Sc 376471

Total protein extracts were harvested and subjected to WB as described previously [23 (link)]. Primary antibodies against the following proteins were used for WB: GLI2 (ab26056; Abcam), PTCH1 (ab55629; Abcam), FOXM1 (sc-376,471; Santa Cruz Biotechnology, CA, USA), TPX2 (12,245; Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (MAB374; Millipore, Billerica, MA, USA). This was followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, namely normal goat anti-mouse IgG (31,430; Thermo Scientific Pierce) or normal goat anti-rabbit IgG (31,460; Thermo Scientific Pierce), and the membranes were probed with SuperSignal™ West Femto Maximum Sensitivity Substrate ECL (34,095; Thermo Fisher Scientific Inc). The immunoblot films were digitalized with Epson V700 scanner, and intensity of major bands were quantitated using Image J (National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated at least thrice.

Cartilage was fixed in 10% neutral buffered formalin and decalcified in 14% EDTA (pH 7.4). The tissues were embedded in paraffin and sectioned to a thickness of 5 μm. Safranin O staining was performed after rehydration, and the Mankin's score was measured. For immunohistochemistry, antigen retrieval using 0.01 M sodium citrate buffer (pH 6.0) was performed and sections were blocked with normal horse serum. Sections were incubated overnight with the following primary antibodies: C1,2C (1:100, 50-1035, IBEX Pharmaceuticals, Canada), FOXM1 (1:100, sc-376471, Santa Cruz, USA), MMP13 (1:200, 3533, BioVision, USA), Aggrecan Neoepitope (1:200, 100-74350, NOVUS, USA), and P16INK4A (1:100, ab189034, abcam, UK). After incubation with a peroxidase-conjugated secondary antibody, detection with the ImmPACT DAB Substrate (Vector Laboratories, USA) was performed.

The PLK1 inhibitor, volasertib, was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies against PLK1 (4513), Bcl‐2 (2876), Bcl‐xl (2762), cleaved caspase‐3 (9664), PARP (9542), CD44 (3570), CD133 (64326), NANOG (4903), Cyclin B1 (4135), phospho‐Bad (9295), Bad (9292) and GAPDH (5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against FoxM1 (sc‐376 471) and caspase‐3 (sc‐56 053) were purchased from Santa Cruz Biotechnology, Inc. Cyclin D1 (ab139260) was obtained from Abcam (Cambridge, MA, USA). Phospho‐CDK1/CDC2 (orb127843) was purchased from Biorbyt Ltd (Cambridge, UK).