Journal: Aging Cell

Article Title: Diosgenin Alleviates Age‐Related Sarcopenia by Promoting Satellite Cell Proliferation and Myogenic Differentiation via Activation of the SIRT1 / PGC ‐1α Signaling Pathway

doi: 10.1111/acel.70651

Figure Lengend Snippet: Diosgenin promoted proliferation and myogenic differentiation of senescent satellite cells. (A) Representative images of Pax7/Ki67 double IF staining in skeletal muscle tissues, with quantitative analysis. Green: Pax7 (satellite cell marker); red: Ki67 (proliferation marker); blue: DAPI (nuclei); yellow: Pax7 + /Ki67 + double‐positive cells. Scale bar = 20 μm. (B) Cytotoxicity of diosgenin in differentiated myotubes (determination of safe concentration range, n = 3). (C) Proliferative activity of diosgenin in undifferentiated C2C12 myoblasts in the absence (left panel) or presence (right panel) of 100 μM D‐gal ( n = 3). (D) Representative WB images and quantitative analysis of the myogenic differentiation markers Myf5, Pax7, and MyHC II in mouse skeletal muscle. GAPDH was used as the internal control. (E) Representative WB images and quantitative analysis of the myogenic differentiation markers Myf5, Pax7, and MyHC II in C2C12 cells. GAPDH was used as the internal control. Data are presented as mean ± SEM. * p ‐value < 0.05, ** p ‐value < 0.01, *** p ‐value < 0.001 versus the YC group; # p ‐value < 0.05, ## p ‐value < 0.01, ### p ‐value < 0.001 versus the OC group. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. All experiments were performed with at least three independent replicates.

Article Snippet: Membranes were blocked with 5% skim milk at room temperature for 1 h and incubated with the following primary antibodies at 4°C overnight: SIRT1 (1:1000; Affinity Biosciences, Liyang, China; Cat. #DF6033), PGC‐1α (1:1000; ABclonal, Wuhan, China; Cat. #AP20542C), p53 (1:1000; ABclonal, Wuhan, China; Cat. #A21630), p21 (1:1000; ABclonal, Wuhan, China; Cat. #A1483), Myf5 (1:1000; ABclonal, Wuhan, China; Cat. #A116227), MyHC II (1:1000; ABclonal, Wuhan, China; Cat. #A15293), Pax7 (1:1000; ABclonal, Wuhan, China; Cat. #A7335), and GAPDH (1:20,000; ABclonal, Wuhan, China; Cat. #AC033).

Techniques: Cell Characterization, Staining, Marker, Concentration Assay, Activity Assay, Control

Journal: Aging Cell

Article Title: Diosgenin Alleviates Age‐Related Sarcopenia by Promoting Satellite Cell Proliferation and Myogenic Differentiation via Activation of the SIRT1 / PGC ‐1α Signaling Pathway

doi: 10.1111/acel.70651

Figure Lengend Snippet: Diosgenin regulated SIRT1 to ameliorate age‐related sarcopenia. (A) Representative WB images and quantitative analysis of SIRT1 in C2C12 cells. (B) Quantitative analysis of PGC‐1α protein expression and PGC‐1α acetylation levels in C2C12 cells. (C) mRNA levels of Cpt1b in C2C12 cells. (D) mRNA levels of Metrnl and Igf1 in C2C12 cells. (E) Protein concentrations of METRNL and IGF‐1 in C2C12 cells. (F) Representative WB images and quantitative analysis of the myogenic differentiation markers Myf5, Pax7, and MyHC II in C2C12 cells. (G) Representative WB images and quantitative analysis of the senescence markers p53 and p21 in C2C12 cells. (H) Representative SA‐β‐gal staining images of C2C12 cells at 100× and 400× magnification, with quantitative analysis. (I) Molecular docking results of diosgenin with the SIRT1 protein. (J) Schematic diagram of the interactions between diosgenin and key residues of SIRT1. (K) Representative WB images reflecting the thermal stability of SIRT1 protein under different temperature gradients and the corresponding thermal stability curve; the y‐axis represents the remaining SIRT1 protein level relative to 37°C. Data are presented as mean ± SEM. * p‐ value < 0.05, ** p ‐value < 0.01, *** p ‐value < 0.001 versus the YC group; # p ‐value < 0.05, ## p ‐value < 0.01, ### p ‐value < 0.001 versus the OC group. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. All experiments were performed with at least three independent replicates.

Article Snippet: Membranes were blocked with 5% skim milk at room temperature for 1 h and incubated with the following primary antibodies at 4°C overnight: SIRT1 (1:1000; Affinity Biosciences, Liyang, China; Cat. #DF6033), PGC‐1α (1:1000; ABclonal, Wuhan, China; Cat. #AP20542C), p53 (1:1000; ABclonal, Wuhan, China; Cat. #A21630), p21 (1:1000; ABclonal, Wuhan, China; Cat. #A1483), Myf5 (1:1000; ABclonal, Wuhan, China; Cat. #A116227), MyHC II (1:1000; ABclonal, Wuhan, China; Cat. #A15293), Pax7 (1:1000; ABclonal, Wuhan, China; Cat. #A7335), and GAPDH (1:20,000; ABclonal, Wuhan, China; Cat. #AC033).

