Anti collagen 1

For TA, DIAPH, and EDL immunofluorescence, cryosections of 10 µm were fixed in 4% paraformaldehyde (Winkler), permeabilized with PBS-0.05% Triton, and blocked for 1 h with blocking buffer (BSA 2%, 0.05% Triton X-100 in PBS). Samples were incubated overnight at 4 °C with: anti-Fibronectin (F3648, Sigma Aldrich), anti-Collagen I (PA1-26204, Invitrogen, Waltham, MA, USA), anti-Slow Myosin (M8421, Sigma Aldrich), anti- nNOSµ (617000, Invitrogen) and anti-IIA myosin heavy chain antibody (SC-71, DSHB, University of Iowa, Iowa City, IA, USA). The corresponding Alexa Fluor- 568 or 488-conjugated anti-IgGs (Invitrogen) were used as secondary antibodies. In addition, fluorescent wheat germ agglutinin (WGA) (Thermo Fisher, Waltham, MA, USA) was used to label cell membranes and Hoescht for nuclear staining. Slices were washed and mounted in Fluoromount G. Samples were visualized on a Nikon Eclipse E600 using NIS Elements software v4.20 or Leica DM2000 epifluorescence using Mshot Image Analysis System software with 20× or 40× objectives.

Liver samples were fixed in 10% neutral formalin fixative and embedded in paraffin, and were sectioned at 4 µm thickness. Histological analysis was performed in hematoxylin-eosin staining and Sirius red staining. For immunohistochemistry, liver sections were incubated with anti-ARK5 (Santa, Santa Cruz, CA, USA, sc-271828), anti-α-SMA (Invitrogen, Waltham, MA, USA, PA5-18292), anti-collagen I (Invitrogen, Waltham, MA, USA, PA5-29569) primary antibodies overnight at 4 °C. Positive staining was visualized with the EnVision immunodetection system (K5007; Dako, Copenhagen, Denmark) according to the manufacturer’s instructions. For immunofluorescence, liver sections or cells were fixed in ice-cold methanol and were incubated with anti-ARK5 (Santa, Santa Cruz, CA, USA, sc-271828), anti-α-SMA (Invitrogen, Waltham, MA, USA, PA5-18292), anti-p-Smad2/3 (Abcam, Cambridge, UK, ab254407), anti-Smurf2 (Santa, Santa Cruz, CA, USA, sc-518164), anti-TβRI (Santa, Santa Cruz, CA, USA, sc-518018), anti-Smad4 (Cell Signaling Technology, Beverly, MA, USA, 46535S) and anti-HNF-4α (Abcepta, Suzhou, China, AP60014) antibodies followed by incubation with Alexa Fluor 488 or 555-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA). Nucleis were counterstained with DAPI. Images were captured using an inverted fluorescence microscope or confocal laser scanning microscope.