HPASMCs (ScienCell Research Laboratories, California, USA) were isolated from human pulmonary arteries  and cultured in the smooth muscle cell (SMC) medium (ScienCell Research Laboratories, Carlsbad, USA) supplemented with 2% fetal bovine serum and 1% SMC growth supplement. This study was approved by the Beijing Hospital Ethics Committee (BHEC) with the registration No. of 2017BJYYEC-108-05. Hela cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 4.5 mg/ml glucose. All the cells were incubated at 37°C in a humidified normoxia condition (21% O2, 5% CO2 and 74% N2).
Construction of plasmids and DNA transfection.
To construct the expression plasmids for LETM-Flag, a DNA fragment containing LETM1was inserted into pCDNA3.1-Flag-c. LETM1C552A mutant was constructed by PCR using the primers (Letm1C552A-F: 5’-CCTCAGCGATGCCGCCTCTAAGCTG-3’ and Letm1C552A-R: 5’-GCGGCATCGCTGAGGATGTCGATTTC-3’) and cloned into pCDNA3.1-Flag-c. LETM1 -Flag and LETM1C552A-Flag were transiently expressed in Hela cells and DNA transfection was performed using lipo3000 (Invitrogen) according to the manufacturer’s instructions.
Antibodies and reagents.
The antibodies used in the present study were as follows:
rabbit anti-LETM1 (dilution 1:2,000; cat. no. 16024-1-AP), rabbit anti-THEM4 (dilution 1:2,000; cat. no. 14692-1-AP), mouse anti-GAPDH (dilution 1:2,000; cat. no. 60004-1-Ig), rabbit anti-Flag (dilution 1:2,000; cat. no. 20543-1-AP), mouse anti-AKT (dilution 1:2,000; cat. no. 60203-1-Ig), mouse anti-pS473-AKT (dilution 1:2000; cat. no. 66444-1-Ig). All of the antibodies were purchased from Protein Tech Group, Inc.
The reagents used in the present study were as follows:
protease inhibitor cocktail (Roche Group), protein (A/G) ultraLink resin (Thermo Fisher Scientific, Inc.), azide agarose beads (Nanocs), thiopropyl sepharose 6B (Sigma‑Aldrich; Merck KGaA), hydroxylamine (Sigma‑Aldrich; Merck KGaA), N‑ethylmaleimide (NEM; Sigma‑Aldrich; Merck KGaA), Tris (2‑carboxyethyl)phosphine hydrochloride (TCEP; Sigma‑Aldrich; Merck KGaA), Tris [(1‑benzyl ‑1H‑1,2,3‑triazol‑4‑yl)methyl]amine (TBTA; Sigma‑Aldrich; Merck KGaA), 8-Br-cGMP (Sigma‑Aldrich; Merck KGaA).
Immunoprecipitation (IP) and western blot analysis.
HPASMCs were harvested and lysed (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail) on ice for 30 min. IP and western blots were performed as previously described .
Protein Spalmitoylation assay in cells.
The HPASMCs were treated with 8-Br-cGMP (1mM) or H2O (control). Hela cells were transfected with pCDNA3.1-LETM1-Flag and pCDNA3.1-LETM1C552A-Flag. After 24 h, both the HPASMCs and Hela cells were harvested for S-palmitoylation assay. The S-palmitoylation assay was performed as previously described  with minor modifications. In brief, the cells were washed in PBS and lysed in lysis buffer [10 mM sodium phosphate, 2 mM Na2-EDTA, 0.32 M sucrose, 1% Triton X-100, protease inhibitor cocktail; pH 7.4] on ice for 30 min. Then, the lysates were added with N-ethylmaleimide (50 mM) and immunoprecipitated at 4˚C overnight utilizing protein A/G resin preloaded with LETM1 or Flag antibody. Next day, the protein A/G resin was washed three times in lysis buffer and incubated with elution buffer (1% SDS, 10 mM sodium phosphate, 2 mM Na2-EDTA, 0.32 M sucrose) at 50˚C for 5 min to release LETM1, LETM1-Flag or LETM1C552A-Flag. Eluted samples were divided into two equal portions. One was treated with 1 M hydroxylamine (pH 7.4) and thiopropyl sepharose 6B and the other was treated with 1M Tris·HCl (pH 7.4) and thiopropyl sepharose 6B as the negative control. After a 2-h incubation at room temperature, sepharose beads were washed three times with washing buffer (10 mM sodium phosphate, 2 mM Na2-EDTA, 0.32 M sucrose, 1% Triton X-100, 500 mM NaCl and 0.2% SDS). Western blots was performed to determine the presence of LETM1, LETM1-Flag or LETM1C552A-Flag.
