Puromycin

Low-passage murine Ba/F3 cells, kindly provided by Connie J. Eaves (Terry Fox Laboratory, British Columbia Cancer Agency and University of British Columbia, Vancouver, BC, Canada), were cultured in RPMI-1640 (Wisent Bio Products, QC, Canada) supplemented with 10% FBS (Wisent Bio Products), 100 units/mL penicillin and streptomycin (Wisent Bio Products, QC, Canada) and 5 ng/ml of recombinant murine IL3 (Peprotech, NJ, USA) at 37 °C in a humidified atmosphere at 5% CO₂. Cells infected with lentiviral plasmids were selected by adding puromycin (1 μg/mL) (Wisent Bio Products, QC, Canada) to the cell culture medium. Stable infection of Ba/F3 cells was performed as described previously [27 (link)]. Expression of FLT3 was confirmed by RT-qPCR and Western blot as described elsewhere [28 (link)]. The following antibodies were used: anti-FLT3 (Cell Signaling, #3462, Danvers, MA, USA), anti-phospho ERK1/2 (Cell Signaling, #4370, Danvers, MA, USA), anti-ERK1/2 (Cell Signaling, #4695, Danvers, MA, USA), anti-phospho STAT5 (Cell Signaling, #9359, Danvers, MA, USA) and anti-STAT5a/b (Abcam, EPR16671-40, Cambridge, UK) and anti-GAPDH (sc-32233) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Splenic B cells were purified from wild-type C57BL/6 mice with a negative selection mouse B cell enrichment kit (Stemcell Technologies). Cells were then cultured in complete RPMI media (Gibco) with 25 ng/mL LPS for 24 hours, followed by lentiviral transduction in the presence of 8 ng/mL polybrene (Sigma-Aldrich). Positively transduced cells were then selected with 0.6 μg/mL puromycin (Wisent Bio Products) and stimulated with 25 ng/mL recombinant mouse IL-4 (R&D Systems) for 4 days, and then analyzed by flow cytometry. Experimental protocols with mice were carried out in accordance with the University of Toronto Division of Comparative Medicine guidelines and regulations and were approved using protocol number 20011472.

A shRNA, targeting the GALNT3 sequence 5'-TACTGCTGAAGGAAATCAT-3', was designed using the siRNA Ambion Target Finder software (http://www.ambion.com/techlib/misc/siRNA_finder.html), and subcloned into the pSilencer 4.1 puro vector (Ambion). A2780s cells were stably transfected with the shRNA-GALNT3 plasmid using the ExGen 500 transfection reagent (Fermentas Canada Inc., Burlington ON), according to the manufacturer's instructions. Cells were consecutively grown for 2 weeks in selection medium containing 5 μg/ml puromycin (Wisent, Canada) to isolate stable clones. Cells were also mock-transfected with the pSilencer 4.1 puro vector, and the stably-transfected clones were isolated as controls. Stable clones with inhibited GALNT3 expression were evaluated and validated by semi-quantitative RT-PCR and Western blot.

RUNX1, TSP1 TGFβRII knockdown was achieved using lentiviral shRNA vectors from the Mission TRC genome-wide shRNA collections purchased from Sigma-Aldrich Corporation with the following catalog numbers; Scrambled shRNA#: SHC016, RUNX1#1: TRCN0000338428, RUNX1#2: TRCN0000338427, TSP1#1: TRCN0000226402, TSP1#2: TRCN0000219072, TGFβRII#1: TRCN0000000831 and TGFβRII#2: TRCN0000000834. Lentiviral supernatants were generated using the calcium phosphate method as described^{118 (link)}. Cancer cells were incubated with lentivirus-containing media with polybrene (8 µg/ml) and incubated for 72 h at 37 °C with 5% CO₂ followed by 1 µg/ml of Puromycin (Wisent Inc., 450-162-XL) selection for 15 days.

Lentivirus was produced by transfecting HEK293FT cells (Thermo Fisher Scientific) with psPAX2, pMDG1.vsvg, and pLentiCRISPR transfer vector. The supernatant, which contains lentivirus, was collected at 72 h post transfection and filtered before being used^{64 (link)}. Efficient retrovial infection required addition of 5 μg/ml Polybrene (Millipore) for HEK293, CFPAC1, HPAF-II, and Hs766T. Selection was performed using puromycin (5 μg/ml) (Wisent) or hygromycin (100 μg/ml) (Invitrogen) 24 h after infection and polyclonal populations were generated.

Reh (Human B cell precursor leukemia; ATCC CRL-1567) and NALM-6 (Human B cell precursor leukemia; DSMZ ACC-128) cells were cultured in RPMI 1640 medium (Wisent) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Wisent). Reh_shRNA control and Reh_shRNA-RP11-137H2.4 cells were cultured in RPMI 1640 medium (Wisent) supplemented with 10% heat-inactivated FBS and 1µg/mL puromycin (Wisent). Reh_shRNA-RP11-137H2.4_RP11-137H2.4 cells (“Rescue” cell line) were cultured in the same media as Reh_shRNA RP11-137H2.4, but supplemented with 5µg/mL blasticidin (Wisent). Human embryonic kidney 293T cells (HEK293T; ATCC CRL-3216) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Wisent) supplemented with 10% heat-inactivated FBS. All culture media were supplemented with 1% penicillin/streptomycin (Wisent), and all cell lines were cultured in a 37 °C incubator.