L lysine and l arginine

Human epidermoid carcinoma A431 cells were cultured as previously described^{1 (link), 32 (link)}. For SILAC experiments, cells were maintained in SILAC medium comprising Dulbecco’s modified Eagle medium (DMEM; Invitrogen) supplemented with10% dialyzed FBS (Invitrogen). L-lysine and L-arginine(Sigma-Aldrich) or [¹³C₆]-L-lysine and [¹³C₆]-L-arginine (ISOTEC; Sigma-Aldrich) were added at 0.1 g/L for light or heavy stable isotope labeling, respectively, and cells were cultured for at least 10 doubling times to achieve 95% incorporation^{63 (link), 64 (link)}.

SUM52 and MFM223 cells were cultured at 37 °C, 5% CO₂ in RPMI-1640 or DMEM containing 2 mM L-Glutamine (Lonza), respectively, supplemented with 0.1 mg/ml streptomycin, 100 U/ml penicillin (Sigma-Aldrich), and 10% v/v fetal calf serum (Biosera). For SILAC labelling, SUM52 cells were prepared as previously described^{125 (link)} and MFM223 cells were cultured in SILAC DMEM (Thermo Fisher Scientific) supplemented with 0.798 mM L-lysine and 0.398 mM L-arginine (either isotopically “light” L-lysine and L-arginine (Sigma-Aldrich), “medium” 4,4,5,5-D4 L-lysine and ¹³C₆ L-arginine, or “heavy” ¹³C₆ L-lysine and ¹³C₆¹⁵N₄ L-arginine (CK Isotopes)), supplemented with 10% dialyzed FBS (Biosera), 0.1 mg/ml streptomycin, and 100 U/ml penicillin at 37 °C with 5% CO₂.

For the stiffness proteome, HUVECs were SILAC‐labeled in custom‐made EGM‐2 medium without arginine and lysine (Lonza) and supplemented with ¹³C₆¹⁵N₂ L‐lysine and ¹³C₆¹⁵N₄ L‐arginine (heavy, Cambridge Isotope Laboratories) or L‐lysine and L‐arginine (light, Sigma) amino acids. Forward and reverse experiments were performed, where labeling conditions were swapped. For the proteome of HUVECs silenced for CCN1 and those cultured on Matrigel, a heavy SILAC‐HUVEC standard was mixed at a 1:1 ratio with each of the lysates from the non‐labeled samples (triplicates for each condition). Cell lysates were collected in 2% SDS, 100 mM Tris–HCl pH 7.6, subsequently reduced with DTT, and boiled before being run through a 4–12% gradient NuPAGE Novex Bis‐Tris gel (Life technologies) in MOPS running buffer (Life technologies). Proteins were digested in‐gel (stiffness and siCCN1 experiments) (Shevchenko et al, 2006), or digested on filter using the FASP protocol with trypsin (Promega) and peptides separated into six fractions using on‐tip strong anion exchange (SAX) chromatography (Wisniewski et al, 2009) (Matrigel experiment). Digested peptides were desalted using StageTip (Rappsilber et al, 2007). After removal of acetonitrile (ACN) using speed vacuum, peptides were resuspended in 1% TFA and 0.2% acetic acid buffer for MS data analysis.