Bars represent mean values SD ( em n /em ?=?4). MstnPP metabolites were predominantly retained within the endoplasmic reticulum (ER), also evident in sIBM histology. MstnPP cleavage Tripelennamine hydrochloride products formed insoluble high molecular weight aggregates, a process that was aggravated by experimental ER stress. Importantly, ER stress also impaired secretion of mature myostatin. Reduced secretion and aggregation of MstnPP metabolites were not simply caused by overexpression, as both events were also observed in wildtype cells under ER stress. It is tempting to speculate that reduced circulating myostatin growth factor could be one explanation for the poor clinical efficacy of drugs targeting the myostatin pathway in sIBM. Electronic supplementary material The online version of this article (10.1007/s12035-018-0997-9) contains supplementary material, which is available to authorized users. oxidase, and myophosphorylase stainings. Stainings were conducted by standard protocols. Immunohistochemistry, with antibodies against major histocompatibility complex I (MHC-I; 1:1000; W6/32; DAKO), membrane attack complex of complement (MAC, C5b9; 1:100; aE11; DAKO), CD3 (1:50; T3-4B5; DAKO), and CD 68 (1:80; EBM11; DAKO), were included whenever an inflammatory myopathy was clinically suspected or suggested by the standard histology listed above. The respective diagnosis was based on established histological criteria. The control samples were from patients without specific myopathologic changes (e.g., suspected mitochondrial cytopathy cases) or with nonspecific muscular complaints (typically muscle pain or stiffness). Control patients were ultimately declared free of muscle disease. Chronic neurogenic conditions were diagnosed based on fiber type grouping, grouped atrophy, and a bimodal fiber size distribution without major inflammatory or structural pathology as encountered in sIBM. All sIBM samples showed the canonical pathological features [39], i.e., inflammatory myopathy with partial invasion of non-necrotic fibers, rimmed vacuoles, and intracellular congophilic deposits. Antibodies and Chemicals Antibodies were from the following companies; mouse mAb anti-myostatin (MstnPP) (6H12) (Abcam & ThermoFischer Scientific); goat pAb anti-human myostatin (amino acid residues 268C376) (R & D systems); mouse mAb anti-APP 6E10 against A epitope RHDSGYE (BioLegend); mouse mAb anti-APP 22C11 against the aminoterminal residues 66C81 (Merck Millipore); rabbit pAb anti-Giantin ab24586 (Abcam); rabbit pAb anti-LC3B NB100-2220 (Novus biological); rabbit pAb anti-Lamp1 ab24170 (Abcam); rabbit pAb anti-GRP-78 H-129 (Santa Cruz); rabbit pAb anti-GFP A-6455 (ThermoFischer Scientific); rabbit mAb anti-Calreticulin ERP3924 (Merck Millipore); rabbit pAb anti-Calnexin C4731 (Sigma); rabbit pAb anti-Ubiquitin Z0458 (DAKO); mouse mAb anti-Actin (MP Biomedicals); Alexa Fluor-conjugated secondary antibodies (Molecular Probes); HRP-coupled secondary goat antibodies (Dianova). Chemicals were purchased from Sigma or Roth. Histological Examination of Muscle Biopsies Cryostat sections of patient material were studied immunohistochemically according to routine diagnostic techniques. Briefly, 7?m thick transverse cryosections were transferred onto silaned glass slides, air-dried and fixed in 4% paraformaldehyde for 10?min at RT. Serial sections to those stained for immunohistochemistry were stained with hematoxylin-eosin and modified Gomori trichrome [40] to identify fibers with rimmed vacuoles. Images were captured using ?20C40 objectives and a Nikon H800 microscope (Nikon, Germany) with a SPOT FLEX 64 Mp Shifting Pixel CCD-camera (Visitron Systems GmbH) Tripelennamine hydrochloride and SPOT software (version 4.6, Visitron Systems). Confocal Microscopy of Muscle Biopsies Cryosections were fixed in 4% paraformaldehyde in PBS for 10?min at room temperature (RT). Unspecific binding was blocked with 5% BSA and 10% horse serum in phosphate Tripelennamine hydrochloride buffered saline (PBS) for 30?min at RT. Muscle tissue was incubated with anti-Mstn 6H12, anti-APP 6E10 or 22C11, or anti-Calreticulin antibodies overnight at 4?C. Samples were rinsed extensively with PBS and incubated with secondary antibodies for 60?min at RT. After additional washing with PBS, nuclei were counterstained with bis-benzimide (1:10,000 in PBS 0.5?g/ml; Sigma-Aldrich) for 2?min Tripelennamine hydrochloride at RT. Specimen were mounted in a Mowiol 4C88 (Calbiochem, Merck Chemicals) and glycerol mix in pH?8.5 Tris buffer with 0.1% DABCO (1,4-Diazabicyclo (2,2,2) octane; Sigma-Aldrich). Confocal laser scanning microscopy was carried out using 40 oil lenses and an LSM 700 laser-scanning microscope (Zeiss). Cross-reactivity of secondary antibodies was excluded by control stainings EIF4EBP1 without primary antibodies (not shown). Single optical planes are shown. Cell Lines The human rhabdomyosarcoma cell line CCL 136 (American Type Culture Collection, Rockville, MD, USA) was used for all experiments. Cells were maintained in DMEM with GlutaMAX (Gibco) supplemented with 10% fetal calf serum (FCS) (Biochrom) and.