Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: Murine Myoblasts Exposed to SYUIQ-5 Acquire Senescence Phenotype and Differentiate into Sarcopenic-Like Myotubes, an In Vitro Study

doi: 10.1093/gerona/glae022

Figure Lengend Snippet: Sarcopenic features of myotubes differentiated from senescent SYUIQ-5 treated myoblasts. ( A ) Representative western blot images of MURF1 and ATROGIN-1 proteins expressed in myotubes lysates differentiated for 7 d from SYUIQ-5 untreated (NT) or treated (0.4 μM and 1.0 μM) myoblasts and relative histograms of protein levels quantification normalized on VINCULIN (MURF1 histogram: n = 5–7, NT vs 1.0 μM ** p = .059, 1-way ANOVA Tukey’s multiple comparison test; ATROGIN-1 histogram: n = 6, NT vs 1.0 μM * p = .0370, 1-way ANOVA Tukey’s multiple comparison test). ( B ) Representative western blot images of TOTAL UBIQUITIN protein expressed in myotubes differentiated for 7 d from SYUIQ-5 untreated (NT) or treated (1.0 μM) myoblasts and relative histogram of protein levels quantification normalized on β-ACTIN ( n = 3, NT vs 1.0 μM * p = .0395, unpaired t test). ( C ) Representative confocal images of myotubes differentiated for 7 d from SYUIQ-5 untreated (NT) or treated (0.4 μM and 1.0 μM) myoblasts and incubated with MitoTracker (red channel) before being fixed. Cells have been immunolabeled with anti-MyHC (green channel) to select differentiated myoblasts and nuclei have been stained with DAPI (blue channel). Objective 20×, scale bar 20 μM. Histogram referred to mitochondria content measured as mean fluorescence intensity signal of MitoTracker ( n = 47–128, NT vs 0.4 μM **** p < .0001, NT vs 1.0 μM **** p < .0001, 0.4 μM vs 1.0 μM * p = .0423, Kruskal–Wallis Dunn’s multiple comparison test). ( D ) Histogram of MYOSTATIN quantification in supernatant of myotubes differentiated from nontreated myoblasts (NT) and from myoblasts treated with 0.4 μM and 1.0 μM of SYUIQ-5. MYOSTATIN levels are expressed as pg/ml and detected by ELISA assay ( n = 5, NT vs 1.0 μM * p = .0226, 1-way ANOVA Tukey’s multiple comparison test).

Article Snippet: The following primary antibodies were used: anti-TUBULIN (1:400, #T4026 Merck); anti-phosphorylated H2AX (Ser139, ƴ-H2AX, 1:800, #2577 Cell Signaling Technologies); anti-MyHC (1:200, #MAB4470, Bio-Techne); anti-MURF1 (1:200, #AF5366, Bio-techne); anti-ATROGIN1 (1:100, #AM3141, ECM Biosciences).

Techniques: Western Blot, Comparison, Incubation, Immunolabeling, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Classification of node-positive melanomas into prognostic subgroups using keratin, immune, and melanogenesis expression patterns

doi: 10.1038/s41388-021-01665-0

Figure Lengend Snippet: a The tree trained on the 469 TCGA samples. Classification of a new sample into one of the four subtypes is done by traversing the tree from its root to one of its leaves (representing an assignment to a subtype). Three biomarkers (shown in black) are used to determine the route along the tree: overexpression of KLK8 distinguishes the “Keratin” subtype, overexpression of TIGIT distinguishes the “Immune” subtype, and finally, overexpression of TRIM63 distinguishes the “Melanogenesis-high” from the “Melanogenesis-low” subtype. b Threshold values and surrogate genes for the three decision tree predictors as identified by the algorithm. Threshold values are used to distinguish between high and low values (based on normalized expression values), and surrogates can be used as alternatives for the respective predictor gene.

Article Snippet: Slides were first blocked and incubated with various combinations of primary antibodies including LCK (AF3704, R&D Systems), TIGIT (A700-047, Bethyl Laboratories), TRIM63 (bs2539R, Bioss), OCA2 (bs15510R, Bioss), GPNMB (AF2550, R&D Systems), HMB45 (ab732, Abcam), and KLK8 (MAB1719, R&D Systems).

