Puromycin

High glucose Dulbecco’s Modified Eagle’s Medium (DMEM), L-glutamine enriched Roswell Park Memorial Institute medium (RPMI) and Dulbecco’s Phosphate Buffered Saline (DPBS) were purchased from Invitrogen. Hydroxylamine, glycine, sodium hydroxide, dibasic sodium phosphate and dimethyl sulfoxide (DMSO) were obtained from Sigma. Acetonitrile was obtained from Fisher. Hydrochloric acid, trifluoroacetic acid (TFA) mass spectroscopy grade and C-18 spin columns were purchased from Pierce Thermo Scientific. MG132, MG262 and clasto-Lactacystin β-lactone were purchased from Boston Biochem. AM114, butabindide and puromycin were purchased from Tocris Bioscience. Other inhibitors and their commercial sources were bortezomib (LC Laboratories), MLN2238 (Selleckchem), carfilzomib (ChemieTek), bestatin (Sigma), and bestatin methylester (Calbiochem). Recombinant human puromycin-sensitive aminopeptidase was purchased from R&D Systems. Suc-Leu-Leu-Val-Tyr-AMC, Ala-Ala-Phe-AMC, Leu-AMC and Ala-AMC were procured from Bachem. The isotopic labeling reagents 4-trimethylammoniumbutyryl-N-hydroxysuccinimide (TMAB-NHS) containing either 0, 3, 6, or 9 atoms of deuterium (D0-, D3-, D6-, and D9-TMAB-NHS, respectively) or 9 atoms of deuterium and three ¹³C atoms (D12-TMAB-NHS) were synthesized as described [30] .

Cells were treated with 5 μM puromycin (Tocris #4089) for 15 min, then washed twice and immediately lysed in polysomal lysis buffer supplemented with protease inhibitor cocktail.

GA101 was kindly provided by Roche Glycart AG. Thapsigargin and tunicamycin were purchased from Merck Millipore (Fontenay s/s bois, France). BTP2, Ned-19 trans, and puromycin were supplied by Tocris Bioscience (Lille, France). Hoechst 33258 and siramesine were purchased from Sigma-Aldrich (L’Isle d’Abeau, France). Tetramethylrhodamine methyl ester (TMRM) and lysotracker red DND-99 were from ThermoFisher Scientific (Courtaboeuf, France), and Fluo2-leak resistant (LR)- acetoxymethyl ester (AM) was from Euromedex (Mundolsheim, France). FAM-FLICA in vitro caspase 3 detection kits were supplied by AbD Serotec (Kidlington, UK).
Anti-human Orai1 rabbit polyclonal antibody was from Alomone Labs (Jerusalem, Israel). The anti-human CD19-PE, CD19-488 (clone HIB19), and anti-human CD20-FITC (clone 2H7) and their respective isotype controls were provided by eBiosciences (San Diego, CA, USA). Anti-cathepsin B and anti-BIM were supplied by Santa Cruz Biotechnology (Heidelberg, Germany). The anti-phospho eIF2α and anti-eIF2α were from Cell Signaling Technology (Ozyme, France). Alexa 594-conjugated donkey anti-goat came from Life Technologies (Saint Aubin, France). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit were from ThermoFisher Scientific.

Retrovirus for the expression of green fluorescent protein (GFP) or the GFP-chimera of the PKA inhibitory peptide (PKI) was produced by co-transfecting GP2-293 cells with p-vesicular stomatitis virus (VSV)-G vector (encoding the pantropic VSV-G envelope protein) and either pLPCX-GFP or pLPCX-PKI-GFP. Culture media containing viral particles were harvested after 48 h of transfection and used to infect primary human ASM cell cultures as described previously [22 (link), 23 (link), 28 (link)]. Human ASM cell cultures were subsequently selected for homogeneity with puromycin (5 µg/ml) (Tocris Bioscience, UK).

Q7Q7, Q7Q111 and Q111Q111 cells were obtained from Dr. Erik Snapp (Albert Einstein College of Medicine) and cultured as described [36 (link)]. High glucose Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco’s Phosphate Buffered Saline (DPBS), penicillin-streptomycin, and M-MLV Reverse Transcriptase were purchased from Invitrogen. Fluorescent substrates containing the 7-amino-4-methylcoumarin (AMC) group were procured from Bachem. Hydroxylamine, G-418, glycine, sodium hydroxide, dibasic sodium phosphate, dimethyl sulfoxide (DMSO), and bestatin were purchased from Sigma. Hydrochloric acid, mass spectroscopy grade trifluoroacetic acid (TFA), and C-18 spin columns were purchased from Pierce Thermo Scientific. Other reagents and their sources were puromycin (Tocris Bioscience), TriPure Reagent (Roche), Power SYBR Green Master Mix (Applied Biosystems), acetonitrile (Fisher), fetal bovine serum (Seradigm), epoxomicin (Calbiochem), and bortezomib (LC Laboratories). The isotopic labeling reagents 4-trimethylammoniumbutyryl-N-hydroxysuccinimide (TMAB-NHS) containing either 0, 3, 6, or 9 atoms of deuterium (D0, D3-, D6-,and D9-, respectively) or 9 atoms of deuterium and three ¹³C atoms (D12-) were synthesized as described [37 (link),38 (link)].

Human Fetal Foreskin Fibroblasts (HFFF2) (ECACC, Salisbury, UK) and Human Epithelial Kidney cells (HEK 293) were grown in D-MEM. Cell medium was supplemented with 10% fetal bovine serum (FBS), 10,000 units/mL penicillin, 10 mg/mL streptomycin and 2 mM L-glutamine (Euroclone, Milan, Italy). All cell lines were maintained in a humidified incubator at 37 °C, with 95% relative humidity and 5% CO₂.
As described by Counter et al. [65 (link)], pBabe-Puro-hTERT (Addgene plasmid #1771) vector plasmid containing the cDNA of hTERT protein was transfected into HEK 293 using Lipofectamine 2000 (Thermo Fisher Scientific, USA) and Ampho Retrovirus Packaging Vector (Novus Biological, Englewood, CO, USA) to obtain retroviral vector particles. Four days later, the supernatant from these cells were used to infect HFFF2 cells to establish the HF-TERT cell line. After the infection, cells were selected with puromycin (2 μg/mL) (Tocris, Ellisville, MO, USA) for 5 days. Human glioblastoma astrocytomas (U251MG) were grown in MEM, supplemented with 10% fetal bovine serum (FBS), 10,000 units/ml penicillin, 10 mg/ml streptomycin, 2 mM L-glutamine, 1% Non-Essential Amino Acids and 1 mM Sodium Pyruvate (Euroclone, Milano, Italy). U251 were used in RQ-TRAP assay as positive control of telomerase activity.