Oci ly3

Human DLBCL cell lines OCI‐Ly19, OCI‐Ly1, OCI‐Ly3, OCI‐Ly10 and SU‐DHL‐4 were purchased from ATCC (Rockefeller, MD, USA) and DLBCL cell lines were grown in RPMI‐1640 medium (Gibco, Billings, MT, USA). OCI‐Ly3 cells were cultured in IMDM (Gibco). Supplemented with 10% fetal bovine serum (HyClone, Thermo Scientific, Waltham, MA, USA) at 37 °C in a humidified CO₂ incubator. The primary DLBCL samples (n = 12) were obtained from the Department of Hematology, the First Affiliated Hospital, College of Medicine, Zhejiang University (Hangzhou, China) between 2021 and 2022. This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University. The study methodologies conformed to the standards set by the Declaration of Helsinki and the experiments were undertaken with the understanding and written consent of each subject.

HeLa, DU145, THP-1, RKO, SKM1, A549, OCI-LY3, G361, and HL60 human cancer cell lines were obtained from ATCC (Manassas, VA, USA). Roswell Park Memorial Institute (RPMI) 1640 medium, penicillin-streptomycin and 0.05% trypsin were from Gibco (Carlsbad, CA, USA). Heat-inactivated fetal bovine serum (FBS) was purchased from Hyclone (South Logan, UT, USA). Anti-pAKT (S473) #9271, anti-AXL (C44G1) #4566, anti-MER (D21F11) #4319, anti-TYRO3 (D38C6) #5585, and anti-rabbit Alexa 488 antibody were purchased from Cell Signaling Technology (Danvers, MA, USA).

Two DLBCL cell lines U2932 and OCI-Ly3 were purchased from ATCC and cultured in DMEM medium supplemented with 10% FBS. Cell transfection was conducted by using Lipofectamine 2000 (Invitrogen, CA, USA). For constructing the cell lines stably silencing TUC338, two validated shRNAs targeting TUC338 were inserted into psi-LVRU6GP lentiviral vector (GeneCopoeia, CA, USA), followed by transfection into U2932 and OCI-Ly3 cells. After 48 h, puromycin was added to culture medium to screen stable cell lines.

The 16 DLBCL cell lines (DOHH2, HT, OCI-LY19, DB, OCI-LY1, SUDHL-4, SUDHL-5, SUDHL-10, NUDHL-1, OCI-LY7, WSU-DLCL2, SUDHL-6, NUDUL-1, U2932, OCI-LY3, and RI-1) were purchased from the American Type Culture Collection or from DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany). They were grown in RPMI-1640 Glutamax medium (Gibco, Invitrogen, Cergy Pontoise, France), supplemented with 10% fetal bovine serum (FBS) (PAA laboratory GmbH, Pasching, Austria) (U2932, SUDHL-4, HT, DOHH2, SUDHL-10, RI-1, and WSU-DLCL2 cells), 20% FBS (OCI-LY3, DB, SUDHL-5, NUDHL-1, and SUDHL-6 cells), or 15% FBS (NUDUL-1 cells). OCI-LY1, and OCI-LY7 cells were cultured in IMDM Glutamax (Gibco, Invitrogen, Cergy Pontoise, France), supplemented with 20% FBS, and OCI-LY19 cells in MEM alpha modified Glutamax (Gibco, Invitrogen, Cergy Pontoise, France) with 20% FBS. Cultures were maintained at 37 °C in a humidified atmosphere with 5% CO₂. Contamination by Mycoplasma species was regularly monitored.

The human DLBCL cell lines BJAB, U2932, OCI-Ly3 were obtained from DSMZ, SUDHL-4, and SUDHL-6 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). All cell lines were cultured in RPMI 1640 medium supplemented with 20% FBS and 100 U/ml penicillin/streptomycin (Gibco). OCI-Ly10 was purchased from Cobioer Biosciences Co., LTD (Nanjing, China) and cultured in IMDM with 20% FBS, 100 U/ml penicillin/streptomycin (Gibco) and 50 μM β-mercaptoethanol (Sigma-Aldrich). All cell lines were cultured at 37°C in a humidified atmosphere of 5% CO₂.
z-VRPR-fmk (Enzo Life Sciences) was dissolved in ddH₂O at a concentration of 50 μM throughout all experiments. MI-2, BPTES, QNZ, CPI-613 (Selleck), and PMA/Iono (Sigma-Aldrich) were reconstituted in DMSO (final DMSO concentration 0.1%) and their final concentrations were 1 μM, 2 μM, 5 μM, 100 μM, and 50/500 ng/ml, respectively.

The cell lines OCI-LY7, OCI-LY3 and HEK293T were obtained from American Type Culture Collection (ATCC). OCI-LY7 is germinal center B-cell (GCB)-subtype cell line; OCI-LY3 is an activated B-cell (ABC)-subtype cell line; and HEK293T is an embryonic kidney cell line. OCI-LY7 was maintained in complete Iscove’s modified essential medium (IMDM; GIBCO, Carlsbad, CA, USA) with 2-mercaptoethanol (1:10000) and 10% fetal bovine serum (GIBCO). OCI-LY3 was cultured in IMDM with 20% fetal bovine serum. HEK293T cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO) supplemented with 10% fetal bovine serum. All of the cell lines were supplemented with 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). All of the cell cultures were maintained at 37°C under 5% CO₂ and 95% air.