Ab57834

Deparaffinized sections were incubated with antibodies to CD44 (ab157107, Abcam, Cambridge, UK); STRO-1 (ab57834, Abcam), osteocalcin (OC, ab198228, Abcam), osteopontin (OPN, ab218237, Abcam), dentin sialophosphoprotein (DSPP, ab216892, Abcam), alkaline phosphatase (ALP, ab216892, Abcam), pan-cytokeratin (pan-CK, CF190321, TrueMAB™ Thermo Fisher Scientific), vimentin (Vim, MA5-11883, Thermo Fisher Scientific). Antigen demasking was performed before immunohistochemical staining using low pH EnVison FLEX Target Retrieval Solution (Dako, Glostrup, Denmark A/S) at 97 °C for 20 min. Endogenous peroxidase and host IgG were blocked. Primary antibodies were diluted according to the manufacturer’s recommendations. Incubation was carried out for 12 h at 4 °C. An EnVision FLEX detection system (Dako, Glostrup, Denmark A/S) with 2,3-diaminobenzidine DAB chromogen (DAB Chromogen Solution, Dako) was used for imaging. The nuclei were counterstained with hematoxylin. We used incubation without primary antibodies as a negative control.

STIM1 expression was assayed in EBV-negative and -positive NPC cells. EBV-positive cells were transfected with recombinant plasmid vector GV248 encoding short hairpin RNA against STIM1 (shRNA-STIM1) or negative control short hairpin RNA (shRNA-NC). Lentivirus transfections were carried out according to the instructions provided by the manufacturer (GeneChem Technology, Shanghai, China). STIM1 expression was assayed by Western blotting with an anti-STIM1 antibody (1 mg/mL, cat. no. ab57834, Abcam, Cambridge, UK). β-actin was assayed as an internal control using an antibody (1 mg/mL, cat. no. ab8226, Abcam).

The lentiviral vector GV248, carrying a short hairpin RNA against STIM1 (shRNA-STIM1) or negative control short hairpin RNA (shRNA-Ctrl), was used in this study (GeneChem Technology Co. Ltd., Shanghai, China). The shRNA sequence against STIM1 was GGGAAGACCTCAATTACCA, and the nonsense sequence TTCTCCGAACGTGTCACGT was used as the scrambled shRNA control. The 5–8F cells were transfected with the lentivirus vector according to the manufacturer’s instructions, and puromycin (1 µg/mL, Thermo Fisher Scientific) was used to screen the cell clones for a stable infection. The sequence encoding green fluorescence (GFP) was simultaneously introduced into the NPC cell line 5–8F. The percentage of GFP-expressing cells was determined to evaluate the transfection efficiency. A real-time quantitative polymerase chain reaction (RT-qPCR) was applied to detect the relative expression levels of STIM1, and the primer sequences and product lengths are shown in Supplementary Table S1. The expression of STIM1 was also detected by Western blotting using an antibody against STIM1 (1:1000, cat. No. ab57834, Abcam, Cambridge, UK). The GAPDH expression was used as the internal reference (1:1000; cat. no. ab8245, Abcam).