Effectence transfection reagent

Transfection of HEK293T cells were performed with the Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA). Transfection of HUVECs was performed with the Effectence transfection reagent (Qiagen, Suzhou, Jiangsu, China). SP600125 were purchased from Selleck Chemicals (Shanghai, China). Antibodies used were anti-SPAG9 rabbit antibody from Abcam (Cambridge, MA, USA), anti-p-JNK rabbit antibody and anti-JNK rabbit antibody from Beyotime Institute of Biotechnology (Nantong, Jiangsu, China), anti-Flag rabbit antibody from Cell Signaling Technologies (Beijing, China), anti-α-Tubulin mouse antibody, anti-GAPDH mouse antibody, anti-Actin mouse antibody, and anti-VEGFA rabbit antibody from Santa Cruz Biotechnology (Dallas, TX, USA). The polyclonal rabbit anti-vIRF1 antibodies were kindly provided by Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine.

Based on the pHAGE-vIRF1-Flag expressing plasmid described in the previous studies [45 (link)], the vIRF1 domain-mutant plasmids (vIRF1-MD1-Flag, vIRF1-MD2-Flag), and the vIRF1 single lysine-mutant plasmids were constructed to replace the indicated lysine site(s) with arginine by Tsingke Biotechnology Co., Ltd. (Beijing, China), as well as the Lys406 and Lys442 co-mutant vIRF1 plasmid (vIRF1-K406R+K442R-Flag). The sirtuins expressing plasmids (SIRTs-1~7-Myc), USP10-Flag, IRF3-Myc and STING-Myc were generated based on the pCDH vector (Tsingke Biotechnology Co., Ltd., China). pcDNA3.1-p300-HA was kindly provided by Dr. Xiaoming Wang (Nanjing Medical University), and pcDNA3.1-CBP-HA was constructed based on a generous gift from Dr. Xiao Han (Nanjing Medical University). A 300 bp fragment of IFN-β promoter (-280 to +20) was amplified and subcloned into pGL3-basic vector containing the firefly luciferase reporter gene (Promega, USA). The mpCDH plasmid was used as the short hairpin RNA (shRNA) expressing lentiviral vector, and the shRNA sequences to USP10 were listed in S1 Table. All plasmids were prepared using Vazyme FastPure Plasmid Mini Kit and confirmed by DNA sequencing. HEK293T cells were transfected with Lipofectamine 2000 Reagent (Invitrogen, USA), and EA.hy926 cells was transfected with Effectence transfection reagent (Qiagen, China).

HUVECs were transfected using the Effectence transfection reagent (Qiagen, Suzhou, Jiangsu, China), while HEK293T cells were transfected using the Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA). 5-aza (Decitabine), a potent inhibitor of DNA methylation, was from Selleck Chemicals (Shanghai, China). siRNAs were synthesized from Genepharma (Suzhou, China), the sequences of siRNAs are listed in S1 Table. LncRNA Smart Silencer was obtained from RiboBio (Guangzhou, China). Antibodies against KSHV LANA, HMGB2, CMPK1, DNMT1 and Dicer were from Abcam (Cambridge, MA, USA). Anti-Flag was obtained from Cell Signaling Technologies (Beijing, China). Anti-Myc, anti-α-Tubulin, and anti-GAPDH were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-rabbit immunoglobulin G (IgG), anti-mouse IgG, anti-phosphorylated p53, anti-acetylated p53, anti-p53, and anti-p21 were purchased from Beyotime Institute of Biotechnology (Nantong, Jiangsu, China). Western-blotting analysis was conducted as previously described [81 (link), 82 (link)]. In this study, all Western blotting results were independently repeated at least three times unless otherwise stated.