Project description:The cytoplasmic actin proteins, beta- and gamma-actin, are 99% identical but perform non-redundant functions. Genes encoding the cytoplasmic actins, Actb and Actg1, respectively, are less similar but still share 89% of their nucleotide sequences. Knockout (KO) of Actb by deletion of first coding exons 2 and 3 in mice is embryonic lethal while KO embryonic fibroblasts (MEFs) fail to proliferate. In contrast, KO of Actg1 is viable but mice lacking Actg1 present with increased perinatal lethality and Actg1 KO MEFs present with a much milder defect in cell proliferation. Recent studies have identified important protein-independent functions for both Actb and Actg1 and demonstrate that deletions within the Actb nucleotide sequence, and not loss of the beta-actin protein, cause the most severe phenotypes in KO mice and cells. Here, we use a multi-omics approach to better understand what drives the phenotypes of Actb KO MEFs. RNA-sequencing and mass spectrometry of Actb KO MEFs reveal largescale changes to the transcriptome, proteome, and phosphoproteome. Pathway analysis of genes and proteins differentially expressed upon Actb KO shows widespread dysregulation of genes involved in the cell cycle. Together, these data suggest novel, protein-independent roles for Actb in regulating gene expression associated with control of cell proliferation.
Project description:RNA-Sequencing data of patient derived normal fibroblasts (NFs), cancer associated fibroblasts (CAFs) and tumor spheroid samples performed in the context of the characterization of an IL1R1 positive CAF population in CRC tumour samples.
Project description:The anterior silk gland in the silkworm plays an important role in the process of liquid fibroin to solid silk fiber .In view of this,the proteomics analysis was applied to to study the relationship between the function of proteins in the anterior silk gland and the mechanism of spinning. The anterior silk glands on the 3rd day of fifth instar were dissected.Aftter 1D SDS-PAGE ,one gel lane was cut into 10 bands and each band further sliced into small pieces was subjected to in-gel tryptic digestion for 20 hours.The digested peptides were separated by RP nanoscale capillary liquid chromatography and analyzed using a surveyor LC system (Thermo Figgigan, San Jose, CA).The eluate from the RP column was analyzed by Finnigan LTQ(Thermo Electron Corporation)linear ion trap Mass equipped with a nanospray souce in the positive ion mode. The MS analysis was performed with one full MS scan followed by three MS/MS scans on the most intense ions from the MS spectrum with the dynamic exclusion settings: repeat count 2, repeat duration 30s, exclusion duration 90s. Data were acquired in data-dependent mode using Xcalibur software.Ten raw datasets from LC-MS/MS were searched against the predicted silkworm database by Xia.et al which consists of 21312 silkworm proteins.The searching was carried out with the Turbo SEQUEST(Bioworks version 3.2, Thermo Electron).
Project description:Transformed AC16 cells were acquired from Millipore and used at P10-15 and expanded in DMEM/F-12 with 10% FBS (Gibco). Primary human ventricular cardiac fibroblasts were acquired from Promocell and expanded in FGM-3 (Promocell) and used at P3-5. For the experiment, cells were separately placed on trans-wells/inserts (0.4 µm pore size, Greiner # 657641) at a density of 3,500 – 3,560 cells/cm2 for AC16 cells and plated in normal six-well tissue-culture coated plates at a density of 3,350 – 3,550 cells/cm2 for human ventricular cardiac fibroblasts. The cells are separately cultured in fibroblast growth medium FGM-3 (Promocell), which was tested in previous experiments to support the growth of AC16 cells for at least 48 hours. Once the AC16 cells achieved 40% confluency a 0.1 µM doxorubicin and vehicle FGM-3 media were prepared to replace the media from four six-well plates with transwell inserts. Once the primary human fibroblasts achieved >10% confluence, 5ng/mL of TGF-β1 and vehicle FGM-3 media were prepared and used to replace the media from six separate 6-well-plates. All cells were cultured at 37°C in 5% CO2. To initiate co-culture, the media in the AC16 cells were removed then the cells were washed with PBS twice; 1.5mL of FGM-3 was added to each trans-well. The media for the six fibroblast plates were changed with fresh corresponding TGF-β1 or vehicle treated FGM-3 media. The transwells were nested into each well of the fibroblast plates following the experimental design. Primary cardiac ventricular fibroblasts were lysed using RIPA buffer and Protease Inhibitor (Pierce Halt) followed by sonication (Bioruptor) to solubilize proteins. Protein concentration in each sample was measured using BCA assay (Thermo) against a standard curve of BSA. For mass spectrometry, proteins were digested using a filter-assisted sample preparation protoco: 25 µg protein from each sample was added to 10 kDa MWCO 0.5 mL volume PES protein concentrator (Thermo Scientific), washed with 250 µL of 8 M urea centrifuged (10 min, 22°C, 14,000 g) 2x to remove detergent, then exchanged to 300 µL 100 mM TEAB 2x followed by centrifuged (10 min, 22°C, 14,000 g), reduced with 10 mM TCEP and alkylated with 40 mM CAA, digested with sequencing grade trypsin (Promega) O/N at 37 °C. Peptides were labeled with TMT 10-plex tags for 1 hr, followed by quenching with 1 µL 5% hydroxylamine. The samples were dried, desalted with C18 clean-up columns (Thermo), then fractionated using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo) following manufacturer’s protocol. The samples were analyzed on a Q-Exactive HF Orbitrap mass spectrometer coupled to an Easy-nLC 1200 liquid chromatograph (Thermo) in data-dependent acquisition mode using conventional settings. Raw spectra were converted to mzML format using ThermoRawFileParser, then searched using Comet v.2022_01 against the UniProt SwissProt human protein sequence canonical + isoform database appended with common contaminant protein sequences (retrieved 2023-03-22 using Philosopher; 42,435 forward entries). Post-processing and calculation of false discovery rate of identification was performed using Percolator (crux v.4.1 distribution) [8], with 1% FDR accepted for ID. Spectrum TMT intensity was extracted using pyTMT v.0.4.1.
Project description:Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the reprogramming of human patient’s fibroblasts into iPSCs can be enhanced to an unprecedented efficiency via the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNAs-367/302s delivered as mature miRNA mimics. This synergism is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to primary human fibroblasts. As a result, we have developed a novel, integration- and feeder-free reprogramming approach that generates up to 4,019 TRA-1-60 positive colonies from only 500 starting human primary neonatal fibroblasts and reprograms up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant iPSCs from a variety of human patient’s fibroblasts and opens up new horizons for studying the biology of reprogramming.