Cell Culture and Stable Isotope Labeling

MV4-11 (male), IMR5 (male) and HLE (male) cells were grown in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS (Capricorn Scientific) and 1% penicillin/streptomycin solution (Sigma). HEK293 (female) and U2OS (female) cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin/streptomycin. For stable isotope labeling of MV4-11 cells, cells were cultured in RPMI 1640 medium for SILAC (Thermo Fisher Scientific) with L-lysine and L-arginine (light) or [^{2 (link)}H₄]-L-lysine and [^{13 (link)}C₆]-L-arginine (medium labeled) or [^{13 (link)}C₆, ^{15 (link)}N₂]-L-lysine and [^{13 (link)}C₆, ^{15 (link)}N₄]-L-arginine (heavy labeled) for at least five generations. All light, medium and heavy SILAC media were supplemented with 10% dialyzed FBS and 1% penicillin/streptomycin solution. Moreover, medium and heavy SILAC media were also enriched with proline to prevent arginine-to-proline conversion. Cells were analyzed for labeling efficiency before SILAC proteomics.
Cells were cultured at 37 °C in 5% CO₂. Cells were routinely screened for mycoplasma contamination in a PCR-based assay and found negative.