Journal: Frontiers in Neurology

Article Title: Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

doi: 10.3389/fneur.2018.00846

Figure Lengend Snippet: Messenger RNA levels in cultured CCL-136 cells treated for 24 h.

Article Snippet: Pools of three target-specific 19–25 nucleotide silencing RNAs (siRNAs), purchased from SantaCruz Biotechnology, were used: siRNA SLS5A3 (sc-44516), siRNA SLC6A12 (sc-95904), and siRNA AKR1B1 (sc-37119).

Techniques: Cell Culture

Journal: Frontiers in Neurology

Article Title: Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

doi: 10.3389/fneur.2018.00846

Figure Lengend Snippet: Messenger RNA levels in cultured normal human myotubes treated for 24 h.

Article Snippet: Pools of three target-specific 19–25 nucleotide silencing RNAs (siRNAs), purchased from SantaCruz Biotechnology, were used: siRNA SLS5A3 (sc-44516), siRNA SLC6A12 (sc-95904), and siRNA AKR1B1 (sc-37119).

Techniques: Cell Culture

Journal: Frontiers in Neurology

Article Title: Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

doi: 10.3389/fneur.2018.00846

Figure Lengend Snippet: Messenger RNA levels in cultured normal human myotubes treated with cytokines or added NaCl for up to 3 days.

Article Snippet: Pools of three target-specific 19–25 nucleotide silencing RNAs (siRNAs), purchased from SantaCruz Biotechnology, were used: siRNA SLS5A3 (sc-44516), siRNA SLC6A12 (sc-95904), and siRNA AKR1B1 (sc-37119).

Techniques: Cell Culture

Journal: Frontiers in Neurology

Article Title: Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

doi: 10.3389/fneur.2018.00846

Figure Lengend Snippet: Immunofluorescent cytostaining. (A) Staining with mouse anti-SLC6A12 (AlexaFluor 594, red) and rabbit anti-AKR1B1 (AlexaFluor488, green) in CCL-136 cells after 24 h treatment. Untreated control cells shows low levels of SLC6A12 and AKR1B1. Treatment with 30 ng/ml TNFα markedly increases AKR1B1 levels. Treatment with 300 u/ml IFNγ and 30 ng/ml TNFα strongly increases both SLC6A12 levels and AKR1B1 protein levels. Addition of 100 mM NaCl to the medium also increases both SLC6A12 and AKR1B1 protein expression. (B) Staining with rabbit anti-SLC6A12 (AlexaFluor 488, green) and goat anti-AKR1B1 (AlexaFluor594, red) in cultured healthy human myotubes at different time points. Myotubes treated with 300 u/ml IFNγ and 30 ng/ml TNFα display low levels of SLC6A12 that is increased at the 48 h time point. Levels return back to constitutive low levels after 72 h, with staining levels similar to those in untreated cells. Staining for AKR1B1 shows continuously high levels between 24 and 72 h. In untreated cells, AKR1B1 expression levels are substantially lower. Scale bar = 50 μm.

Article Snippet: Pools of three target-specific 19–25 nucleotide silencing RNAs (siRNAs), purchased from SantaCruz Biotechnology, were used: siRNA SLS5A3 (sc-44516), siRNA SLC6A12 (sc-95904), and siRNA AKR1B1 (sc-37119).

