Bleomycin-Induced Fibrosis Model in Mice

The procedure to induce fibrosis by bleomycin in mice was published previously (2 (link), 37 (link)). In C57BL/6 mice (The Jackson Laboratory, 000664), 100 μL of bleomycin (1 mg/mL) or PBS was injected s.c. into a single location on the shaved back once every day for 2 weeks. JQ1 (50 mg/kg; MedChemExpress) or vehicle control (20% DMSO, 50% PBS, 30% PEG from J.T.Baker) was given daily by oral gavage. In a separate experiment, BRD4 inhibitors AZD5153 (5 mg/kg; Cayman Chemical) or ARV825 (5 mg/kg, MedChemExpress) or vehicle control (22% DMSO, 48% PBS, 30% PEG) were given daily i.p. At the end of the experiment, fixed skin sections were stained with Masson’s trichrome. Dermal thickness was measured by analyzing the distance between the epidermal–dermal junction and the dermal–fat junction in 3 fields in 2 or more skin sections from each animal. Immunofluorescence was performed on sections using anti–α-SMA (Abcam ab5694) or anti-F4/80 (Invitrogen MA5-16630; clone CI:A3-1) Abs after antigen-retrieval. Collagen content in the skin was measured using the Hydroxyproline Kit (Abcam). All animal protocols were approved by the IACUC at the University of Michigan.