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MASH human liver - secreted proteome

ABSTRACT: An approximate 1 cm3 wedge liver biopsy was collected from the left lobe of the liver during surgery. The liver was cut into two portions. One portion was placed in formalin and transported to TissuPathTM (Mount Waverley, Victoria), paraffin embedded and processed for histological analysis. Samples were graded according to the Clinical Research Network (CRN) NAFLD activity score (NAS) [36] and Kleiner classification of liver fibrosis [4] by TissuPath or Alfred Pathology. The main outcome was the diagnosis of NASH CRN where steatosis, inflammation, and ballooning scores are ≥1. The other portion of liver was placed in oxygenated Medium 199 media (M199; Gibco, USA) with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (P/S; Gibco, USA), embedded in 3% SeaPlaque agarose (Lonza BioScience, USA) using a Tissue Embedding Unit (Alabama Research and Development, USA). Embedded livers were then sliced on the Alabama R&D Tissue Slicer (Alabama Research and Development, USA) in 500 mL of oxygenated phenol red-free Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) with 1% P/S at 300 µm thickness, as previously described [24, 37, 38]. The liver slices were washed in Phosphate Buffered Saline (PBS; Gibco USA) and cultured in oxygenated M199 medium containing 1% P/S for 1 h. Subsequently, the liver slices were washed with PBS and cultured in 1 mL EX-CELL 325 protein free medium (Sigma-Aldrich, Australia) for 16 h at 37oC. The following day, the liver slices were weighed and snap-frozen in liquid nitrogen, while the incubation medium was collected and centrifuged at 300 x g at 4 ºC for 10 min. The supernatant was snap-frozen and stored at -80 ºC for subsequent proteomics analysis.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Liver

SUBMITTER: Benjamin Parker

LAB HEAD: Benjamin Parker

PROVIDER: PXD052787 | Pride | 2025-03-25

REPOSITORIES: Pride

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			Action	DRS
	20191016_QEHFX4_BenP_A1.raw	Raw
	20191016_QEHFX4_BenP_A10.raw	Raw
	20191016_QEHFX4_BenP_A11.raw	Raw
	20191016_QEHFX4_BenP_A12.raw	Raw
	20191016_QEHFX4_BenP_A2.raw	Raw

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Project description:Myotonic dystrophy type 1 (DM1, MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in DM1 patients resemble the appearance of a multisystem accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. Transcriptomic analysis of fibroblasts derived from DM1 patients and healthy individuals revealed a decrease in cell cycle activity, cell division, and DNA damage response in DM1, all of which were required for the accumulation of cellular senescence. Serial passage studies in vitro confirmed the accelerated increase in senescence and the acquisition of a senescence-associated secretory phenotype in DM1 fibroblasts. Moreover, functional studies highlighted the impact of BMI-1/p16INK4a pathway dysregulation in DM1-associated cellular phenotypes. Importantly, treatment with the senolytic compounds, quercetin, dasatinib, or navitoclax, reversed the accelerated aging phenotypes in both DM1 fibroblasts in vitro and in fruit flies in vivo. Our results identified the accumulation of senescence-related processes as a major driver of DM1 pathophysiology and therefore, demonstrated the efficacy of senolytic compounds in a pre-clinical setting. This approach warrants further assessment as a novel therapeutic strategy for DM1. Cell isolation and culture: For the isolation of primary fibroblasts from healthy donors and DM1 patients (age range, 34 to 71), punch skin biopsies were cut into 2–3 mm3 fragments and placed on a surface moistened with modified Eagle’s medium, containing 13% newborn calf serum, 0.4% penicillin/streptomycin (Gibco, Waltham, MA, USA) and 2 mM L-glutamine (Gibco). Flasks were incubated vertically for 3–6 h at 37ºC under 5% CO2 and then returned to the horizontal position. Human fibroblasts were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA), 1% L-glutamine (Gibco), and 1% penicillin–streptomycin (Gibco). Fibroblasts were tested regularly for mycoplasma contamination. Seven cultures from different DM1 patients and five from healthy controls were established. Transcriptomic study and data analysis: RNA was extracted from the fibroblasts from six DM1 patients and four age- and sex-matched controls at an early passage using TRIzol (Life Technologies, Carlsbad, CA, USA). Large-scale gene expression analysis was performed using the Human Clariom D microarray (Affymetrix, Santa Clara, CA, USA). RNA integrity was confirmed using an RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). Those samples with an RNA integrity value above 9 were used in the analysis. A total of 300 ng of RNA from each sample was used for microarray analysis, following the manufacturer's instructions. Microarray data analysis: The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using the log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab (MathWorks™). The GO terms were taken from the AMIGO gene ontology database. The significance of the GO terms of the DEGs was addressed calculating the P-values using an enrichment approach based on the hypergeometric distribution. All the sets of GO terms were back-propagated from the final term appearing in the gene annotation to the root term of each GO.

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MASH human liver - secreted proteome

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