Techniques: Staining, Control, Expressing, Cell Culture

Journal: Frontiers in Neurology

Article Title: Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

doi: 10.3389/fneur.2018.00846

Figure Lengend Snippet: Western blotting of protein extracts preepared from cultured normal human myotubes. (A) SLC6A12 and AKR1B1 protein levels in myotubes treated with cytokine mixtures. SLC6A12 and AKR1B1 proteins are visualized in control cells (C) and in cells treated for 24 h-48 h-72 h with either 300 u/ml IFNγ+30 ng/ml TNFα, or 20 ng/ml IL1β+30 ng/ml TNFα. SLC6A12 is undetectable in untreated- and in 300 u/ml IFNγ+30 ng/ml TNFα-treated cells, but is induced by treatment with 20 ng/ml IL1β+30 ng/ml TNFα from the 48 h time point on. Low constitutive levels of AKR1B1 in control cells are increased in myotubes treated with both cytokine mixtures, peaking at the 48 h time point. Relative protein densities of AKR1B1, normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels as an internal standard, have been indicated. (B) SLC6A12 and AKR1B1 protein levels in myotubes treated with added NaCl. Protein bands for SLC6A12 and AKR1B1, and the internal standard glyceraldehyde 3-phosphate dehydrogenase (GAPDH), are given in an untreated control (C) and in cells treated with different concentrations of added NaCl. A time-dependent increase is observed in cells treated with 25 and 50 mM added NaCl, while more elevated NaCl concentrations show the highest levels at the 48 h timepoint. The corresponding relative protein densities are listed in Table .

Article Snippet: Pools of three target-specific 19–25 nucleotide silencing RNAs (siRNAs), purchased from SantaCruz Biotechnology, were used: siRNA SLS5A3 (sc-44516), siRNA SLC6A12 (sc-95904), and siRNA AKR1B1 (sc-37119).

Techniques: Western Blot, Cell Culture, Control

Journal: Frontiers in Neurology

Article Title: Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

doi: 10.3389/fneur.2018.00846

Figure Lengend Snippet: Protein densities in cultured normal human myotubes treated with added NaCl for varying periods of time.

Article Snippet: Pools of three target-specific 19–25 nucleotide silencing RNAs (siRNAs), purchased from SantaCruz Biotechnology, were used: siRNA SLS5A3 (sc-44516), siRNA SLC6A12 (sc-95904), and siRNA AKR1B1 (sc-37119).

Techniques: Cell Culture

Journal: Frontiers in Neurology

Article Title: Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

doi: 10.3389/fneur.2018.00846

Figure Lengend Snippet: Knockdown studies. (A) Hematoxylin&Eosin staining of CCL-136 and healthy human myotube cultures treated with silencing RNAs. Representative images are shown of cell cultures exposed to 300 u/ml IFNγ+20 ng/ml IL1β or to 50 mM of added NaCl. Culture densities decrease substantially in siSLC6A12- and siAKR1B1-treated cultures challenged with 50 mM of added NaCl, both in CCL-136 cells and in normal human myotubes. Addition of siSLC5A3 does not apparently inhibit cell growth, only resulting in mildly reduced densities of CCL-136 cells which could equally be observed in controls treated with 50 mM NaCl. Scale bar = 100 μm. (B) Western blots visualizing SLC6A12 and AKR1B1 protein in CCL-136 cells treated with silencing RNAs. The severe reduction of the AKR1B1 protein band in siAKR1B1-treated cells (arrow) shows the efficiency of expression knockdown. C represents the control sample, and equal loading is shown by β-actin protein bands.

Article Snippet: Pools of three target-specific 19–25 nucleotide silencing RNAs (siRNAs), purchased from SantaCruz Biotechnology, were used: siRNA SLS5A3 (sc-44516), siRNA SLC6A12 (sc-95904), and siRNA AKR1B1 (sc-37119).

Techniques: Knockdown, Staining, Western Blot, Expressing, Control

Journal: Frontiers in Neurology

Article Title: Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

doi: 10.3389/fneur.2018.00846

Figure Lengend Snippet: Immunofluorescent histostaining. Staining of SLC6A12 and AKR1B1 in muscle sections from patients diagnosed with inflammatory myopathy. Polymyositis (PM): A subset of CD68+ cells (AlexaFluor488, green), representing mostly M1 phenotype macrophages, express SLC5A3 and SLC6A12 (CY3, red). Dermatomyositis (DM): Two small regenerating muscle fibers (arrows), identified as strongly positive for CD56 (AlexaFluor488, green), express high levels of SLC6A12 protein (CY3, red). Scale bar =50 μm.

Article Snippet: Pools of three target-specific 19–25 nucleotide silencing RNAs (siRNAs), purchased from SantaCruz Biotechnology, were used: siRNA SLS5A3 (sc-44516), siRNA SLC6A12 (sc-95904), and siRNA AKR1B1 (sc-37119).

Techniques: Staining