Ab151796

TMJ blocks were fixed, decalcified, dehydrated, and embedded using conventional methods. The 4 µm thick sections were used for histomorphology and immunohistochemical staining. The serial sections were stained with Safranin O as previously reported in order to observe histological and proteoglycan changes in the articular cartilage [26 (link),32 (link)]. IHC staining with anti-Ihh antibody (13388-1-AP, Proteintech, Philadelphia, IL, USA), anti-Smo antibody (ab113438, Abcam, Cambridge, UK), anti-PTHrP antibody (sc-20728, Santa Cruz, CA, USA), anti-PTH1R antibody (sc-20749, Santa Cruz, CA, USA), anti-Aggrecan antibody (ab36861, Abcam, Cambridge, UK), anti-ALP antibody (ab108337, Abcam, Cambridge, UK), anti-Collagen X antibody (ab58632, Abcam), anti-MMP13 antibody (18165-1-AP, Proteintech), and anti-Gli1 antibody (ab151796, Abcam) was performed as a standard, three-step, avidin-biotin complex staining procedure. Images were captured by a Leica light microscope (Leica 2500, Hessen, Germany). For negative controls, nonimmune goat serum was substituted for the primary antibody. The Sox9 expression in primary chondrocytes was detected with anti-Sox9 antibody Alexa Fluor @647 (ab196184, Abcam).

DLBCL cells were collected, washed with cold PBS buffer, and lysed on ice for 30 min using lysis buffer (RIPA buffer, 89900, Thermo Fisher). Proteins were harvested from the lysates, and protein concentrations were quantified. Then, equal amounts of proteins from each sample were loaded into SDS–polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk for 1 h at room temperature (RT). Next, the membrane was incubated with primary antibodies at 4 °C overnight. Primary antibodies against GLI1 (ab151796), cyclin D1 (ab226977), CDK4 (137675), p27 (ab45872), MYC (ab39688) and β-actin (ab8227) were purchased from Abcam. Antibodies against GAPDH were purchased from ProteinTech (Chicago, USA). The membrane was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. The bands were detected with ECL reagent purchased from Millipore Corp.

Western blot was carried out to analyze activation-states of the Hh- and PI3K/AKT/mTOR pathways. Tumor tissue samples from the same animals used in the gene expression analysis were homogenized in RIPA Lysis and Extraction Buffer (Thermo Scientific) using the TissueLyser LT (Qiagen) and Bioruptor® (Diagenode). Cell debris was removed by centrifugation and the protein extract was stored at −20 °C. Protein extracts (100 μg) were run on SDS-PAGE using Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad) and transferred to nitrocellulose membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Antibodies specific to GLI1 (ab151796, Abcam), GLI2 (LS-C313075, LifeSpan BioSciences), S6 (#2217, Cell Signaling Technology), AKT (#9272, Cell Signaling Technology), p-AKT (#9271, Cell Signaling Technology) and GAPDH (ab9485, Abcam, used as control) were detected using Amersham ECL Rabbit IgG (NA934VS, GE Healthcare Life Sciences). SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used for detection and digitalized images were acquired using Fujifilm Luminescent Image Analyzer LAS-1000 (Fujifilm, Tokyo, Japan).

For immunohistochemistry, sections were incubated with antibody against Bcl-2 (1:100, #ab32124, Abcam), Ki-67 (1:100, #ab16667, Abcam), proliferating cell nuclear antigen (PCNA) (1:100, #ab92552, Abcam), β-catenin (1:200, #ab22656, Abcam), Gli1 (1:100, #ab151796, Abcam), NICD1 (1:100, #07-1232, Millipore, Billerica, MA, USA), phospho-Akt (Ser473) (1:100, #ab81283, Abcam), phospho-GSK3β (Ser9) (1:100, #ab75814, Abcam) at 4 °C overnight. Primary antibodies were detected by the Dako EnVision Kit (Dako, Glostrup, Denmark) according to the manufacturer’s protocol. The staining intensity was evaluated and scored by two independent pathologists. The extent of staining was scored as 0, 0% of cells stained; 1, 1–25% of cells stained; 2, 26–50% of cells stained; 3, 51–75% of cells stained; or 4, >75% of cells stained. Staining intensity was scored as 0, negative; 1, weak; 2, intermediate or 3, strong. The final staining score was defined as the product of the extent and intensity scores.