Techniques: Over Expression, Expressing

Journal: Oncogene

Article Title: Classification of node-positive melanomas into prognostic subgroups using keratin, immune, and melanogenesis expression patterns

doi: 10.1038/s41388-021-01665-0

Figure Lengend Snippet: a Hematoxylin and eosin (H&E) staining of lymph nodes containing melanoma metastases from six different patients taken at 20× magnification. Patients 1–3 had good survival, while patients 4–6 survived poorly. b Immunohistochemical staining of the three proteins of the decision tree on the lymph node samples of the six patients. Nuclei were stained blue using DAPI. Row 1: using KLK8 (pink) as a predictor for the Keratin subgroup. Row 2: using TIGIT (Green) as a predictor of the Immune subgroup. Row 3: using TRIM63 (Pink) as a predictor of the Melanogenesis-high subgroup. The assignments of the specimens from the six patients to subtypes based on the expression levels of the three predictor genes are summarized as a label at the bottom bar. c Immunohistochemical staining of additional biomarkers for general prognosis. Row 1: LCK, an immune protein indicative of good prognostic outcome. Row 2: melanogenesis protein OCA2. Row 3: melanogenesis protein GPNMB. d Color matrix quantifying the fluorescence intensity of immunohistochemistry across biomarkers and patients. For each protein, values were independently normalized across the samples. (The KLK staining of all samples is negative, as they are all nonprimary, so KLK is not included here).

Article Snippet: Slides were first blocked and incubated with various combinations of primary antibodies including LCK (AF3704, R&D Systems), TIGIT (A700-047, Bethyl Laboratories), TRIM63 (bs2539R, Bioss), OCA2 (bs15510R, Bioss), GPNMB (AF2550, R&D Systems), HMB45 (ab732, Abcam), and KLK8 (MAB1719, R&D Systems).

Techniques: Staining, Immunohistochemical staining, Expressing, Fluorescence, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Calycosin inhibited autophagy and oxidative stress in chronic kidney disease skeletal muscle atrophy by regulating AMPK/SKP2/CARM1 signalling pathway

doi: 10.1111/jcmm.15514

Figure Lengend Snippet: Effect of calycosin on muscle histology, apoptosis, and MuRF1 and MAFbx expression levels in muscle of 5/6 Nx rats. A, Representative images of H&E stained rat muscles (200x). B‐E, Representative images and scores of IHC staining of MuRF1 and MAFbx in rat muscles (×200). Bar = 50 µm. * P < 0.05 compared to the sham group. ## P < 0.01 compared to the 5/6 Nx model group

Article Snippet: Equal amounts of protein were loaded onto 10% SDS polyacrylamide gels, subjected to electrophoresis (at constant pressure, 60 V for 30 minutes, 120 V for 60 minutes), transferred to PVDF membranes (Millipore, Billerica, MA, USA) under a constant current of 350 mA for 120 minutes, blocked with 5% BSA or 5% milk in 1× TBST for 1 hour, then incubated with the following primary antibodies at 4°C overnight: GAPDH (1:2000; EarthOx, USA), MuRF1 (1:1000, Boster, USA), MAFbx (1:1000, Affinity), Myogenin (1:1000, Affinity), LC3A/B (1:800, Affinity), ATG7 (1:800, Affinity), SKP2 (1:1000, Affinity), CARM1 (1:1000, Affinity), H3R17me3a (1:1000, Affinity), AMPK (1:1000, Affinity, AF6423), p‐AMPK (1:1000; Affinity, AF3424), FOXO3a (1:1000, Affinity, AF5418), and p‐FOXO3a (1:1000, Affinity).

Techniques: Expressing, Staining, Muscles, Immunohistochemistry

Journal: Theranostics

Article Title: Ablation of NLRP3 inflammasome attenuates muscle atrophy via inhibiting pyroptosis, proteolysis and apoptosis following denervation.

doi: 10.7150/thno.74831

Figure Lengend Snippet: Figure 3. Denervation induces NLRP3 inflammasome activation accompanied by pyroptotic molecules and UPS ligases upregulation in atrophic GAS. (A) Western blot analysis of expression levels of NLRP3, GSDMD, GSDMD-N, Caspase 1, Caspase 1 p20, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1 at 0, 14, 21 and 28 d after denervation. (B) Quantification of NLRP3, GSDMD, GSDMD-N, Caspase 1, Caspase 1 p20, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1. (C) Quantitative PCR analysis of mRNA levels of NLRP3, GSDMD, Caspase 1, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1 at 0, 14, 21 and 28 d after denervation. (D) Representative immunofluorescence staining images of NLRP3 at 0, 14, 21 and 28 d after denervation. Scale bar, 50 μm. (E) Quantitative ELISA analysis of IL-1β and IL-18 in GAS at 0, 14, 21 and 28 d after denervation. Data are expressed as mean ± SD. n = 6 per group. *p < 0.05, **p < 0.01, ns p > 0.05 vs Sham group. Den, denervation. GAS, gastrocnemius.

