A 485

Here, 2 × 10⁶ CDS1 cells were resuspended in 100 uL of 1X PBS prior to bilateral subcutaneous injection in the subcutaneous suprascapular region of NSG mice. The experiment included a cohort of eleven mice, which were allocated between the control-treated (n = 6) or the drug-treated (n = 5) group to normalize their size before starting the treatment. A-485 (MedChemExpress, Cat# HY-107455, Monmouth Junction, NJ, USA) was prepared daily and administered intraperitoneally at a dose of 50 mg/kg twice daily. When the first tumors started to be visible, mice were treated with either 50 mg/kg of A-485 or solvent for 15 days. All mice were monitored daily for signs of distress and were weighed three times a week. Tumor size was measured three times a week with a caliper, and tumor volume was calculated according to the following equation: (length × width²)/2 = tumor volume [mm³]. Control-treated mice received the solvent used for oral administration. Mice were sacrificed, as described above, the day after the last dose of A-485.

Jurkat, CEM, C8166/45, MT-2, SLB-1, and ATL-2 cells were cultured in Isocove’s modified Dulbecco medium (IMDM). Primary CD4⁺ lymphocytes, TL-Om1, and HTLV-1-immortalized lymphocyte cell lines [47 (link)] were cultured in Roswell Park Memorial Institute (RPMI) medium. HeLa and HeLa-HBZ clonal cell lines [48 (link)], CHO-LFA-1 clones [38 (link)], and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM). All cells were supplemented with 10% FBS or 10% FetalPlex (GeminiBio) and 2 mM L-glutamine, 100 U/mL penicillin, and 50 μg/mL streptomycin. Jurkat pminLuc-viral CRE cells [38 (link)] and Jurkat-HBZ cells [38 (link)] were supplemented with 1.5 mg/mL of G418. HeLa and CHO clones were supplemented with 0.5 mg/mL of G418. Primary lymphocytes and lymphocyte cell lines were cultured with IL-2. Primary lymphocytes were activated in culture wells coated with anti-CD3 and anti-CD28 antibodies. Where indicated, cells were treated with 10 μM A485 (MedChem Express) or DMSO for 3 h.

Primary mouse hepatocytes were isolated from C57BL/6 mice by a two-step perfusion technique as described^{17 (link)}. Cells were treated with 100 μM 8-Bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, Sigma), 3 μM A-485 (MedChemExpress), 10 μM MG132 (Beyotime Biotechnology), or 10 μg/ml cycloheximide (CHX, Sigma).