Ab228402

Immunohistochemical analysis was performed as described previously (Pakharukova et al., 2019b (link); Kovner et al., 2019 (link)). Antibodies and dilutions used in this study were as follows: anti–alpha-smooth muscle actin (α-SMA; 1:200; cat. # ab7817, Abcam), anti-MMP9 (1:200; cat. # ab228402, Abcam), anti-collagen 1 alpha (1:200; cat. # ab34710; # ab53444, Abcam), anti-E-cadherin (1:200; cat. # ab76055, Abcam), and anti–N-cadherin (1:200; # ab76011, Abcam) antibodies with the respective secondary antibodies: a cyanine 3–conjugated goat anti-mouse IgG (H + L) cross-adsorbed antibody (1:500; cat. # M30010, Invitrogen, USA) or a GFP-conjugated AffiniPure goat anti-rabbit IgG (H + L) antibody (1:1000; cat. # 111–095-003, Jackson AB, USA) and a mouse anti-rabbit IgG-CFL 555 antibody (sc-516,249, Santa Cruz, USA). The slices were coverslipped with the Fluoro-shield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; cat. # F6057, Sigma-Aldrich, USA) and examined under an Axioplan 2 microscope (Zeiss, Germany).

The total proteins were extracted from SKOV3 and HO8910 cells and quantified by BCA protein analysis kit (Beyotime), then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After separation, each protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA), which was blocked with 5% skimmed milk at room temperature for 2 h and then incubated with the corresponding primary antibody overnight at 4 ℃. The membrane was washed three times with phosphate buffer solution with 0.1% Tween-20 (PBST) for 10 min each time, and incubated with secondary antibody at 37 ℃ for 1 h. Protein bands were visualized using an imaging system and enhanced chemiluminescence (ECL) reagents (Millipore, USA). β-actin was used as an internal control, and the results were analyzed using ImageJ software. The primary antibodies used were: β-actin (1:500, ab115777, Abcam), Ki67 (1:1,000, ab16667, Abcam), matrix metalloproteinase (MMP)9 (1:1,000, ab228402, Abcam), MMP2 (1:1,000, ab92536, Abcam), and ESR1 (1:1,000, ab108398, Abcam).

Cells were lysed in cell lysis buffer (Thermo Fisher Scientific, Pittsburgh, USA). The protein concentration in the cell homogenate was determined using a bicinchoninic acid (BCA) assay kit (Beyotime, Shanghai, China). SDS-PAGE separated the proteins (30 μg), which were subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Biorbyt, Cambridge, UK). After blocking with 5 % skim milk for 2 h, the membranes were incubated overnight at 4 °C with rabbit antibodies against GBP5 (1:2000, 13,220–1-AP, Proteintech, Wuhan, Hebei, China), IRF1 (1:1000, 8478s, CST, Boston, USA), MMP9 (1:1000, ab228402, Abcam, MA, USA), MMP13 (1:1000, bs10581 R, Bioss, Boston, MA, USA), COL2A1 (1:1000, ab34712, Abcam), aggrecan (1:1000, AF6126, Beyotime), Sox9 (1:1000, BS1597, Bioworld, Nanjing, Jiangsu, China), NLRP3 (1:1000, bs1001 R, Bioss), Caspase1 (1:1000, BS5641, Bioworld), GSDMD (1:1000, ab219800, Abcam), Pro-IL-1β (1:1000, ab216995, Abcam), Pro-Caspase1 (1:1000, ab179515, Abcam), and GAPDH (1:1000, 5174s, CST, Boston, USA). Subsequently, the membranes were incubated with secondary antibodies (1:20,000, BS13278, Bioworld) at room temperature for 1.5 h. After washing the membranes in TBST, chemiluminescent signals were detected using a Bio-Rad Molecular Imager ChemiDocTM XRS + system (Bio-Rad, Hercules, CA, USA), with GAPDH serving as the loading control.