Fig. 4

Metformin accelerates fibrosis resolution in the bleomycin model in mice. a Schematic representation of the Acta2-Cre-ERT2 and tdTomato^flox constructs. b Schematic representation of the timeline of the experiment. Bleomycin was administered intratracheally at day 0. Between days 5 and 14, mice were fed tamoxifen-containing pellets and starting at day 14, metformin (1.5 mg/mL) or vehicle was administered through drinking water. Mice were sacrificed at day 28. c–f Hematoxylin and eosin and Masson’s trichrome staining of metformin- and vehicle-treated lungs. g Quantification of fibrosis in metformin- and vehicle-treated lungs. h, i Immunofluorescence for COL1A1 (green). Endogenous tdTomato signal (red) and DAPI (blue) are also shown. j LipidTOX staining (green) and tdTomato⁺ cells (red) are shown. The box in (j) is magnified in (k). Arrowheads indicate LipidTOX⁺ tdTomato⁺ cells. l–s Gating strategy (to detect CD45⁻ CD31⁻ EpCAM⁻ tdTomato⁺ and/or LipidTOX⁺ cells) and quantification of various cell populations based on tdTomato and LipidTOX detection. Scale bars: c–f 1 mm, h, i 50 μm, and j 25 µm. Each data point within a given group corresponds to one animal and error bars indicate s.e.m. n = 5 per group. Mann–Whitney test was used in (g, q–s). *P < 0.05, **P < 0.01. IF: immunofluorescence, ns: not significant