Journal: The Journal of Clinical Investigation

Article Title: Wilms tumor 1 impairs apoptotic clearance of fibroblasts in distal fibrotic lung lesions

doi: 10.1172/JCI188819

Figure Lengend Snippet: ( A ) Schematic representation of the animal experiment involving PDGFR α CreERT (control) and PDGFR α CreERT WT1 OE (cWT1 OE ) mice. Mice were treated repeatedly with bleomycin via intratracheal administration and with tamoxifen via intraperitoneal injection. ( B ) Quantification of Wt1 gene transcripts by RT-PCR in total lung RNA isolated from control and cWT1 OE mice ( n = 6/group). ** P < 0.01, by 2-tailed Student’s t test. ( C ) Representative confocal images of lung sections from control and cWT1 OE mice co-immunostained for WT1 (red), vimentin (green), and DAPI (blue). Scale bars: 20 μm. ( D ) Representative images of Masson’s trichrome–stained lung sections from control and cWT1 OE mice. Original magnification, ×4 and ×20, respectively. Scale bars: 1,500 μm and 200 μm, respectively. ( E ) The percentage of fibrotic area was quantified in control and cWT1 OE mice using BZ-X image analysis ( n = 6/group). **** P < 0.0001, by 2-tailed Student’s t test. ( F ) Hydroxyproline levels were measured in the right lungs of control and cWT1 OE mice ( n = 6/group). *** P < 0.001, by 2-tailed Student’s t test. ( G ) Lung resistance was assessed using FlexiVent in control and cWT1 OE mice treated with bleomycin ( n = 6/group). * P < 0.01, by 2-tailed Student’s t test. ( H ) Proliferation of fibroblasts was quantified using a BrdU incorporation assay in lung cultures from control and cWT1 OE mice treated with bleomycin ( n = 3/group). * P < 0.05, by 2-tailed Student’s t test. ( I ) Fibroblasts from the lung cultures of control and cWT1 OE mice treated with bleomycin were treated with anti-Fas antibody for 24 hours, followed by TUNEL staining (red). Representative confocal images are shown; nuclei are stained with DAPI (blue). Original magnification, ×20. Scale bars: 100 μm. ( J ) Percentage of TUNEL + fibroblasts in total DAPI + fibroblasts ( n = 3/group). * P < 0.05 and ** P < 0.01, by 1-way ANOVA. Data are representative of 2 independent experiments with similar findings.

Article Snippet: Cell proliferation was evaluated using the BrdU kit (Cell Signaling Technology) as described previously ( ).

Techniques: Control, Injection, Reverse Transcription Polymerase Chain Reaction, Isolation, Staining, BrdU Incorporation Assay, TUNEL Assay