Ar1176

The proteins Nrf2 and PP65 were assessed in vitro. ATDC5 cells were seeded in 6-well plates and divided into the control, IL-1β, and IL-1β plus CDDO-Me (0.6 μM) groups. ATDC5 cells were pretreated with CDDO-Me for 24 h followed by stimulation with IL-1β (10 ng/mL) for 30 min. To assess Sox9 and MMP13 expression, ATDC5 cells were divided into the control, CDDO-Me (0.6 μM), IL-1β, and IL-1β plus CDDO-Me (0.6 μM) groups. The cells were treated with CDDO-Me (0.6 μM) or IL-1β or were treated with IL-1β plus CDDO-Me for 48 h. The cells were washed three times in PBS and then fixed with 4% paraformaldehyde for 30 min, followed by permeabilization using 0.1% Triton X-100 diluted in PBS for 20 min. Then, the sample was blocked with goat serum for 1 h at 37 °C and incubated overnight at 4 °C with the following primary antibodies: anti-MMP13 (1:1000, Cat.# AF5355, Affinity), anti-Sox9 (1:1000, ab185966, Abcam), anti-phospho-p65 (1:1000, Cat.# AF2006, Affinity), and anti-Nrf2 (1:1000, Cat.# AF0639, Affinity). Next, the samples were incubated with fluorescent secondary antibodies (1:100, Cat. BA1127, Boster) for 1 h at room temperature and labeled with DAPI (Cat. AR1176, Boster) for 30 min. Finally, the slide was sealed with 50% glycerin, and the fluorescence intensity was measured using ImageJ.

A549 cells were seeded (5 × 10⁴/ml) on glass bottom cell culture dishes (NEST, Wuxi, China), incubated overnight, and then treated as described previously. Cells were rinsed two times with 1× PBS (pH 7.4) and fixed in 4% paraformaldehyde at room temperature for 30 min. Fixed cells were washed three times and then permeabilized with 1 ml of 0.3% Triton X-100 for 20 min. After blocking with PBS containing 4% BSA for 30 min, cells were incubated with primary antibody (phospho-p38 MAPK, 1:50 dilution; phospho-NF-κB p65, 1:50 dilution; and Nur77, 1:50 dilution) in 4% BSA/PBS overnight at 4°C and then washed and incubated with FITC-conjugated anti-rabbit IgG antibody (1:150 dilution) for 1 h at room temperature. Cells were washed, dried, and mounted in medium containing DAPI (AR1176; Boster) and imaged on an Olympus Fluoview 500 IX71 Confocal Laser Scanning Microscope.

The cells were treated with different methods, and then the basal culture medium was heated to 37 °C. Next, the MitoTracker probe solution (M9940, Solarbio) was added to a final concentration of 100 nM, following the manufacturer’s recommendations. The cells were then incubated with the staining solution for 30 min. Next, the cell nuclei were stained with DAPI (AR1176, Boster Biotechnology). Cells were observed and photographed using a fluorescent microscope.

Frozen sections of liver tissue or KCs in each group were fixed by immersion in 4% buffered formaldehyde at room temperature for 10 minutes, permeabilized with 0.2% Triton X-100 at room temperature for 30 minutes, and blocked with 1% BSA at room temperature for 1 h. PBS was used to wash the sections at room temperature for 5 minutes. Frozen sections of liver tissue or KCs were next incubated in the dark at 4°C overnight with primary antibodies against STING (1 : 300, 19851-1-AP, Proteintech), F4/80 (5 μg/mL, ab6640, Abcam), and caspase 1 (1 : 300, 14F468, Novus). Next, the frozen sections or KCs were washed in the dark at room temperature for 5 minutes. The frozen sections or KCs were then incubated in the dark with a secondary antibody (1 : 500, Abcam) at room temperature for 1 h. The frozen sections or KCs were next washed in the dark at room temperature for 5 minutes. Finally, the frozen sections or KCs were incubated in the dark with DAPI (AR1176, BOSTER) at room temperature for 8 minutes. After mounting using an antifluorescence quenching agent (AR0036, BOSTER), the staining was observed using a laser scanning confocal microscope (LSCM).

Cells cultured on hydrogel with different stiffness were fixed in 4% paraformaldehyde (P1110, Solarbio) for 30 min and permeabilized with 0.2% Triton X-100 (T8200, Solarbio) for 20 min. The cells were then blocked with 10% goat serum (AR1009, Boster) for 1 h and incubated with anti-Ki67 antibody (27309-1-AP, Proteintech) or anti-YAP antibody (13,584–1-AP, Proteintech) overnight at 4 ℃. Subsequently, the cells were incubated with CoraLite488-conjugated secondary antibody (SA00013-2, Proteintech) or rhodamine-conjugated secondary antibody (SA00007-2, Proteintech) for 1.5 h in the dark. F-actin was stained with rhodamine-phalloidin (CA1610, Solarbio). Nuclei were counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (AR1176, Boster), and images were captured by fluorescence microscopy (Olympus, IX73). For tissue sections, after deparaffinization and dehydration, the remaining protocol was the same as that for cells.