human lung fibroblast cell line ccd8lu  (ATCC)

Journal: The Journal of Clinical Investigation

Article Title: Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation

doi: 10.1172/JCI122106

Figure Lengend Snippet: (A) A representative Western blot showing protein expression of CFIm25, FN, and GAPDH from 6 healthy and 7 IPF lung specimens. CFIm25 densitometric analysis shows significantly downregulated CFIm25 levels in IPF lungs. **P < 0.01, by unpaired t test versus healthy control. (B) Immunohistochemistry for CFIm25 (brown) and α-SMA (pink) showing cellular localization in control and IPF lung specimens. Arrow indicates CFIm25-positive cells. Arrowhead indicates α-SMA–positive but CFIm25-negative cells. Scale bars: 100 μm. Original magnification ×100. (C) Western blot shows CFIm25, FN, and β-actin protein expression levels in primary fibroblast lines derived from healthy or IPF lungs. (D–F) Mice were i.p. injected with PBS or bleomycin biweekly for 4 weeks. (D) Western blotting was performed to analyze the protein expression of CFIm25 in whole-lung lysates on day 33 after PBS, or 7, 17, 28, and 33 days after the first bleomycin exposure. The different lanes represent samples collected from distinct mice. β-Actin was used as a protein loading control. (E) Immunofluorescence was carried out to determine CFIm25 (pink) and α-SMA (green) colocalization in lungs from mice exposed to PBS or bleomycin for 33 days. Arrows indicates CFIm25-positive cells; arrowheads indicatesα-SMA–positive but CFIm25-negative cells. Scale bars: 100 μm. (F) Primary fibroblasts were isolated from day-33 PBS or bleomycin-injected mouse lungs. Western blotting was performed to determine CFIm25, FN, and β-actin protein levels.

Article Snippet: Human lung fibroblast lines derived from healthy lungs (CCD8-Lu) or IPF lungs (LL97A) were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Control, Immunohistochemistry, Derivative Assay, Injection, Immunofluorescence, Isolation

Journal: The Journal of Clinical Investigation

Article Title: Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation

doi: 10.1172/JCI122106

Figure Lengend Snippet: (A) Western blot analysis of CFIm25, COL1, and FN expression in healthy human fibroblasts (CCD8-Lu) transfected with control siRNA (si-Con) or siRNAs targeting CFIm25 (si-CFIm25). GAPDH was used as a loading control. (B) Diagram shows pPAS and dPAS, and 2 sets of primers designed to target the translated region (P1) and distal region (P2) of the 3′-UTR. qRT-PCR was performed to demonstrate COL1A1 and FN1 transcript expression (lower left panel) and dPAS usage (lower right panel) after knockdown of CFIm25 using 2 different siRNAs (no. 1 and no. 2). Results in the lower left panel are shown as log2 (fold changes vs. control siRNA–transfected samples) ± SEM (n = 3 biological replicates), and results in the lower right panel are shown as the log2 ratio of (percentage of long transcript in si-CFIm25/percentage of long transcript in si-Con). *P < 0.05, by 1-sample t test versus 0. (C) Scatterplot of percentage PDUIs in control and CFIm25-knockdown cells, in which mRNAs were significantly shortened (n = 808) or lengthened (n = 29) after CFIm25 knockdown in CCD8-Lu cells. (D) Functional annotation assay of CFIm25 targets. (E) RNA-Seq read density for 3′-UTR, terminal exon, and upstream exon(s) of VMA21 in control or CFIm25 siRNA–transfected CCD8-Lu cells. Numbers on the x axis indicate the RNA-Seq read coverage. (F) qRT-PCR was performed to demonstrate dPAS usage of VMA21. n = 3. *P < 0.01, by 1-sample t test versus 0. (G) Western blotting was used to verify VMA21 expression after CFIm25 knockdown in CCD8-Lu cells. KD, knockdown.

Article Snippet: Human lung fibroblast lines derived from healthy lungs (CCD8-Lu) or IPF lungs (LL97A) were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Transfection, Control, Quantitative RT-PCR, Knockdown, Functional Assay, RNA Sequencing

Journal: The Journal of Clinical Investigation

Article Title: Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation

doi: 10.1172/JCI122106

Figure Lengend Snippet: (A) The dPAS usage of CFIm25 targets involved in TGF-β (TGFBR1) and Wnt (WNT5A and FZD2) pathways was verified by qRT-PCR. n = 3 biological replicates. *P < 0.05 one sample t test versus 0. (B) RNA-Seq read density for a representative target (FZD2) is shown in control and CFIm25-knockdown CCD8-Lu cells. n = 3 biological replicates. P < 0.05, by 1-sample t test versus 0. Western blotting was performed to determine protein levels of (C) CFIm25 and TGF-βR1 and (D) Wnt5A and FZD2 in CFIm25-knockdown CCD8-Lu cells. (E) The dPAS usage of CFIm25 targets was determined using qRT-PCR in primary healthy (CCD8-Lu) or IPF fibroblasts (LL97A). n = 3 biological replicates. *P < 0.05, by 1-sample t test versus 0. (F and G) Western blotting was performed to determine protein levels of CFIm25, TGF-βR1, Wnt5A, and FZD2 in primary healthy (CCD8-Lu) and IPF fibroblasts (LL97A) (F) and IPF lungs with different levels of CFIm25 (G).

Article Snippet: Human lung fibroblast lines derived from healthy lungs (CCD8-Lu) or IPF lungs (LL97A) were purchased from the American Type Culture Collection (ATCC).

Techniques: Quantitative RT-PCR, RNA Sequencing, Control, Knockdown, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation

doi: 10.1172/JCI122106

Figure Lengend Snippet: Primary fibroblasts isolated from healthy and IPF lungs were electroporated with empty or CFIm25-overexpressing pCDNA3.1 plasmids. Two or three days after transfection, cells were collected for (A) Western blotting to determine the protein levels of CFIm25 and its target genes and (B) qRT-PCR to determine the dPAS usage of the CFIm25 targets COL1A1, TGFBR1, WNT5A, and FZD2. n = 3 biological replicates. *P < 0.05, by 1-sample t test versus 0.

Article Snippet: Human lung fibroblast lines derived from healthy lungs (CCD8-Lu) or IPF lungs (LL97A) were purchased from the American Type Culture Collection (ATCC).

Techniques: Isolation, Transfection, Western Blot, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation

doi: 10.1172/JCI122106

Figure Lengend Snippet: Four- to six-week-old Col1a1-CreER-CFIm25fl/fl mice and age- and sex-matched littermate controls were administrated 75 mg/kg (i.p.) tamoxifen daily for 5 days to induce Cre expression. n = 4 biological replications. One week later, fibroblasts were isolated from the lungs of these mice. (A) Western blotting was used to confirm the expression of CFIm25, Cre, and CFIm25 targets in fibroblasts, and (B) qRT-PCR was performed to determine the dPAS usage of CFIm25 targets (Col1a1, Tgfbr1, Fzd2, and Wnt5a). n = 5 biological replicates. *P < 0.05, by 1-sample t test versus 0.

Article Snippet: Human lung fibroblast lines derived from healthy lungs (CCD8-Lu) or IPF lungs (LL97A) were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Isolation, Western Blot, Quantitative RT-PCR