Techniques: Expressing, Cell Characterization, Staining

Journal:

Article Title: Role for Actin Filament Turnover and a Myosin II Motor in Cytoskeleton-driven Disassembly of the Epithelial Apical Junctional Complex

doi: 10.1091/mbc.E04-02-0163

Figure Lengend Snippet: Inhibition of mammalian nonmuscle myosin (MNMM) II prevents F-actin reorganization, disassembly of the AJC, and barrier dysfunction in calcium-depleted cells. (A). T84 cells were preincubated for 60 min with blebbistatin (50 μM) or vehicle followed by 60-min incubation in S-MEM containing the same concentrations of the inhibitor. Localization of F-actin, E-cadherin, and occludin was determined by fluorescence labeling and confocal microscopy. Blebbistatin, which selectively inhibits activity of MNMM II, completely blocks formation of contractile F-actin rings and disassembly of AJs and TJs in calcium-depleted cells. (B). T84 cells were either incubated for 120 min in T84 complete medium containing blebbistatin (50 μM) or pretreated with blebbistatin for 60 min followed by 60 min of calcium depletion in the presence of the inhibitor. Appropriate controls were incubated with vehicle, and TEER in all groups was measured. Blebbistatin slightly decreases resistance in T84 monolayers at normal concentration of calcium, but significantly attenuates the drop in TEER during calcium depletion. Data are presented as mean ± SE (n = 3); *p < 0.05; **p < 0.01 comparing to vehicle-treated controls.

Article Snippet: The following primary polyclonal (pAb) and monoclonal antibodies (mAb) were used to detect TJ, AJ, and cytoskeletal proteins by immunofluorescence labeling and Western blotting: antioccludin, ZO-1 and JAM-1 pAbs (Zymed Laboratories, San Francisco, CA); anti-JAM-1 mAb (1H2A9; Liu et al., 2000 ); anti-E-cadherin mAb (HECD-1; Zymed); anti-β-catenin and actin pAbs (Sigma, St. Louis, MO), anti-β-catenin mAb (BD PharMingen, San Diego, CA); antimammalian nonmuscle myosin (MNMM) IIA and IIB pAbs (Covance, Berkley, CA); anti-mono- and diphosphorylated regulatory myosin light chain and antiphospho-cofilin-1 pAbs (Cell Signaling Technology, Beverly, MA); anticofilin pAb (Cytoskeleton, Denver, CO); anticortactin mAb (Upstate Biotechnology, Lake Placid, NY).

Techniques: Inhibition, Incubation, Fluorescence, Labeling, Confocal Microscopy, Activity Assay, Concentration Assay

Journal:

Article Title: Role for Actin Filament Turnover and a Myosin II Motor in Cytoskeleton-driven Disassembly of the Epithelial Apical Junctional Complex

doi: 10.1091/mbc.E04-02-0163

Figure Lengend Snippet: Mammalian nonmuscle myosin IIA isoform is predominantly expressed in T84 epithelial cells and colocalizes with F-actin rings during calcium depletion. (A) Expression of different MNMM II isoforms in T84 total cell lysates was determined by Western blotting. The MNMM IIA is highly expressed in T84 cells, whereas expression of the IIB isoform is weak and MNMM IIC is undetectable. (B). Control T84 cells and calcium-depleted (30 min) cells were double-labeled for MNMM IIA (green) with F-actin (red). As can be seen, in normal cells MNMM IIA is enriched in apical brush-border, whereas in calcium-depleted cells MNMM IIA colocalizes with F-actin rings (arrows). (C). Control T84 cells and cells incubated in S-MEM for 60 min were fractionated using a TX-100/phalloidin-containing buffer as described in MATERIAL AND METHODS, and distribution of MNMM IIA between TX-100-soluble (S) and -insoluble (I) fractions was determined. Calcium depletion increases amount of TX-100-insoluble (F-actin-associated) fraction of MNMM IIA. Bar, 5 μm.

Article Snippet: The following primary polyclonal (pAb) and monoclonal antibodies (mAb) were used to detect TJ, AJ, and cytoskeletal proteins by immunofluorescence labeling and Western blotting: antioccludin, ZO-1 and JAM-1 pAbs (Zymed Laboratories, San Francisco, CA); anti-JAM-1 mAb (1H2A9; Liu et al., 2000 ); anti-E-cadherin mAb (HECD-1; Zymed); anti-β-catenin and actin pAbs (Sigma, St. Louis, MO), anti-β-catenin mAb (BD PharMingen, San Diego, CA); antimammalian nonmuscle myosin (MNMM) IIA and IIB pAbs (Covance, Berkley, CA); anti-mono- and diphosphorylated regulatory myosin light chain and antiphospho-cofilin-1 pAbs (Cell Signaling Technology, Beverly, MA); anticofilin pAb (Cytoskeleton, Denver, CO); anticortactin mAb (Upstate Biotechnology, Lake Placid, NY).

Techniques: Expressing, Western Blot, Control, Labeling, Incubation