Article Snippet: After incubated for two hours at room temperature, the membrane was blocked with 5% skim milk and sequentially incubated with primary antibodies against NLRP3 (Cell Signaling Technology, Danvers, MA, USA), GSDMD (Biorbyt, St Louis, MO, USA), ASC (Biorbyt), Caspase 1/p20/p10 (Proteintech, Rosemont, USA), MuRF1 (Biorbyt), Atrogin-1 (Biorbyt), Caspase 3 (Proteintech), IL-1β (Biorbyt), IL-18 (Sigma-Aldrich, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioworld Technology, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Activation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Staining, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Ablation of NLRP3 inflammasome attenuates muscle atrophy via inhibiting pyroptosis, proteolysis and apoptosis following denervation.

doi: 10.7150/thno.74831

Figure Lengend Snippet: Figure 4. NLRP3 cKO attenuates muscle atrophy and pyroptosis after denervation. (A) Outline of the scheme to obtain WT and NLRP3 cKO mice. (B) General observation of the GAS in each group. (C) Quantification of GAS muscles weight / body weight in WT and NLRP3 cKO mice in each group. (D) The wet weight ratio (operational side weight / contralateral side weight) of GAS muscles in each group. (E) Morphological observation of GAS by HE staining in each group. Scale bar, 50 μm. (F) Fibre size distribution of GAS muscles in each group. (G) Western blot analysis of expression levels of NLRP3, GSDMD, GSDMD-N, Caspase 1, Caspase 1 p20, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1 in each group. (H) Quantification of NLRP3, GSDMD, GSDMD-N, Caspase 1, Caspase 1 p20, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1. Data are expressed as mean ± SD. n = 6 per group. *p < 0.05, **p < 0.01, ns p > 0.05. WT, wild type. Den, denervation.

Article Snippet: After incubated for two hours at room temperature, the membrane was blocked with 5% skim milk and sequentially incubated with primary antibodies against NLRP3 (Cell Signaling Technology, Danvers, MA, USA), GSDMD (Biorbyt, St Louis, MO, USA), ASC (Biorbyt), Caspase 1/p20/p10 (Proteintech, Rosemont, USA), MuRF1 (Biorbyt), Atrogin-1 (Biorbyt), Caspase 3 (Proteintech), IL-1β (Biorbyt), IL-18 (Sigma-Aldrich, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioworld Technology, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Muscles, Staining, Western Blot, Expressing

Journal: Theranostics

Article Title: Ablation of NLRP3 inflammasome attenuates muscle atrophy via inhibiting pyroptosis, proteolysis and apoptosis following denervation.

doi: 10.7150/thno.74831

Figure Lengend Snippet: Figure 5. NLRP3 cKO attenuates muscle atrophy and pyroptosis after denervation. (A) Quantitative PCR analysis of mRNA levels of NLRP3, GSDMD, Caspase 1, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1 in each group. (B) Representative immunofluorescence staining images of NLRP3 in each group. Scale bar, 50 μm. (C) Quantitative ELISA analysis of IL-1β and IL-18 in each group. Data are expressed as mean ± SD. n = 6 per group. *p < 0.05, **p < 0.01, ns p > 0.05. WT, wild type. Den, denervation.

