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HEK293T LC-MS/MS

ABSTRACT: Leucine plays crucial roles in protein proteolytic autophagy process, but the underlying mechanisms are not fully understood. Here, we combined in vitro reconstitution with comparative proteomics technology to identify the potential regulators for leucine-mediated autophagy pathway. We established a in vitro cell-free systems to mimic the activation of autophagy by monitoring the translocation of readout protein ATG14 to the autophagosome membrane. Based on the system, we further screened the potential regulators involved in the leucine-mediated autophagy pathway using comparative proteomics strategy.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney

SUBMITTER: Guokai Yan

LAB HEAD: Xianghua Yan

PROVIDER: PXD007845 | Pride | 2022-03-02

REPOSITORIES: Pride

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Project description:Cancer-associated fibroblasts (CAFs) are acknowledged key determinants in the progression of cancer and thereby represent important targets for cancer therapies. Given the increase attention that ablative radiotherapy is gaining in the clinics, in this study we have aimed at identifying the transcriptional responses occurring in primary CAFs exposed to high-dose irradiation. Established primary cancer-associated fibroblasts (CAFs) obtained from non-small-cell lung cancer (NSCLC) patient material were irradiated with a single dose of 18 Gy and total RNA was isolated 24 hrs after treatment. Radiation-induced transcriptional alterations were investigated by gene expression analysis using genome-wide microarrays. Obtained results were verified by qRT-PCR of relevant genes. To confirm the data achieved by microarrays, diverse functional assays were performed including DNA damage response foci assay, measurements of reactive oxygen species (ROS) by flow cytometry and senescence-associated beta-galactosidase assays were applied. Irradiation resulted in differential expression of 680 genes of which 553 were up- and 127 down-regulated. 153 genes were differentially expressed with a fold-change greater than 1 and an adjusted p-value less than 0.05 across different comparisons (non-irradiated vs. irradiated). Expression patterns revealed profound changes in biological functions and processes involved in DNA repair, apoptosis, p53 pathway, autophagy, senescence, ROS production and immune response. Conclusions: CAFs display pro- and anti-tumorigenic effects after having received a single high-dose radiation. The effects might have an impact on the tumor microenvironment in respect to tumor growth and metastasis. The complexity of the observed differential gene expression patterns and implicative biological effects are interesting future objectives for further elucidation. Established primary cancer-associated fibroblasts (CAFs) obtained from non-small-cell lung cancer (NSCLC) patient material were irradiated with a single dose of 18 Gy and total RNA was isolated 24 hrs after treatment.

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HEK293T LC-MS/MS

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