Journal: Nature medicine

Article Title: Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division

doi: 10.1038/nm.3990

Figure Lengend Snippet: Dystrophin regulates PAR polarity protein localization. ( a ) Immunostaining for Dmd C-terminal (green), Dag1 (red), itga7 (cyan) and DAPI (blue) of cultured myofiber at 36 h. ( b ) Quantification of Dmd expression (rod domain) and localization in satellite cells of cultured myofibers of WT mice at 0, 12, 24, and 36 h. Only undivided cells were quantified. n = 3 mice, approx. 50 cells per mice. ( c ) Representative micrograph ( n = 10 micrographs per condition) of proximity ligation assay (PLA) for Dmd (Dy4/6D3 clone) and Mark2 (red) and for ( d ) Dmd and Pard3 (red), along with immunostaining for itga7 (green) and DAPI (blue) on cultured myofibers from WT and mdx mice at 36 h. n = 3 mice. ( e ) Representative micrograph ( n = 10 micrographs per condition) of proximity ligation assay (PLA) between Dag1 and Mark2 (red), and between ( f ) Dag1 and Pard3 (red), along with immunostaining for itga7 (green) and DAPI (blue) on cultured myofibers from WT and mdx mice at 36 h. n = 3 mice. ( g ) Example ( n = 10 micrographs per condition) of polarity protein distribution from immunostaining for Mark2 (green), Pard3 (red), itga7 (white) and DAPI (blue) of cultured myofiber from WT and mdx mice at 36 h. n = 3 mice. ( h , i ) Quantification for ( h ) Mark2 and ( i ) Pard3 expression and localization in satellite cells of cultured myofibers of WT and mdx mice at 36 h. n = 4 mice, 30–50 cells per mice. ( j ) Schematic representation of the polarity establishment in WT and mdx satellite cells. Error bars represent means ± SEM. P * < 0.05, ** P < 0.01, *** P < 0.005. Statistical significance was calculated by Student’s t test. Scale bars, 5 μm.

Article Snippet: We used the following antibodies: mouse anti-Pax7 (DSHB), chicken anti-GFP (cat# ab13970, Abcam), mouse anti-Dmd C-terminal (MANDRA1, clone 7A10, DSHB), rabbit anti-Dmd C-terminal (cat# ab15277, Abcam), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# VP-D508, vector labs), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# DYS1-CE-S, Leica microsystems), mouse anti-Dmd N-terminal (MANEX1011B, clone 1C7, DSHB), mouse anti-Dag1 (cat# ab49515, Abcam), rabbit anti-Dag1 (cat# D1945, Sigma), rabbit anti-Mark2 (cat# 9118S, Cell signaling technology), rabbit anti-phospho-Mark2 AF647-conjugated (cat# bs-5742R, Bioss), rabbit anti-Pard3 (cat# 07-330, Millipore), rabbit anti-phospho-Aurk (cat# 2914S, Cell Signaling technology), rat anti-itga7 AF647-conjugated (clone R2F2, cat# 67-0010-10, AbLab), mouse anti-itga7 (cat# K0046-3, Cedarlane), rat anti-laminin (cat# L0663, Sigma), rabbit anti-Myod1 (C-20, cat# sc-304, Santa Cruz), rabbit anti-Myog (M225, cat# sc-576, Santa Cruz).

Techniques: Immunostaining, Cell Culture, Expressing, Proximity Ligation Assay

Journal: Nature medicine

Article Title: Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division

doi: 10.1038/nm.3990

Figure Lengend Snippet: PAR polarity proteins are required for muscle stem cell asymmetric divisions. ( a , b ) Myofibers from Myf5-Cre:R26R-YFP mice cultured for 42 h and immunostained for YFP (green), Pax7 (blue) and Dmd (Dy4/6D3, red; left panel), or Mark2 (red; middle panel), or Pard3 (red; right panel) in ( a ) asymmetric cell pairs (YFP-positive/YFP-negative pairs) and ( b ) symmetric YFP-negative cell pairs (YFP-negative/YFP-negative pairs). Representative pictures from n = 10–20 pictures per condition. ( c , d ) Quantification of asymmetric divisions relative to total satellite stem cell divisions in cultured myofibers of Myf5-Cre:R26R-YFP mice following knockdown of ( c ) Mark2 (si Mark2 ) or ( d ) Pard3 (si Pard3 ) compared to scramble siRNA (siSCR). ( e ) Quantification of Myog-expressing cells per fiber and ( f ) total myogenic cells (Pax7-expressing or Myog-expressing cells) per fiber from WT myofibers cultured 72 h treated with si Mark2 , si Pard3 , or siSCR. ( c – f ) n = 3 mice except for c where n = 5 and d where n = 4, 30–40 myofibers per mice. ( g ) Representative pictures ( n > 10 pictures) from WT and Mark2 −/− section from TA muscle immunostained for laminin (green), Pax7 (red), and DAPI (blue). n = 3 mice. ( h ) Distribution of minimal fiber feret size from the TA muscle of Mark2 −/− mice (blue) and WT littermate (red). n = 3 mice, >300 fibers counted per mouse. ( i ) Representative pictures ( n = 5-10 pictures per condition) of immunostaining for Mark2 (green), Pard3 (red), itga7 (white), and DAPI (blue) of cultured myofiber from WT and Mark2 −/− mice at 36 h. n = 3 mice. Error bars represent means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005. Statistical significance was calculated by Student’s t test. ( a , b , i ) Scale bars, 5 μm and ( g )10 μm.

