Arv825

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.

Chemicals, including ARV-825 and JQ1, were from Medchemexpress (Catalog #s: HY-16954 and HY-13030, respectively) and their stocks were in DMSO. The G12A antibody was used to detect APP full length and C-terminal fragments (APP-CTFs) (rabbit polyclonal, clone C7 targeting amino acid residues 732–751 of APP751, custom made by Thermo Fisher Scientific) and were previously reported (57 (link), 58 (link), 59 (link)). The sAPPβ antibody was purchased from IBL (Catalog #: 18975). The 6E10 antibody (BioLegend) is a monoclonal antibody (mAb) reactive to amino acid residues 1–16 of Aβ from the N-terminal sequence and can detect sAPPα in medium. The nicastrin (NCT) antibody (Catalog #: 5665), BACE1 antibody (Catalog #: 5606), and LC3 a/b antibody (Catalog #: 12741) were purchased from Cell Signaling. The BRD4 antibody was from BETHYL (Catalog #: A700-004-T). The β-actin antibody was from Sigma and used as a protein control. The antibody for pTau (S396) was from Cell Signaling (Catalog #: 9632). The HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit) were from Pierce. The dilution factors for primary and secondary antibodies were 1: 1000 and 1: 10,000, respectively.

In Fig. 5 and Supplementary Figs. 10 and 11, HFD-feeding and immunofluorescence staining of cell competition model mice were performed as previously described^{18 (link),27 (link)}. For in vivo experiments, 6–10 weeks old Villin-Ras^V12-GFP mice were given a single intraperitoneal injection of 2 mg of tamoxifen in corn oil (Sigma, #C8267), and were then sacrificed days after Cre activation. For some experiments, the senolytic drug ARV825 (MedChemExpress, #HY-16954)^{31 (link)} was intraperitoneally administrated for 5 consecutive days (total of 2 weeks). For culturing intestinal organoids, isolated crypts from the mouse small intestine were entrapped in Matrigel (Corning, #356231) and plated in a non-coated 35-mm glass-bottom dish as previously described^{48 (link)}. The crypts embedded in Matrigel were covered with Advanced DMEM/F12 supplemented with N2 (Gibco, #17502-048), B27 (Gibco, #17504-044), 50 ng ml⁻¹ EGF (Peprotech, #315-09), 100 ng ml^–1 Noggin (Peprotech, #250-38), 1.25 mM N-Acetylcystein (Sigma-Aldrich, #A7250), and R-spondin conditioned medium collected from 293T-HA-Rspol-Fc cells kindly provided by Dr. Calvin Kuo (Stanford University). After 96 h of culture, organoids were incubated with tamoxifen (Sigma, #T5648) for 24 h to induce transgenes. Subsequently, tamoxifen was washed out, and organoids were cultured for 24 h for analyses.