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TAS1440, an LSD1 inhibitor, activates TGF-β and NOTCH signaling in neuroendocrine small-cell lung cancer

ABSTRACT: NCI-H1417 cells were electrotransfected with either a pcDNA3-3×Flag-myc-empty or pcDNA3-3×Flag-myc-LSD1. Cells were treated with different LSD1 inhibitors for 2 h, harvested. Lysates were immunoprecipitation by using ANTI-Flag® M2 Affinity Agarose Gel (Cat. No.‎ A2220, Sigma-Aldrich) overnight at 4°C. After washing and eluting by 3× FLAG® peptide (Cat. No.‎ F4799, Sigma-Aldrich). Eluted samples were precipitated using acetone. The pellet was dissolved in 1× SDS sample buffer and resolved by SDS–PAGE. The Bands were excised from the gel, and trypsin-digested samples were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Orbitrap Velos Pro (Thermo Fisher Scientific). The resulting LC-MS/MS dataset was analyzed using Mascot software (version 2.5.1, Matrix Science, Boston, MA, USA). The Mascot score indicates the probability that the observed match was a random event (a Mascot score > 100 indicates that the absolute probability is <1 × 10−10).

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tomoaki Tanaka

PROVIDER: PXD053516 | JPOST Repository | Mon Jun 30 00:00:00 BST 2025

REPOSITORIES: jPOST

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			Action	DRS
	orb_220530_OO-TT-56_%231-A.raw	Raw
	orb_220530_OO-TT-56_%231-B.raw	Raw
	orb_220530_OO-TT-56_%231-C.raw	Raw
	orb_220530_OO-TT-56_%231-D.raw	Raw
	orb_220530_OO-TT-56_%231-E.raw	Raw

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Project description:Human keratinocytes, HaCaT cells were transfected with the dsRNA-analogue polyinosinic-polycytidylic acid (poly I:C) (Sigma-Aldrich) using Lipofectamine 2000 reagent (Invitrogen) for intracellular delivery. The cells were transfected with 7 ug/ml poly I:C for 1 h or left untreated. The cells were collected in PBS with a cell scraper and washed once with PBS before they were lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The cell lysates were centrifuged 2,264 x g for 15 min at 4°C, divided into smaller fractions and centrifuged again 11,686 x g for 15 min at 4°C . The supernatant was collected and the protein content was measured with Bio-Rad DC protein assay (Bio-Rad). For the samples, 8 mg of protein was used. The proteins were precipitated with 10 % TCA/acetone at -20°C overnight. Samples were centrifuged 10,733 x g for 20 min at 4°C and the supernatant was removed. The precipitation was washed once with acetone and dissolved in urea buffer (8 M urea, 400 mM NH4CO3, 20 mM DL-dithiotheitol, 1 mM EDTA, pH 8.5). The proteins were reduced, alkylated, and enzymatically digested in-solution with lysyl endopeptidase (Wako Chemicals) and trypsin (Promega). The peptides were fractionated by strong cation exchange chromatography. Phosphopeptide enrichment was performed with immobilized-metal affinity chromatography (IMAC) using PHOS-Select Iron Affinity Gel (Sigma Aldrich). The enriched phosphopeptides were analyzed by nanoLC-MS/MS using an Ultimate 3000 nano-LC (Dionex) coupled to a QSTAR Elite hybrid quadrupole TOF-MS (Applied Biosystems/MDS Sciex) with nano-ESI ionization.23,24 The LC-MS/MS data were submitted through the ProteinPilot 4.0 interface (Applied Biosystems/MDS Sciex) to an in-house Mascot database search engine version 4.0 (Matrix Science), and to the ProteinPilot algorithm Paragon. The data were searched against the human canonical sequences in the Swiss-Prot database (decoy version 08132012 for the Mascot searches and version 04012013 with 539,829 sequences for the Paragon searches). The Mascot search criteria were: fixed modifications: carbamidomethyl of cysteine, variable modifications: oxidation (M), phospho (ST), phospho (Y), acetylation (K), 1 missed cleavage allowed, enzyme: trypsin. The Paragon search criteria were: modifications: cystein alkylation with iodoacetamide, ID focus: biological modifications, phosphorylation emphasis, identification threshold: 95 % confidence (Unused ProtScore (Conf) > 1.3), Thorough search (ID), enzyme: trypsin.

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TAS1440, an LSD1 inhibitor, activates TGF-β and NOTCH signaling in neuroendocrine small-cell lung cancer

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