Ab8191

The cultured HFs were fixed in 4% paraformaldehyde (China) for 24 h and then cut into 3-μm sections. After dewaxing, the sections were soaked with 3% H₂O₂ for 10 min and preincubated with normal goat serum (DAKO, Denmark) for 30 min. Then, the paraffin sections were incubated with primary antibodies against Ki67 (1:200, Abcam, ab8191, USA), β-catenin (1:200, Abcam, ab32572, USA), GSK3β (1:200, Abcam, ab32391, USA), p-GSK3β (1:100, Abcam, ab75814, USA), or androgen receptor (AR, 1:600, CST, #5153, USA) at 4°C overnight. Afterwards, the sections were incubated with secondary FITC- or CY3-conjugated goat anti-mouse/rabbit antibody for 50 min. Next, the sections were stained with 10 μg/ml DAPI (Abcam, USA). These sections were observed under an inverted fluorescence microscope (Nikon, Japan), and images were collected (FITC green excitation wavelength 465–495 nm, emission wavelength 515–555 nm; CY3 red light excitation wavelength 510–560, emission wavelength 590 nm). The relative expression levels of Ki67, β-catenin, GSK3β, and p-GSK3β were analyzed by the Image pro-plus 6.0 software: first of all, the immunofluorescence image is converted into a black and white image; then the integrated optical density (IOD) of the positive region is analyzed; afterwards, calculate the area of positive region (AREA); finally, mean density is obtained as follows: Mean density = IOD/AREA.

Cells were lysed with RIPA buffer mixed with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Total protein (25 μg) was separated by 10% SDS–polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). After being blocked in 5% non‐fat milk in Tris‐buffered saline containing 0.1% Tween‐20 for 1 hour at room temperature, the membranes were incubated overnight at 4°C with primary antibodies as following: anti‐TRPV4 (ab39260; Abcam), anti‐Ki67 (ab8191; Abcam), anti‐IL‐6 (ab9324; Abcam), anti‐RANKL (ab45039, Abcam), anti‐osteoprotegerin (OPG) (ab11994, Abcam), anti‐phospho‐p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (4370, Cell Signaling Technology), anti‐p44/42 MAPK (ERK1/2) (4695, Cell Signaling Technology), and anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (3683S, Cell Signaling Technology). Then, the membranes were incubated with a horseradish peroxidase‐conjugated mouse or rabbit IgG (1:5000; Zhongshanjinqiao), and protein bands were detected by enhanced with a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The relative density of three comparable results was measured using ImageJ software. Each experiment was repeated three times.

Mice tumour tissues were collected and made into 5‐μm paraffin‐embedded sections. Sections were dewaxed at 67°C, and then endogenous peroxidase was inactivated with 3% hydrogen peroxide methanol solution. Non‐specific antigens were blocked with 10% goat serum at 37°C for 1 hour. After washing thrice with PBS, sections were incubated at 37°C for 1.5 hours with primary antibodies, and then incubated with secondary antibody for 1 hour at room temperature. Then sections were washed with PBS and incubated with 3, 3′‐Diaminobenzidine (DAB; Sigma‐Aldrich) for 5 minutes in a dark room. After being washed with PBS, sections were then counterstained for 1 minute with hematoxylin (Sigma‐Aldrich). Permount^™ Mounting Medium (Abcam) was utilized to mount the sections. Primary antibodies used were as listed below: rabbit anti‐SERPINE1 (5 μg/mL, ab66705; Abcam), rabbit anti‐p53 (20 μg/mL, ab1431; Abcam), rabbit anti‐MDM2 (1:100, ab131355; Abcam), rabbit anti‐cleaved Caspase3 (10 μg/mL, ab2302; Abcam) and rabbit anti‐ki67 (1:50, ab8191; Abcam). Secondary antibody was HRP‐labelled goat anti‐rabbit IgG (1:2000, ab205718; Abcam).

Immunofluorescence staining was performed as described previously.²³ Tissue sections were double‐stained with anti‐CD146 (1:200, ab‐75769, Abcam) and anti‐Ki67 (1:200, ab8191; Abcam) or anti‐TRPV4 (1:200, ab39260; Abcam) antibodies. Other sections were stained with anti‐interleukin (IL)‐6 (1:200, ab9324; Abcam) and anti‐IL‐1β (1:200, ab9722, Abcam) antibodies. Next, the sections were incubated with fluorescein isothiocyanate‐conjugated or tetramethylrhodamine isothiocyanate‐conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories). Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole. Confocal microscopic images were processed with LSM 5 Release 4.2 software following acquisition with a laser‐scanning microscope (LSM 510; Zeiss).

Tumor tissues were fixed in 10% formalin (Sigma) at room temperature and embedded in paraffin. Paraffin-embedded samples were then processed for immunohistochemistry; Ki67 (1:100, 0.2 mg/mL, ab8191; Abcam) was used as a measure of cell proliferation. Scoring for the expression of Ki67 was performed as follows: the percentage of Ki67⁺ cells was calculated from three randomly selected regions of the samples by counting an average of 1,600–2,000 cells per slide using the ImageJ software.

Paraffin‐embedded slides and tissue microarrays were dewaxed with dimethylbenzene (I and II) and gradient alcohol (100%, 95%, 85% and 75%). The antigen retrieval was performed by microwaving sections in citrate buffer for 2 minutes. Slides were incubated with 3% hydrogen peroxide (H₂O₂) for 20 minutes and then incubated with goat serum for 30 minutes at room temperature. Without washing, primary antibodies SQSTM1/p62 (1:1000; ab109012; Abcam), Ki67 (1:50; ab8191; Abcam) and caspase‐7 p20 (1:50; SC‐28295; Santa Cruz Biotechnology, Santa Cruz, DE, USA) were applied at 4°C overnight. Biotinylated anti‐IgG was added and incubated at room temperature for 1 hour. After incubating with streptomycin‐HRP for 30 minutes, the slides were stained with DAB and then counterstained with haematoxylin. After washing with flowing water, the slides were dehydrated with gradient alcohol and dimethylbenzene. Finally, the slides were sealed with neutral balsam and coverslips.