Dataset Information

Ubiquitinylation in cancer - LATS1 but not LATS2 represses autophagy by a kinase-independent scaffold function

ABSTRACT: Samples were subjected to sonication using Bioruptor (program mode – 30 sec on, 30 sec off, 10 cycles = 10 min). Samples were sonicated till a clear solution was obtained. After a short spin (5,000 rpm for 10 sec), the samples were heated for 10 minutes at 95°C at 300 rpm in a PCR 96 heating block. Samples were allowed to cool down and spun at 5,000 rpm for 10 sec. 1μl of chloroacetamide was added to the eluate and incubated at 37°C for 30 minutes at 500 rpm. Later the samples were spun down at 5,000 rpm for 10 sec. Proteins were subjected to endoproteinase LysC (1:100 (w/w), Wako) digestion at 37°C for 4 hrs. Later, the proteins were subjected to trypsin digestion (0.5 μg/ μl; 1:50; w/w) at 37 °C overnight. Digestion was stopped by adding 50 μl of 5% TFA (Applied Biosystems) (v/v) that lowered the pH of the solution to below pH 2.0. Subsequently, peptides were cleaned up using the Phoenix 96x (https://preomics.com) kit following the manufacture’s instructions. After drying the peptides in a SpeedVac, samples were stored at -80°C.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Alexander Schmidt

LAB HEAD: Alexander Schmidt

PROVIDER: PXD013159 | Pride | 2020-01-13

REPOSITORIES: Pride

ACCESS DATA

Publications

LATS1 but not LATS2 represses autophagy by a kinase-independent scaffold function.

Tang Fengyuan F Gao Ruize R Jeevan-Raj Beena B Wyss Christof B CB Kalathur Ravi K R RKR Piscuoglio Salvatore S Ng Charlotte K Y CKY Hindupur Sravanth K SK Nuciforo Sandro S Dazert Eva E Bock Thomas T Song Shuang S Buechel David D Morini Marco F MF Hergovich Alexander A Matthias Patrick P Lim Dae-Sik DS Terracciano Luigi M LM Heim Markus H MH Hall Michael N MN Christofori Gerhard G

Nature communications 20191217 1

Autophagy perturbation represents an emerging therapeutic strategy in cancer. Although LATS1 and LATS2 kinases, core components of the mammalian Hippo pathway, have been shown to exert tumor suppressive activities, here we report a pro-survival role of LATS1 but not LATS2 in hepatocellular carcinoma (HCC) cells. Specifically, LATS1 restricts lethal autophagy in HCC cells induced by sorafenib, the standard of care for advanced HCC patients. Notably, autophagy regulation by LATS1 is independent of ...[more]

PMID: 31848340

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.

Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.

