Fig. 3.

Metformin improves IPF lung structure ex vivo. a Schematic representation of the experimental setup. b–e Bright-field imaging of PCLS treated with metformin or vehicle for 5 days. f, g Hematoxylin and eosin staining and COL1A1 immunostaining of PCLS prepared from a non-IPF donor lung. h–m Hematoxylin and eosin staining, Masson’s trichrome staining and COL1A1 immunostaining of PCLS prepared from an IPF lung and treated with metformin or vehicle for 5 days. n, o 3D reconstruction of z-stacks of metformin- and vehicle-treated PCLS stained for COL1A1 (green) and lipid droplets (red). p Gating strategy for flow cytometry-based quantification of LipidTOX⁺ cells that are negative for hematopoietic (CD45), endothelial (CD31), and epithelial (EpCAM) cell markers. q Quantification of flow cytometry measurements on metformin- and vehicle-treated cells. r Total collagen assay for metformin- and vehicle-treated cells. Scale bars: b–e 2 mm, f 500 μm, g, l, m 50 μm, and h–k 200 µm. Each data point within a given group corresponds to one patient and error bars indicate s.e.m. q n = 4 per group. r n = 3 per group. Mann–Whitney test was used in (q, r). * P < 0.05, **P < 0.01