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. 2021 May 12;321(1):L174–L188. doi: 10.1152/ajplung.00323.2020

Figure 4.

Figure 4.

Loss of SRF in lung mesenchymal cells attenuates bleomycin-induced fibrosis. Tbx4LE-rtTA/TetO-Cre/tdTom or Tbx4LE-rtTA/TetO-Cre/tdTom/SRFf,f were given doxycycline (2 mg/mL + 50 mg/mL sucrose) or sham control (50 mg/mL sucrose) in water 2 days before intratracheal treatment with bleomycin (1 U/kg suspended in normal saline, NS) or NS control, as indicated. A: 8 days after bleomycin (and 10 days after start of doxycycline) treatment, Tbx4LE-rtTA/TetO-Cre/tdTom (SRF+/+) or Tbx4LE-rtTA/TetO-Cre/tdTom/SRFf,f (SRF−/− in LMC) mouse lungs were digested to single-cell suspension and subjected to fluorescence-activated cell sorting (FACS) for expression of tdTomato, separating lung mesenchymal cells (LMCs) from the rest of the cell populations. tdTom-positive LMCs were immediately lysed for RNA and subject to RT-PCR against Srf, along with a housekeeping gene (n = 3 for each condition). B: 5 days after start of doxycycline treatment, Tbx4LE-rtTA/TetO-Cre/tdTom/SRFf,f primary mouse lung fibroblasts (MLFs) were derived. Following expansion, MLFs were subjected to FACS against tdTomato. tdTom-positive LMCs (SRF−/−) and tdTom-negative control fibroblasts (SRF+/+) were lysed for RNA and subject to RT-PCR for expression of Srf and housekeeping gene (n = 3 for each condition). Column bars are representing means ± SE, and Unpaired Student’s t test (*P < 0.05) was used for statistical analyses in A and B. C: quantification of collagen accumulation was done 14 days after bleomycin or NS installation (and 16 days after the start of doxycycline/sham treatment) via hydroxyproline assay of total lung homogenates (both lungs) in indicated groups (C; N indicated in figure). One-way ANOVA with Holm–Šídák test was used for statistical analyses (*P < 0.05). D: lung fibrosis was semiquantitatively assessed via lung fixation, paraffin embedding, and Ashcroft fibrosis scoring of Masson’s trichrome stains in Tbx4LE-rtTA/TetO-Cre/tdTom/SRFf,f mice treated with doxycycline (SRF−/− in LMC) or sham control (SRF+/+) and intratracheal bleomycin (n = 5 for each group). Mixed model ANOVA (*P < 0.05) was used for statistical analysis. E: a set of representative images of data from D are displayed (scale bar = 500 µm). Bleo, bleomycin; dox, doxycycline; SRF, serum response factor.