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TARGETING LIPA INDEPENDENT OF ITS LIPASE ACTIVITY IS A THERAPEUTIC STRATEGY IN SOLID TUMORS VIA INDUCTION OF ENDOPLASMIC RETICULUM STRESS

ABSTRACT: pCW57-LIPA-TurboID transduced cells were treated with or without 1mg/mL doxycycline for 48h, then were treated with 50mM biotin for 15min and then harvested and lysed in RIPA buffer (Thermo Scientific) supplemented with Halt protease inhibitor cocktail (Thermo Scientific) and Phosphatase Inhibitor Cocktail set I and set II (MilliporeSigma) on ice for 15min, followed by 20min of 20,000g centrifuge at 4 degrees C. Cleared cell lysates were incubated with M-270 streptavidin Dynabeads overnight. Then the beads were washed twice with RIPA buffer (2min), once with 1M KCl (2min), once with 0.1M Na2CO3 (10s), once with 2M urea in 10mM Tris-HCl (pH 8.0) (10s), and twice with RIPA buffer (2min). Enriched material was eluted from beads by boiling samples in 4x Laemmli buffer supplemented with 2mM biotin and 20mM DTT at 95 degrees C for 10min. Eluted samples were resolved in SDS-PAGE and followed by mass spectrometry by Lumos. biological replicate 1, 929193 (clone 1, no Dox.), 929194 (clone 1, with Dox.), 929196 (clone 2, no Dox.) and 929197 (clone 2, with Dox.). biological replicate 2, 932804 (clone 1, no Dox.), 932805 (clone 1, with Dox.), 932807 (clone 2, no Dox.) and 932808 (clone 2, with Dox.).

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ganesh Raj

PROVIDER: MSV000089108 | MassIVE | Wed Mar 23 09:31:00 GMT 2022

REPOSITORIES: MassIVE

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Project description:Human Umbilical Vein Endothelial Cells pooled from several donors, were purchased from Lonza and cultured in FBS reduced Endopan 3 media. HUVEC cells of passages 5-10 were used for experiments. MPO treatment was performed with protein purified from human blood (Planta) and at 1 μg/ml final concentration. Immunoprecipitation was performed from isolated nuclei (after 8 hours MPO treatment), non-treated cells were used as negative control. For immunoprecipitation antibody against MPO (Calbiochem, 475915) was used. Cell nuclei were isolated by incubating cells for 15 min on ice in NIB buffer (15 mM Tris-HCL pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM sucrose) containing 0.3% NP-40. Nuclei were pelleted for 5 min 800 × g at 4 °C, washed twice in the same buffer, lysed for 10 min on ice in IP buffer (150 mM LiCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% Empigen) freshly supplemented with 2 mM sodium vanadate, 1× protease inhibitor cocktail (Roche), PMSF (10 µl), 0,5 mM DTT, and 50 units Benzonase per ml of IP buffer, before preclearing cell debris by centrifugation at >15,000 × g at 4 °C. Finally, 1 mg of the lysate was incubated with anti-MPO antisera overnight at 4 °C. Magnetic beads (Active Motif) were then washed once with 1× PBS-Tween and combined with the antibody-lysate mixture. Following a 2-h incubation at 4 °C, beads were separated on a magnetic rack and washed 5×, 5 min each in wash buffer (150 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl pH 7.5, 0.5% NP-40) and another two times in wash buffer without NP-40. Captured proteins were predigested and eluted from the beads using digestion buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 1 mM DTT) supplemented with trypsin and eluted from the beads with elution buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 5 mM chloroacetamide) supplemented with trypsin and LysC, before subjected to mass-spectrometry on a Q-Exactive Plus Orbitrap platform coupled to an EASY nLC (Thermo Scientific). Peptides were loaded in solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm length, 75 µm I.D., filled with 2.7 µm Poroshell EC120 C1; Agilent)

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TARGETING LIPA INDEPENDENT OF ITS LIPASE ACTIVITY IS A THERAPEUTIC STRATEGY IN SOLID TUMORS VIA INDUCTION OF ENDOPLASMIC RETICULUM STRESS

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