Article Snippet: We used the following antibodies: mouse anti-Pax7 (DSHB), chicken anti-GFP (cat# ab13970, Abcam), mouse anti-Dmd C-terminal (MANDRA1, clone 7A10, DSHB), rabbit anti-Dmd C-terminal (cat# ab15277, Abcam), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# VP-D508, vector labs), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# DYS1-CE-S, Leica microsystems), mouse anti-Dmd N-terminal (MANEX1011B, clone 1C7, DSHB), mouse anti-Dag1 (cat# ab49515, Abcam), rabbit anti-Dag1 (cat# D1945, Sigma), rabbit anti-Mark2 (cat# 9118S, Cell signaling technology), rabbit anti-phospho-Mark2 AF647-conjugated (cat# bs-5742R, Bioss), rabbit anti-Pard3 (cat# 07-330, Millipore), rabbit anti-phospho-Aurk (cat# 2914S, Cell Signaling technology), rat anti-itga7 AF647-conjugated (clone R2F2, cat# 67-0010-10, AbLab), mouse anti-itga7 (cat# K0046-3, Cedarlane), rat anti-laminin (cat# L0663, Sigma), rabbit anti-Myod1 (C-20, cat# sc-304, Santa Cruz), rabbit anti-Myog (M225, cat# sc-576, Santa Cruz).

Techniques: Cell Culture, Expressing, Immunostaining

Journal: Nature medicine

Article Title: Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division

doi: 10.1038/nm.3990

Figure Lengend Snippet: Dystrophin-deficient satellite cells display impaired mitotic spindle orientation and loss of apicobasal division. ( a ) Representative pictures ( n > 50 pictures) of immunostaining for phospho-Aurora kinase (p-Aurk; green), Pax7 (red), and DAPI (blue) of cultured myofibers from WT mice at 36 h. ( b ) Representative images ( n > 20 pictures) of abnormal mitotic events of cultured myofibers from mdx mice at 36 h immunostained for p-Aurk (green), Pax7 (red), and DAPI (blue). ( a , b ) Mitotic centrosomes (arrowheads) are stained with anti-p-Aurk antibody in prophase and metaphase cells. ( c ) Proportion of mitotic satellite cells on cultured myofibers from WT and mdx mice at 36 h. ( d ) Quantification of abnormal, versus planar, and apicobasal orientated mitotic spindles in satellite cells on myofibers from WT and mdx mice after 36 h of culture. ( c , d ) n = 3 WT mice and n = 4 mdx mice, approximately 100 cells total per condition. ( e ) Example of immunostaining ( n = 10 pictures) for Dmd rod domain (green), itga7 (red), p-Aurk (white) and DAPI (blue) in a mitotic satellite cell undergoing an apicobasal division. ( f ) Examples of mitotic satellite cells ( n > 10 pictures) from immunostaining for itga7 (green), p-Aurk (red), and DAPI (blue) of cultured myofiber from Mark2 −/− mice at 36 h. n = 3 mice. ( g ) Quantification of abnormal, versus planar, and apicobasal orientated mitotic spindles in satellite cells on myofibers from WT and Mark2 −/− mice after 36 h of culture. n = 3 mice, >20 fibers counted per condition. ( c , d , g ) Error bars represent means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005. Statistical significance was calculated by Student’s t test. Scale bars, 5 μm.

Article Snippet: We used the following antibodies: mouse anti-Pax7 (DSHB), chicken anti-GFP (cat# ab13970, Abcam), mouse anti-Dmd C-terminal (MANDRA1, clone 7A10, DSHB), rabbit anti-Dmd C-terminal (cat# ab15277, Abcam), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# VP-D508, vector labs), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# DYS1-CE-S, Leica microsystems), mouse anti-Dmd N-terminal (MANEX1011B, clone 1C7, DSHB), mouse anti-Dag1 (cat# ab49515, Abcam), rabbit anti-Dag1 (cat# D1945, Sigma), rabbit anti-Mark2 (cat# 9118S, Cell signaling technology), rabbit anti-phospho-Mark2 AF647-conjugated (cat# bs-5742R, Bioss), rabbit anti-Pard3 (cat# 07-330, Millipore), rabbit anti-phospho-Aurk (cat# 2914S, Cell Signaling technology), rat anti-itga7 AF647-conjugated (clone R2F2, cat# 67-0010-10, AbLab), mouse anti-itga7 (cat# K0046-3, Cedarlane), rat anti-laminin (cat# L0663, Sigma), rabbit anti-Myod1 (C-20, cat# sc-304, Santa Cruz), rabbit anti-Myog (M225, cat# sc-576, Santa Cruz).

Techniques: Immunostaining, Cell Culture, Staining

Journal: International Journal of Molecular Sciences

Article Title: Effects of Periostracum Cicadae on Cytokines and Apoptosis Regulatory Proteins in an IgA Nephropathy Rat Model

doi: 10.3390/ijms19061599

Figure Lengend Snippet: TLR4, MCP-1, IgA, and caspase-3 expression in rat kidney tissues. ( a ) TLR4, IgA, MCP-1, and caspase-3 expression levels were analyzed by Western blotting. ( b ) Quantitative analysis of TLR4, IgA, MCP-1, and caspase-3 protein levels. A: Healthy, normal controls; B: IgAN model group; C: prednisone acetate group; D: tripterygium glycoside tablet group; E–G: treatment group that received 0.5, 1, and 2 g/kg periostracum cicadae. Data represent the mean ± standard deviation (** p < 0.01 vs model group).

Article Snippet: Rabbit anti-TLR4 polyclonal antibody (Boster Biological Technology, Pleasanton, CA, USA), rabbit anti-IgA polyclonal antibody (Boster Biological Technology), rabbit anti-MCP-1 polyclonal antibody (Boster Biological Technology), and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody (Boster Biological Technology) were used in the detection of the corresponding target proteins.

Techniques: Expressing, Western Blot, Standard Deviation