Chemidoc imaging system

For WB analysis, 30 μg of protein
lysate was resolved by SDS-PAGE, transferred to nitrocellulose, blocked
in Tris-buffered saline containing 5 mg/mL nonfat milk and 1% Tween-20
for 1 h at RT under horizontal rotation, and incubated with primary
and secondary antibodies as outlined in Supplementary Table S1B. Chemiluminescence generated using
the Clarity Western ECL Substrate (Bio-Rad; #1705061) was detected
using either a ChemiDoc Imaging System (Bio-Rad, Cat # 12003153) or
on film. Films were scanned, and WB figures were assembled using Adobe
Photoshop and Adobe Illustrator.

Total protein samples were extracted from treated cells using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, # 89901) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, # 78430) and phosphatase inhibitor cocktail (Thermo Fisher Scientific, # 78420). The proteins were separated using SDS–polyacrylamide gel electrophoresis gel electrophoresis and then transferred to methanol (Aladdin)–prewetted polyvinylidene difluoride (PVDF) membranes (Millipore) using Trans-Blot Turbo (Bio-Rad). Subsequently, the PVDF membranes were blocked with Beyotime blocking buffer (#P0252) for 1 hour to prevent nonspecific binding. Primary antibodies—including COX2 (1:1000; Abcam, ab188183), HDAC3 (1:5000; Abcam, # ab32369), and GAPDH (1:2000; Abcam, # ab8245)—were diluted in blocking buffer and incubated with the PVDF membranes for 12 hours at 4°C. The membranes were washed thrice with TBST (5 min each time) before being incubated with the appropriate horseradish peroxidase (HRP)–conjugated secondary antibody (diluted at 1:2000 in blocking buffer) for 1 hour at room temperature. The following secondary antibodies were used: goat anti-rabbit antibody conjugated to HRP (1:2000; Bio-Rad, #1662408EDU) and goat anti-mouse igg (H+L)-HRP conjugate (1:2000; Bio-Rad, #1706516). The membranes were washed thrice with TBST (5 min each time) following the secondary antibody incubation, then incubated with Clarity Western ECL Substrate (Bio-Rad, #1705061), and imaged by a ChemiDoc imaging system (Bio-Rad). The immunoreactive bands were quantified using ImageJ, and their densities were normalized to the density of GAPDH.

PCLS were homogenized
in a lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM
EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, supplemented
with 1 mM Na₃VO₄, 1 mM PMSF protease inhibitor,
and 1 μg/mL cOmplete protease inhibitor cocktail (Roche Applied
Science, Indianapolis, IN). The detergent-insoluble material was precipitated
by centrifugation at 18,600g for 30 min at 4 °C.
The protein concentration was measured using a Pierce BCA protein
assay kit (Thermo Fisher Scientific). Twenty μg of protein was
separated on a 12% SDS polyacrylamide gel and subsequently transferred
to a PVDF membrane (Roth, Karlsruhe, Germany). The membrane was blocked
with 5% nonfat milk for 1 h at room temperature and then incubated
with a mouse anti-MMP7 (1:500, cat. no. MAB9071, R&D Systems,
Wiesbaden, Germany) or goat anticollagen 1 (1:500, cat. no. 1310-01,
SouthernBiotech, Birmingham, AL) antibody overnight at 4 °C.
β-actin, used as a loading control, was detected using a mouse
anti-β-actin antibody (1:5000, cat. no. A1978, Sigma-Aldrich,
Taufkirchen, Germany). Proteins were detected using either an Amersham
ECL Select Western blotting detection reagent (GE Healthcare, Chicago,
IL) or a Pierce ECL Western blotting substrate (Thermo Fisher Scientific).
All images were acquired using a ChemiDoc imaging system (Bio-Rad,
Hercules, CA).