Actinomicin d

VPA was purchased from Enzo Life Sciences. Stock solutions were prepared in sterile water. Entinostat (MS-275) and vorinostat were from Selleck Chemicals; panobinostat (LBH589) from Novartis International; 5-Azacytidine (azacitidine) was from Sigma; GSK126 was from Active Biochem. Stock solutions were prepared in DMSO. Monoclonal antibodies anti-mouse PD-1 (clone RMP1-14, #BE0146) and anti-mouse CTLA-4 (clone 9H10, #BE0131) were purchased from Bioxcell. Actinomicin D was purchased from Sigma Aldrich.
All media, serum, antibiotics, and glutamine were from Corning.
Primary antibodies (Abs) for western blotting: β-Actin-Ab (Sigma-Aldrich, cod.A5316), Programmed death-ligand 1 (PD-L1)-Ab (Abcam, cod.Ab58810); (PD-L1)-Ab (cod.#13684), acetyl-H3-Ab (cod.#9649), PARP-Ab (cod.#9542) (Cell signaling Technology), and acetyl-H4-Ab (Millipore cod.06946). For IHC: monoclonal anti-mouse Ki67-Ab (Cell signaling Technology; cod.#12202), monoclonal anti-mouse CD4-Ab (Abcam, cod.Ab183685), anti-mouse CD8-Ab (eBioscience, clone 56-6.7). For immunofluorescence on fresh frozen tissues: anti-Foxp3-efluor570 (eBioscience clone FJK-16s, #41-5773-80).

RNA transcript analysis was performed using quantitative reverse transcription PCR (q-PCR) on the 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) with SYBR Green (Thermo Fisher Scientific, 4309155). The reaction mix volume (10 μL) included 0.5 μM of each primer and 1 μL of cDNA diluted at 200 ng/μL. Amplification conditions were: 40 cycles of 50 °C, 2 min; 95 °C, 10 min; 95 °C, 15 seg; 60 °C, 1 °C min), and the primer sequences are listed below. Transcript levels were normalized against the number of parasites and plotted as relative change to the untreated cells. Analysis of all samples were performed in triplicate. Actinomicin D (Sigma-Aldrich, A9415) and α-amanitine (Sigma-Aldrich, A2263) were used as positive controls of inhibition for Pol I and Pol II transcription respectively.