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Human skeletal muscle transcriptome (39 independent analyses)

ABSTRACT: The annotation of the Affymetrix HTA 2.0 array was updated to optimise the detection of RNA in human skeletal muscle samples by removing invalid and low signal-high-variance probes Human primary skeletal muscle cells were isolated from muscle biopsies from healthy adults as previously described (Crossland et al. 2016; Crossland et al. 2017). Myogenic cell enrichment used magnetic-activated cell sorting (MACS) and anti-CD56 microbeads (130-050-401; Miltenyi Biotec). Myogenic purity was assessed through measurement of fractional desmin positivity (Crossland et al. 2016; Crossland et al. 2017). Cells were washed in PBS and fixed in ice-cold 1:1 acetone/methanol, before being blocked in 5% (v/v) goat serum for 30 min at room temperature. Cells were subsequently incubated with rabbit anti-desmin monoclonal antibody (ab32362; Abcam) for 1h at room temperature, washed, and incubated with anti-rabbit TRITC-conjugated secondary antibody as previously detailed (Crossland et al. 2016; Crossland et al. 2017). Finally, cells were washed in PBS and mounted/DAPI-stained using FluoroshieldTM mounting medium with DAPI. Myoblasts at passage 5-6 were cultured on Collagen Type I-coated 6-well dishes in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Life Technologies) containing 20% (v/v) fetal bovine serum (FBS, Sigma-Aldrich), 1% (v/v) antibiotic-antimycotic (AbAm) solution and 4mM L-glutamine (Life Technologies). Cells were maintained at 37oC with 5% CO2. Once cells reached ~95% confluency, differentiation was induced by switching the medium to DMEM/F-12 containing 2% (v/v) horse serum (Sigma-Aldrich), 4mM L-glutamine and 1% (v/v) AbAm solution (Life Technologies). Six days following the initiation of differentiation, a media change was carried out, using differentiation media supplemented with long R3 IGF-1 (10 ng/ml; Sigma-Aldrich), with or without 100 nM rapamycin (Sigma-Aldrich). DMSO (final concentration 0.01% (v/v)) was used as a vehicle control for rapamycin. Samples were collected at 0h baseline and following 4h and 24h treatment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE130789 | GEO | 2019/08/01

REPOSITORIES: GEO

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Project description:CD47 is a transmembrane glycoprotein that is ubiquitously expressed in different organs and tissues (Barclay and Van den Berg 2014; Liu, et al. 2017). In the human immune system, CD47 interacts with some integrins, two counter-receptor signal regulator protein (SIRP) family members, and the secreted thrombospondin-1 (TSP1) (Barclay and Van den Berg 2014; Gao, et al. 2016; Kaur, et al. 2013; Oldenborg, et al. 2000). CD47 has two established roles in the immune system. The CD47-SIRPα interaction was identified as a critical innate immune checkpoint, which delivers an antiphagocytic signal to macrophages and inhibits neutrophil cytotoxicity (Martínez- Sanz, et al. 2021). Its interaction with inhibitory SIRPα is a physiological anti-phagocytic “don’t eat me” signal on circulating red blood cells that is co-opted by cancer cells (Matlung, et al. 2017). Many malignant cells overexpress CD47 (Betancur, et al. 2017; Chao, et al. 2011; Jaiswal, et al. 2009; Majeti, et al. 2009; Oronsky, et al. 2020; Petrova, et al. 2017). CD47/SIRPα-targeted therapeutics have been developed to overcome this immune checkpoint for cancer treatment (Kaur, et al. 2020; Matlung, et al. 2017). Secondly, engagement of CD47 on T cells by TSP1 regulates their differentiation and survival (Grimbert, et al. 2006; Lamy, et al. 2007) and inhibits T cell receptor signaling and antigen presentation by dendritic cells (DCs) (Kaur, et al. 2014; Li, et al. 2002; Liu, et al. 2015; Miller, et al. 2013; Soto-Pantoja, et al. 2014; Weng, et al. 2014). TSP1/CD47 signaling has similar inhibitory functions to limit NK cell activation (Kim, et al. 2008; Nath, et al. 2018; Nath, et al. 2019; Schwartz, et al. 2019) and IL1β production by macrophages (Stein, et al. 2016). CD47 is therefore a checkpoint that regulates both innate and adaptive immunity. The recent understanding of CD47 antagonism associated with increased antigen presentation by DCs (Liu, et al. 2016) and natural killer cell cytotoxicity (Nath, et al. 2019) contributes to the heightened interest in CD47 as a therapeutic target (Kaur, et al. 2020).

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Human skeletal muscle transcriptome (39 independent analyses)

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