Article Snippet: After incubated for two hours at room temperature, the membrane was blocked with 5% skim milk and sequentially incubated with primary antibodies against NLRP3 (Cell Signaling Technology, Danvers, MA, USA), GSDMD (Biorbyt, St Louis, MO, USA), ASC (Biorbyt), Caspase 1/p20/p10 (Proteintech, Rosemont, USA), MuRF1 (Biorbyt), Atrogin-1 (Biorbyt), Caspase 3 (Proteintech), IL-1β (Biorbyt), IL-18 (Sigma-Aldrich, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioworld Technology, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Real-time Polymerase Chain Reaction, Staining, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Ablation of NLRP3 inflammasome attenuates muscle atrophy via inhibiting pyroptosis, proteolysis and apoptosis following denervation.

doi: 10.7150/thno.74831

Figure Lengend Snippet: Figure 6. NIA induces myocyte atrophy accompanied by pyroptotic molecules and UPS ligases upregulation in C2C12 myotubes. (A) Western blot analysis of expression levels of NLRP3, GSDMD, GSDMD-N, Caspase 1, Caspase 1 p20, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1 in C2C12 myotubes treated with NIA for 0, 12 h, 1 d and 3 d. (B) Quantification of NLRP3, GSDMD, GSDMD-N, Caspase 1, Caspase 1 p20, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1. (C) Quantitative PCR analysis of mRNA levels of NLRP3, GSDMD, Caspase 1, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1 in C2C12 myotubes treated with NIA for 0, 12 h, 1 d and 3 d. (D) Representative immunofluorescence staining images of NLRP3 in C2C12 myotubes treated with NIA for 0, 12 h, 1 d and 3 d. Scale bar, 50 μm. (E) Quantitative ELISA analysis of IL-1β and IL-18 in the supernatant of C2C12 myotubes after NIA treatment for 0, 12 h, 1 d and 3 d. Data are expressed as mean ± SD. n = 6 per group. *p < 0.05, **p < 0.01, ns p > 0.05 vs Sham group. NIA, NLRP3 inflammasome activator.

Article Snippet: After incubated for two hours at room temperature, the membrane was blocked with 5% skim milk and sequentially incubated with primary antibodies against NLRP3 (Cell Signaling Technology, Danvers, MA, USA), GSDMD (Biorbyt, St Louis, MO, USA), ASC (Biorbyt), Caspase 1/p20/p10 (Proteintech, Rosemont, USA), MuRF1 (Biorbyt), Atrogin-1 (Biorbyt), Caspase 3 (Proteintech), IL-1β (Biorbyt), IL-18 (Sigma-Aldrich, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioworld Technology, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Staining, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Ablation of NLRP3 inflammasome attenuates muscle atrophy via inhibiting pyroptosis, proteolysis and apoptosis following denervation.

doi: 10.7150/thno.74831

Figure Lengend Snippet: Figure 7. Knockdown of NLRP3 ameliorates NIA-induced myocyte atrophy and pyroptosis in C2C12 myotubes. (A) The transfection of sh-NLRP3 in vitro. Scale bar, 50 μm. (B) Quantitative PCR analysis confirmed the mRNA level of NLRP3 knockdown in C2C12 myotubes. (C) Western blot analysis confirmed the expression level of NLRP3 knockdown in C2C12 myotubes. (D) Bright field photomicrographs of C2C12 myotubes in each group. Scale bar, 50 μm. (E) Quantitative ELISA analysis of IL-1β and IL-18 in the supernatant of C2C12 myotubes in each group. (F) Western blot analysis of expression levels of NLRP3, GSDMD, GSDMD-N, Caspase 1, Caspase 1 p20, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1 in each group. (G) Quantification of NLRP3, GSDMD, GSDMD-N, Caspase 1, Caspase 1 p20, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1. (H) Quantitative PCR analysis of mRNA levels of NLRP3, GSDMD, Caspase 1, ASC, IL-1β, IL-18, MuRF1 and Atrogin-1 in each group. (I) Representative immunofluorescence staining images of NLRP3 in each group. Scale bar, 100 μm. Data are expressed as mean ± SD. n = 6 per group. *p < 0.05, **p < 0.01, ns p > 0.05 vs Sham group. NIA, NLRP3 inflammasome activator.

Article Snippet: After incubated for two hours at room temperature, the membrane was blocked with 5% skim milk and sequentially incubated with primary antibodies against NLRP3 (Cell Signaling Technology, Danvers, MA, USA), GSDMD (Biorbyt, St Louis, MO, USA), ASC (Biorbyt), Caspase 1/p20/p10 (Proteintech, Rosemont, USA), MuRF1 (Biorbyt), Atrogin-1 (Biorbyt), Caspase 3 (Proteintech), IL-1β (Biorbyt), IL-18 (Sigma-Aldrich, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioworld Technology, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Knockdown, Transfection, In Vitro, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Staining