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(a-c) Quantitative RT-PCR analysis of Nlrp3 (a) , Asc (b) , and Caspase-1 (c) mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (** P < 0.01, *** P < 0.001). (d) Immunostaining of gastrocnemius muscle sections 3 days after notexin injection using anti-NLRP3, anti-ASC, and anti-Caspase-1 antibodies on serial sections. The upper panels show control muscles, and the lower panels show notexin-treated muscles. Scale bars, 20 μm. Asterisks indicate calcified muscle fibers. (e) Immunofluorescence staining showing the localization of NLRP3 (green), ASC or Caspase-1 (red) around calcified muscle fibers in notexin-injected skeletal muscle. Nuclei are counterstained with DAPI (blue). Scale bar, 20 μm. Asterisks indicate calcified muscle fibers. (f, g) Quantitative RT-PCR analysis of Il-1β (f) <t>and</t> <t>Il-18</t> (g) mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (*** P < 0.001). (h) Serum IL-18 levels measured by ELISA in control and notexin-injected mice (n = 6 (control), n = 9 (notexin)). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (* P < 0.05). (i) Quantitative RT-PCR analysis of Runx2 mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (* P < 0.05). (j) Western blot analysis of Runx2 and tubulin (loading control) protein expression in skeletal muscle from control and notexin-injected mice.
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(a-c) Quantitative RT-PCR analysis of Nlrp3 (a) , Asc (b) , and Caspase-1 (c) mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (** P < 0.01, *** P < 0.001). (d) Immunostaining of gastrocnemius muscle sections 3 days after notexin injection using anti-NLRP3, anti-ASC, and anti-Caspase-1 antibodies on serial sections. The upper panels show control muscles, and the lower panels show notexin-treated muscles. Scale bars, 20 μm. Asterisks indicate calcified muscle fibers. (e) Immunofluorescence staining showing the localization of NLRP3 (green), ASC or Caspase-1 (red) around calcified muscle fibers in notexin-injected skeletal muscle. Nuclei are counterstained with DAPI (blue). Scale bar, 20 μm. Asterisks indicate calcified muscle fibers. (f, g) Quantitative RT-PCR analysis of Il-1β (f) and Il-18 (g) mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (*** P < 0.001). (h) Serum IL-18 levels measured by ELISA in control and notexin-injected mice (n = 6 (control), n = 9 (notexin)). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (* P < 0.05). (i) Quantitative RT-PCR analysis of Runx2 mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (* P < 0.05). (j) Western blot analysis of Runx2 and tubulin (loading control) protein expression in skeletal muscle from control and notexin-injected mice.

Journal: PLOS One

Article Title: In vivo mouse model of calcific myonecrosis induced by injury

doi: 10.1371/journal.pone.0346816

Figure Lengend Snippet: (a-c) Quantitative RT-PCR analysis of Nlrp3 (a) , Asc (b) , and Caspase-1 (c) mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (** P < 0.01, *** P < 0.001). (d) Immunostaining of gastrocnemius muscle sections 3 days after notexin injection using anti-NLRP3, anti-ASC, and anti-Caspase-1 antibodies on serial sections. The upper panels show control muscles, and the lower panels show notexin-treated muscles. Scale bars, 20 μm. Asterisks indicate calcified muscle fibers. (e) Immunofluorescence staining showing the localization of NLRP3 (green), ASC or Caspase-1 (red) around calcified muscle fibers in notexin-injected skeletal muscle. Nuclei are counterstained with DAPI (blue). Scale bar, 20 μm. Asterisks indicate calcified muscle fibers. (f, g) Quantitative RT-PCR analysis of Il-1β (f) and Il-18 (g) mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (*** P < 0.001). (h) Serum IL-18 levels measured by ELISA in control and notexin-injected mice (n = 6 (control), n = 9 (notexin)). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (* P < 0.05). (i) Quantitative RT-PCR analysis of Runx2 mRNA expression in skeletal muscle from control and notexin-injected mice (n = 3). Data are presented as mean ± S.D. Statistical significance was assessed using unpaired t-test (* P < 0.05). (j) Western blot analysis of Runx2 and tubulin (loading control) protein expression in skeletal muscle from control and notexin-injected mice.

Article Snippet: Serum IL-18 concentrations were measured using the Mouse IL-18 DuoSet ELISA Kit (DY7625-05; R&D Systems) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Control, Injection, Immunostaining, Muscles, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Western Blot