Project description:This series represents a group of five samples (from sigmoid colon biopsies of clinically normal Patients A-E) profiled by a publicly available cDNA-based platform (GPL284) and a commercial oligonucleotide-based platform (GPL91). Sample RNA extraction: Biopsies taken during endoscopic analysis were immediately snap-frozen in liquid nitrogen and at no time prior to or during subsequent sample manipulation were the samples allowed to thaw. Standard laboratory procedures to eliminate the presence of RNases, including the use of RNase free plastic-ware, heat baked glassware, molecular biology grade chemicals and DEPC-treated solutions, were adopted. Frozen biopsies were crushed to a fine powder under liquid nitrogen using a manual crusher with a teflon head (Omnilab) and total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturers protocol, with minor modifications that improved RNA yields and purity in our hands. In brief, crushed biopsy samples were lysed in an appropriate volume of Buffer RLT (typically 600 µL/20 mg tissue). To facilitate complete lysis, samples were allowed to stand at room temperature for 5-10 min before vortexing. The lysate was homogenized by addition to a QIA shredder column (Qiagen) and centrifuged at 14,000 rpm for 2 min. The resulting supernatant was centrifuged at 14,000 rpm for 3 min to pellet any cell debris. One volume of 70% ethanol was added to the cleared lysate, added to the RNeasy column and centrifuged at 10,000 rpm for 15 sec. Bound RNA was washed with the addition of RW1 buffer to the column and allowed to stand at room temperature for 10 min followed by centrifugation at 10,000 rpm for 15 sec. DNase 1 incubation mix (Stock: 273 kunits; RNase-free DNase Set, Qiagen) was added directly to the spin column membrane and digestion of any contaminating genomic DNA was carried out at room temperature for 30 min. The column was washed with RWI buffer and the DNase treatment step was repeated. The bound RNA was washed with RW1 buffer and centrifuged at 10,000 rpm for 15 sec three times, followed by two additional wash and centrifugation steps with RPE buffer. Finally, purified RNA was eluted off the column with RNase-free water. RNA concentration and purity was determined spectrophotometrically at A260/A280 and was determined to be intact as assessed by formaldehyde gel electrophoresis. The presence of genomic DNA contamination was assessed by PCR amplification of GAPDH using standard PCR conditions and the following primers: GAPDH_F2-5’- ACCCACTCCTCCACCTTTGAC-3’, GAPDH_R2- 5’-CTGTTGCTGTAGCCAAATTCGT-3’. RNA was re-treated with DNase if necessary. Upon confirming the quality of the RNA (intact and free of genomic DNA), 15 µg of total RNA from each of the five biological replicates was used for expression screening on either the Affymetrix or the clone-based filter platform. Keywords: parallel sample

Project description:Approximately 2x106 CL1-5 and H23 cells were seeded on 10-cm dishes with RPMI-1640 medium containing 10% fetal bovine serum. After the cells covered 80% of the dish, cells were serum-starved for 24 h in RPMI-1640 medium. CL1-5 and H23 cell lysates were harvested, trypsinized and analyzed by using LTQ-Orbitrap XL and LTQ-Orbitrap Discovery, respectively. Halobacterium salinarum NRC-1 (ATCC700922) starter culture was prepared by inoculating a single colony in 5 mL of Halobacterium medium containing trace metals (CM+) and grown at 37 °C with shaking at 225 rpm for one week. An aliquot of 300 μL of the culture was spread on 15-cm diameter CM+ plates containing 2% of agarose. After 7~10 days of incubation at 37 °C, the GVs from cells on 10 plates were harvested using the centrifugation facilitated flotation method as previously described except the GVs were washed for a total of 5 times instead of the suggested 3 times to remove nonspecific bound proteins.(29) After collecting the gas vesicles in the primary cell lysate, the cell membrane and residual GV were removed by ultracentrifugation at 53 000× g, 4 °C for 16 h twice to obtain the GV-depleted lysate (GVD). GVD lysate was harvested, trypsinized and analyzed by using LC-MS/MS ( LTQ-Orbitrap XL). The H. salinarum strain NRC-1 was grown in complex medium (CM+) at 37 oC with shaking (200 rpm) to an OD600 of 0.5 (early exponential phase. The cultures were then subjected to an additional hour of incubation at 37 oC for preparation of proteins. The total lysate was harvested, trypsinized and analyzed by using LC-MS/MS (LTQ-Orbitrap Discovery). All MS raw data in Thermo XCalibur (version 2.0.7) binary format were converted to the mzXML open data format using a modified version of the ReAdW program. SEQUEST (version 27) was applied to search the MS/MS spectra against the forward and reverse (decoy) protein sequences of H. salinarum (2,427 proteins; EMBL-EBI Integr8, www.ebi.ac.uk/integr8/EBI-Integr8-HomePage.do) or human (38,425 proteins) plus bovine (22,863 proteins) (GenBank reference sequences, www.ncbi.nlm.nih.gov). SEQUEST results were further processed using the Trans Proteomic Pipeline.

Dataset Information

Ubiquitinylation in cancer - LATS1 but not LATS2 represses autophagy by a kinase-independent scaffold function

Publications

LATS1 but not LATS2 represses autophagy by a kinase-independent scaffold function.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure