Ab9100

Immunohistochemistry was automatically performed using an AutostainerLink 48 immunostainer (Dako-Agilent, Santa Clara, CA, USA). Briefly, the slides were incubated at room temperature (RT) in: (1) mouse monoclonal antibody to TLR2 (ab9100) (Abcam, Cambridge, UK) at 1:100 for 30 min; (2) EnVision^®+ Dual Link System-HRP (dextran polymer conjugated with horseradish peroxidase and affinity-isolated goat anti-mouse and goat anti-rabbit immunoglobulins) (Dako-Agilent, K4065) for 20 min; (3) DAB+ substrate-chromogen solution (1 mL of substrate buffer solution containing hydrogen peroxide and 20 µL of 3,3′-diaminobenzidine tetrahydrochloride chromogen solution) for 10 min; and (4) EnVision FLEX hematoxylin for 15 min. The intensity of each TLR2 staining was quantified by ImageJ (Rasband, WS, USA) by measuring the inverted DAB signal and by calculating the average with at least thirteen cells for each image.

For the measurements of cell surface markers via flow cytometry, the hTMSCs were plated at a density of 1 × 10⁵ cells/ml into a test tube (BD, Franklin Lakes, NJ) and washed three times with wash buffer (PBS with 3% FBS) as previously described [12 (link)]. The antibodies against CD14 (all anti-human CD from BD Biosciences, San Jose, CA), CD19, CD34, CD73, CD90, CD105, HLA-DR, TLR 2 (ab9100) (all anti-human TLR from Abcam, Cambridge), TLR 3 (ab12085), TLR 4 (ab30667), and TLR 5 (ab13875) were added to the incubation of the hTMSCs as the primary antibody. Cell fluorescence was evaluated by flow cytometry using a FACS-Calibur instrument (BD).

Human NPC cell lines (S26 and 5–8 F) were kindly gifted by Professor Chaonan Qian at SYSUCC (Guangzhou, China). The human embryonic kidney HEK293T cells were obtained from Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Chinese Academy of Sciences. All cells were maintained in DMEM (Dulbecco’s Modified Eagle Medium, Gibco, NY, USA) with 10% fetal bovine serum (FBS, Gibco, NY, USA) and 1% Penicillin-Streptomycin (100 nM Gibco, NY, USA), in the humidified incubator with 5% CO₂ at 37 °C. All cell lines were routinely examined to be mycoplasma-free using the mycoplasma detection kit (Vazyme, Nanjing, China).
Primary antibodies were commercially available: PARP1, Cleaved-PARP1, CCND1, C-MYC, N-Cadherin, Vimentin, γ-H2AX, α-Tubulin (9542, 5625, 2922, 9402, 14215, 5741, 9718, 2144, Cell Signaling Technology, Danvers, USA), Ki-67 (AB_393778, BD Pharmingen™, New Jersey, USA), SRSF3, HA, PMCA, EZH2 (ab125124, ab9100, ab254025, ab191250, Abcam, Cambridge, USA), AMOTL1, YAP1 (16871-1-AP, 16871-1-AP13584-1-AP, Proteintech, Chicago, USA), GAPDH (RM2002, Ray Antibody Biotech, Beijing, China), β-actin (AC004, Abclonal, Wuhan, China), Flag-tag (F1804, Sigma-Aldrich, St. Louis, USA). Secondary antibodies were HRP-linked anti-mouse IgG and anti-rabbit IgG (7076, 7074, Cell Signaling Technology, Danvers, USA).