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 <i>fl/fl</i> it was 343 bp, and for TERT CKO it was 215bp after tamoxifen-induced Cre excision. “Ladder” referred to 100 bp DNA ladder.</p

Effect of TERT overexpression on α-SMA expression.

<p>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.</p

The in vitro excision of MLF TERT by Cre activation.

<p>The MLF were isolated from TERT <i>fl/fl</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142547#pone.0142547.g002" target="_blank">Fig 2</a>. n = 3. (C) The MLF from TERT <i>fl/fl</i> or TERT <i>fl/fl</i>/,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.</p

TERT and telomerase in floxed TERT vs WT mice.

<p>Total RNA or protein lysates were prepared from the lung tissues and MLF from BLM or PBS-injected TERT <i>fl/fl</i> 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.</p

The impairment of pulmonary fibrosis in TERT CKO mice.

<p>(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.</p