Ab185966

For IHC, the paraffin sections were deparaffinized and dehydrated by xylene and a series of graded ethanol according to the procedure described in our previous study [9 (link)]. Paraffin-embedded ccRCC tissues, NATs, orthotopic tumor tissues, and mouse metastasis lungs were subject to IHC analysis to determine the protein expression using antibodies against SOX9 (ab185966, 1:1000, Abcam, Burlingame, CA, USA), FUS (ab243880, 1:300, Abcam), Ki67 (ab92742, 1:500, Abcam), and α-SMA (ab32575, 1:100, Abcam) at 4 °C overnight. Subsequently, the tissues were incubated with secondary antibody (GB23303, 1:400, Servicebio, Wuhan, China) for 1 h at room temperature. Finally, the sections were fixed with neutral balata and photographed by a fluorescence microscope (Olympus, Tokyo, Japan). The staining area score was measured as 0, < 5%; 1, 5–25%; 2, 25–50%; 3, 50–75%; and 4, > 75%. The staining intensity score was measured as 0, no staining; 1, weak staining; 2, moderate staining; and 3, intense staining. The total staining score was determined by two independent pathologists, and the total score was calculated by combination of staining intensity and area, where samples with a score ≥ 6 were considered to have high expression, while those with a score < 6 were considered to have a low expression [25 (link)].

The immunofluorescent staining was performed as described elsewhere (Zheng et al., 2021 (link)). Cells or tissues were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked by incubation in 5% bovine serum albumin (BSA) for 1 h at 37°C. Then, the samples were incubated with primary antibodies against p-PLCγ1 (Tyr783; Affinity; AF3210; 1:100) or SOX9 (Abcam; ab185966; 5 μg/mL) overnight at 4°C. Next, a secondary antibody conjugated to Alexa Fluor^®488 (Abcam; ab150077; 1:500) or Alexa Fluor^®594 (Abcam; ab150080; 1:500) was added and incubated for 1 h at room temperature. After being rinsed with PBS, the samples were stained with DAPI, observed under an Olympus BX53 microscope, and analyzed using ImageJ.

E10.5 embryos were fixed in 4% PFA, cryoprotected in 30% sucrose and embedded in OCT. Then, 10 μm sections were permeabilized with 0.25% Triton X-100 in PBS and blocked with 20% normal goat serum, 0.3% Triton X-100 in PBS. Sections were stained with rabbit monoclonal antibody against SOX9 (ab185966, Abcam) at 1:500 in 2% bovine serum albumin, 0.3% Triton X-100 in PBS overnight. After washing, sections were stained with goat polyclonal antibody against rabbit IgG conjugated to Alexa Fluor 594 (ab150080, Abcam; 1:1000) for 2 h. Sections were counterstained with 1 μg/ml Hoechst 33342 for 15 min, mounted in Vectastain mounting medium for fluorescence (H-1000, Vector Laboratories), coverslipped and cured at 4°C overnight. Sections were imaged with a Nikon A1r confocal microscope using a 20× multi-immersion objective and SOX9-positive cells were quantified using ImageJ software.

After washing the human SW1353 chondrocytes with PBS buffer, cells were fixed using 4% paraformaldehyde (Sigma-Aldrich, USA) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, USA), followed by being incubation with the primary antibody against SOX-9 (ab185966 Abcam, Cambridge, UK) at room temperature for 2 hours. Subsequently, the appropriate secondary antibodies conjugated with TRITC and DAPI (a nuclear marker and the color is blue) were used to incubate with the collected cells. Lastly, the fluorescence density of each group was observed under a fluorescence microscope (Olympus, Toyko, Japan) [23 (link)].

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.

Expanded islet clusters were harvested using cell recovery solution (Corning, 354253) and fixed in 4% paraformaldehyde for 30 min at room temperature, followed with blocking and permeabilizing in PBS with 0.5% Triton X-100 (Solarbio, T8200) and 5% donkey serum (Solarbio, SL050) for 30 min at room temperature. Then, samples were incubated with primary antibody at 4 °C overnight, followed by incubation with secondary antibody for 2 h at room temperature. DAPI (Beyotime, C1002) was used to stain the nucleus and find islets. The following antibodies were used for immunofluorescence: anti-insulin (1:200, sc-9168; Santa), anti-somatostatin (1:600, ab30788; Abcam), anti-glucagon (1:200, G2654; Sigma). anti-PDX1 (1:200, ab47267; abcam), anti-SOX9 (1:200, ab185966; abcam), anti-NKX6.1 (1:200, ab221549; abcam), anti-MAFA (1:200, ab26405; abcam), anti-KI67 (1:200, D3B5; Cell Signaling Technology). Imaging of the expanded islet clusters was performed on Zeiss LSM 780 and processed using ImageJ or Adobe illustrator software.