Gsk591

Human PRMT5 knockdown lentiviral plasmids were as described previously [19 (link)]. The packaging plasmid MD2G and helper plasmid PAX2 were purchased from Addgene. The PRMT5 knockdown targeting sequences are listed below: shRNA1, 5’-GGATAAAGCTGTATGCTGT-3’; shRNA2: 5’-GCCATCTATAAATGTCTGCTA-3’. PRMT5 specific inhibitor GSK591 (cat# SML-1751) was purchased from Sigma. Human PRMT5 cDNA subcloned into Flag vector was purchased from Addgene and verified by sequencing.

Primary human foetal lung fibroblast cells (IMR90, normal cells) were cultured in RPMI1640 medium with 10% (v/v) foetal bovine serum (FBS, Sigma, cat# F2442). Human lung adenocarcinoma cells A549, H1299, H322, H441 and PC14 were used in this study. A549 cells were cultured in F12K medium/Dulbecco’s Modified Eagle’s Medium (1:1) (cat no. 11320033; Thermo Fisher Scientific) supplemented with 10% (v/v) FBS (Sigma) and the other cells were cultured in RPMI 1640 medium (cat no. 11875119; Thermo Fisher Scientific) with 10% FBS. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂% and 95% air. PRMT5 specific inhibitor GSK591 (cat no. SML‐1751) was purchased from Sigma.

U87-MG cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma, Kawasaki, Japan), supplemented with 10% foetal bovine serum. U87-MG spheroids were produced by culture on 1.5% agarose gel following published protocols [15 (link)]. Cells were treated with the following PRMT inhibitors: AdOx, AMI-1, GSK591 (all from Sigma), MS023 and furamidine (both from Tocris Bioscience, Bristol, UK).

HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) already including 1% glutamine and supplemented with 10% fetal bovine serum (FBS; Life Technologies), penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells were cultured at 37°C in a 5% CO2 humidified atmosphere. The cells were tested free of mycoplasma contamination. MS023 was purchased from Cayman chemicals; GSK591 was purchased from Sigma Aldrich. Both MS023 (10 µM) and GSK591 (5 µM) were used for 48 h treatment, together with DMSO as control. For triple SILAC, HeLa were grown in ‘‘Light’’, ‘‘Medium’’ and ‘‘Heavy’’ SILAC DMEM (Thermo Fisher Scientific), supplemented with either L-Arginine, L-Lysine or their medium (Arg6: Sigma-Aldrich; Lys4: Sigma-Aldrich) or heavy (Arg10: Sigma-Aldrich; Lys8: Sigma-Aldrich) isotope-counterparts. Arginine and Lysine were added at a concentration of 84 mg/L and 146 mg/L, respectively. SILAC media were supplemented with 10% dialyzed FBS (GIBCO, Life Technologies), 100 U/ml Penicillin and 100 mg/ml Streptomycin. HeLa cells were grown in the respective heavy-isotopes containing media for at least 9 replication cycles, to ensure full incorporation of isotope-encoded amino acids, with a careful monitoring of growth rate, viability and overall morphology, to guarantee that normal cell physiology was preserved.