Resiquimod (R848) is a Toll-like receptor 7/8 (TLR7/TLR8) agonist that induces cytokine upregulation. Resiquimod is an immunomodulator with antitumor and antiviral activity.

方法: 金绒球头肾淋巴细胞 HKL 用 Resiquimod (0.175-32 µg/mL) 处理 12 h，使用 CCK8 assay 检测细胞活力。
结果: CCK8 测定结果显示，0.25-32 µg/mL Resiquimod 显著促进 HKL 增殖。[1]
方法: 外周血单核细胞 PBM 用 Resiquimod (0.01-100 µM) 处理 1-14 h，使用 Western Blot 方法检测靶点蛋白表达水平。
结果: 1 µM 的剂量足以改变 FcγR 的表达，更高的剂量不会导致更大的变化。FcγRIIa 的增加发生在晚期，而 γ 链的小幅增加出现在 3 h，但在 14 h 时更高。然而，FcγRIIb 蛋白在1 h 内减少，而 FcγRIIb 的转录物保持到 4 h。[2]

方法: 为检测体内抗肿瘤活性，将 Resiquimod (2 mg/kg) 和 4D5 抗 HER2 抗体 (20 mg/kg) 腹腔注射给携带 CT26-HER2/neu 肿瘤的 Balb/cJ 小鼠，每周三次，持续 13 天。
结果: 13 天后，接受 Resiquimod 加抗体的小鼠肿瘤的生长速率显著降低。统计测试显示 4D5 和 Resiquimod 在降低肿瘤生长速率方面具有协同作用。[2]

For luciferase assay, FG-9307 cells are transfected with the firefly NF-κB-specific luciferase reporter vector pNFκB-Met-Luc2. Transfection efficiency is monitored by co-transfection with the pSEAP2 control vector, which constitutively expresses the human secreted enhanced alkaline phosphatase (SEAP). Then the cells are treated with Resiquimod (R848, 1?μg/mL), CQ (10?μM), CQ plus R848 or PBS and incubated at 22°C for 24?h. The culture medium of the transfectants is then analyzed for luciferase activity and SEAP activity using Luciferase Assay Kit and the Great EscAPe? SEAP Chemiluminescence Detection Kit, respectively. The assay is performed three times.

Resiquimod is dissolved in DMSO. For inhibition of lysosomal acidification, cells are incubated with 10?μM CQ for 1?h before Resiquimod (R848) treatment. After treatment, 20?μL of 5?mg/mL MTT is added to the plate. The plate is incubated at 22°C for 4?h, and 200?μL dimethyl sulfoxide is added to the plate to dissolve the reduced formazan. The plate is then read at 490?nm with a microplate reader. To determine the effect of Myd88 inhibition on R848-induced cell proliferation, the Myd88 inhibitor Pepinh-MYD and the control peptide Pepinh-Control are added to PBL at the concentration of 50?μM, and the plate is incubated at 22°C for 6?h. After incubation, the cells are treated with R848 and subjected to MTT assay as above. To determine the effect of NF-κB inactivation on R848-induced cell proliferation, BAY-11-7082, an irreversible inhibitor of IκB-α phosphorylation, is added to the cells at the concentration of 1?μM, and the plate is incubated at 22°C for 1?h. After incubation, the cells are treated with R848 and subjected to MTT assay as earlier. All experiments are performed three times.

Animal Models: Wild-type mice,TLR7-deficient mice,and MyD88-deficient mice. Formulation: saline. Dosages: 50 nmol. Administration: i.p.