Synthesis of palmitate probe, metabolic labeling and click chemistry reaction.
The ω-alkynyl-palmitate acid analogue, Alk-C16, was synthesized as previously described  and dissolved in DMSO to generate a 50-mM stock solution and stored at 80˚C. Before cell treatment, AlkC16 was dissolved in SMC medium supplemented with 5% BSA (fattyacid free) and 1% SMC growth supplement at a final concentration of 100 µM, and then sonicated for 15 min at room temperature. In addition, an equal volume of DMSO was added to the same medium as a negative control. Then, the seeded HPASMCs were incubated in the above medium containing Alk-C16 or DMSO respectively for 24 h at 37˚C with 5% CO2. Then, the cells were harvested and click chemistry reaction were performed as previously described . In brief, the cells were lysed in 500 µl lysis buffer (1% Nonidet P-40, 150 mM NaCl, 100 mM sodium phosphate and protease inhibitor cocktail; pH 7.5) for 30 min at 4˚C. Then, the protein extracts were subjected to the click-chemistry reaction for 1 h at room temperature by the addition of the following reagents in order: 1 mM azide agarose beads, 1 mM TCEP dissolved in water, 0.2 mM TBTA dissolved in DMSO/tert-butanol (20/80% v/v) and 1 mM CuSO4 in PBS. Following the reaction, the Alk-C16-conjugated proteins were bound to the azide agarose beads. The beads were washed three times with lysis buffer at room temperature. Then, the proteins bound to the beads were digested for MS analysis.
NanoLCMS/MS analysis, database searching, data processing and statistical analysis were performed as previously described[17, 19]. The steps were as follows.
An Easy nLC1000 (Thermo Fisher Scientific, Inc.) system combined with the LTQ-Orbitrap-Elite (Thermo Fisher Scientific, Inc.) mass spectrometer were applied for proteomic profiling.
The recorded MS spectra was analyzed by utilizing MaxQuant Software (version 184.108.40.206; https://www.Maxquant.org/). The MS/MS peak list analysis was performed by searching against a forward and reverse version of the UniProtKB/Swiss-Prot human database (generated from version 2017_05; human taxonomy; 20,316 entries; http://www.uniprot.org/). The cutoff of the false discovery rate for peptide and protein identification was set to 0.01, and only peptides with ≥7 amino acidic residues were analyzed. Label-free quantitation (LFQ) was performed using the MaxQuant software on the identified razor and unique peptides in order to quantify the identified proteins.
Protein abundances normalized by the LFQ algorithm integrated in MaxQuant were log2-transformed for further analysis. The filtering steps were performed using Microsoft Excel 2010. DanteR (version 220.127.116.11) and Perseus (version 18.104.22.168) were used to perform various types of statistical analysis including log2 transformation, correlation plot, statistical tests and p-value adjustments.
The functional enrichment analysis of the identified palmitoylated proteins were performed by Metascape web-based platform . Enriched terms with a p-value < 0.01, a minimum count of 3, and an enrichment factor > 1.5 were collected and grouped into clusters based on their membership similarities. "Log10 (P)" is the p-value in log base 10. Protein-protein interaction (PPI) network construction was performed by STRING database (Search Tool for the Retrieval of Interacting Genes/Proteins, http://string-db.org/) .