Dataset Information

Proteome of senescent human lung fibroblasts and Panc-1 cells

ABSTRACT: Proteomic analysis of proliferating and senescent normal healthy lung fibroblasts (NHLFs) and Panc-1 cells. Senescence was induced using 5 uM Palbociclib for 6 days. Cells were lysed in SDS lysis buffer (2% SDS in 100 mM TEAB), boiled (15 min, 95 °C, 500 rpm) and sonicated in a sonication bath (10 min). DNA was sheared with Benzonase® Nuclease (Merck Millipore) and 2 mM MgCl2 at 37 °C (30 min, 500 rpm). Reversibly oxidized cysteines were reduced with 10 mM TCEP followed by alkylation with 20 mM IAA (each step 30 min, 22 °C, 600 rpm, in the dark). Proteins were acetone precipitated and the protein pellet was resuspended in 100 µL 100 mM TEAB. Proteins were digested overnight with LysC/trypsin (1:50 protease:protein ratio, 37 °C, 600 rpm) Peptides were desalted by solid-phase extraction using Oasis HLB cartridges (10 mg; Waters), resuspended in 3% ACN/0.1% TFA and analysed on an Orbitrap Eclipse Tribrid mass spectrometer in combination with the FAIMS Pro Interface (Thermo Scientific).

INSTRUMENT(S): Orbitrap Eclipse

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

SUBMITTER: Karim C. El Kasmi

PROVIDER: MSV000090607 | MassIVE | Fri Oct 28 04:46:00 BST 2022

SECONDARY ACCESSION(S): PXD037794

REPOSITORIES: MassIVE

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Publications

Senescent cells suppress macrophage-mediated corpse removal via upregulation of the CD47-QPCT/L axis.

Schloesser Daniela D Lindenthal Laura L Sauer Julia J Chung Kyoung-Jin KJ Chavakis Triantafyllos T Griesser Eva E Baskaran Praveen P Maier-Habelsberger Ulrike U Fundel-Clemens Katrin K Schlotthauer Ines I Watson Carolin Kirsten CK Swee Lee Kim LK Igney Frederik F Park John Edward JE Huber-Lang Markus S MS Thomas Matthew-James MJ El Kasmi Karim Christian KC Murray Peter J PJ

The Journal of cell biology 20221202 2

Progressive accrual of senescent cells in aging and chronic diseases is associated with detrimental effects in tissue homeostasis. We found that senescent fibroblasts and epithelia were not only refractory to macrophage-mediated engulfment and removal, but they also paralyzed the ability of macrophages to remove bystander apoptotic corpses. Senescent cell-mediated efferocytosis suppression (SCES) was independent of the senescence-associated secretory phenotype (SASP) but instead required direct ...[more]

PMID: 36459066

Similar Datasets

Project description:2×10^7 HL60 cells were treated with 10 uM ATRA for 2 and 5 days, and conducted Hi-C to study the chromosomal architecture. Cells were fixed by 1% formaldehyde (v/v) for 15 min at room temperature (RT) with slowly rotation. Cells were lysed by 550 μl lysis buffer (500 μl 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 and 50 μl protease inhibitors) using a homogenizer, and harvested the chromatin at 5,000 rpm and washed twice. The pellet was added 1% SDS and incubated at 65 oC for 10 min, then quenched by Triton X-100. The chromatin was digested by 600 U MboI (NEB, Ipswich, USA) at 37 oC overnight. The next day, enzymes were inactivated by 10% SDS at 65 oC for 30 min, and the restriction fragment overhangs were filled in using biotin-14-dCTP and Klenow at 37 oC for 45 min, and the ligation was conducted in 7.61 ml ligation mix (745 μl 10% Triton X-100, 745 μl 10x ligation buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT), 80 μl 10 mg/ml BSA, 80 μl 100 mM ATP and 5.96 ml water) for 4 h at 16°C. The blunt-end Hi-C ligation was sonicated and pull down by streptavidin beads, then purified by ethanol. Hi-C library was prepared for paired ending sequencing using NEBNext® UltraTM DNA Library Prep Kit for Illuina® (NEB). For regular 3C ligation, after enzymes inactivation, 10 μl 1 U/μl T4 DNA ligase was added to incubate at 16°C overnight. The interaction was detected by qPCR using the specific 3C primers (Table 1) followed by DNA purification. Hi-C was conducted in PE-150. An iterative method for Hi-C mapping with initial length 30bp, if it fails to map uniquely, the next round of mapping for additional 20 bp continued, this procedure lasts until the full read length reaches 150bp. The Hi-C reads were mapped to the human reference genome (assembly GRCh38) using bowtie2 (v2.2.9). The reads mapped to the same restriction fragment, the reads less than 500bp and PCR duplicates were all removed. Hi-C contact maps were normalized using ICE.