Myc 16286 1 ap

Antibodies used in this study were as follows: BUB1B (4116, Cell Signaling Technology, USA), MYC (16286-1-AP, ProteinTech Group, China), CEP170 (18899-1-AP, ProteinTech Group, China), α-Tubulin (ab7291, Abcam, UK), FLAG (F-4020, Merck KGaA, Germany), β-actin (4970 S, Cell Signaling Technology), rabbit IgG (a7016) and mouse IgG (a7028, Beyotime Institute of Biotechnology, China).
Doxycycline (Dox) was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Puromycin was from Merck KGaA (Darmstadt, Germany). Bortezomib (BTZ) and Adriamycin (ADR) were purchased from Selleck Chemicals (Houston, TX). The rapid Giemsa staining kit was obtained from BBI Life Sciences (Shanghai, China).

Cells were harvested and proteins were extracted with RIPA lysis buffer. The following antibodies were used: NPM1 (FC-61991; Invitrogen, Carlsbad, CA, USA; 1:1000), PD-L1 (GTX104763; GeneTex, Irvine, CA, USA; 1:4000), PD-L1 (GTX31308; GeneTex; 1:1000), PD-L1 (13684; Cell Signaling Technology, Danvers, MA, USA; 1:1000), PARP1 (9532; Cell Signaling Technology; 1:1000), Flag (14793; Cell Signaling Technology; 1:1000), Myc (16286-1-AP; Proteintech, Rosemont, IL, USA; 1:1000), HSP70 (46477; Cell Signaling Technology; 1:1000), GAPDH (10494-1-AP; Proteintech; 1:10,000). Cytokine IFN-γ (EST-IFg-0100; Stemimmune LLC, Richmond, CA, USA) was used to treat cells at the concentration of 25 μg mL⁻¹.

HEK293T cells were seeded in 6-well plates 16 h before transfection. The indicated expression vectors (Myc-CDK9, HA-HDAC1, or HA-HDAC2: 1 μg) were co-transfected with or without the Flag-METTL3 expression vector (1 μg) in HEK293T cells. For the Co-IP of HDAC1/2 and METTL3, total cell lysates were first incubated with DNase1 (200 U/ml) at 37 °C for 30 min to digest genomic DNA. These lysates were then immunoprecipitated with Myc, Flag, or HA beads at 4 °C for 2 h. Immunoblotting was performed using the indicated antibodies. Antibody dilutions were as follows: METTL3 (96391, Cell Signaling Technology), 1:2500; Lamin B1 (12987-1-AP, Proteintech), 1:5000; Tubulin (sc-5286, Santa Cruz), 1:5000; β-actin (60008-1-Ig, Proteintech), 1:5000; CDK9 (11705-1-AP, Proteintech), 1:2500; p-CDK9 (2549, Cell Signaling Technology), 1:2500; CD36 (18836-1-AP, Proteintech), 1:5000; Flag (F1804, Sigma), 1:5000; Myc (16286-1-AP, Proteintech), 1:5000; HDAC1 (5356, Cell Signaling Technology), 1:2500; HDAC2 (5113, Cell Signaling Technology), 1:2500; and Caspase3 (9662, Cell Signaling Technology), 1:2500. Other reagents were listed in Supplementary Table 3.