Journal: Acta Physiologica (Oxford, England)

Article Title: miR‐29c improves skeletal muscle mass and function throughout myocyte proliferation and differentiation and by repressing atrophy‐related genes

doi: 10.1111/apha.13278

Figure Lengend Snippet: miR‐29c overexpression modulates gene markers of skeletal muscle trophicity. mRNA levels of MuRF1, Atrogin‐1 and HDAC4 were determined by qPCR at 4 (A), 7 (B) and 30 (C) days after electrotransfer in control EV and pMIR29c groups; n = 5 per group. The 18s ribosomal RNA was used as an endogenous control gene . Protein levels of MuRF1, Atrogin‐1 and HDAC4 were determined at 4 (D and G), 7 (E and H) or 30 (F and I) days after electrotransfer in control EV and pMIR29c groups. Densitometry (D‐F) or representative western blots bands (G‐I); n = 5 per group . GAPDH protein level was used as a loading control. Data were expressed in arbitrary units (au) as mean and SEM. Statistical analysis included one‐way ANOVA followed by a Tukey's posttest. * P ≤ 0.05 vs the respective EV group

Article Snippet: The following primary antibodies were used: rabbit anti‐HDAC4 (1:1000, cat#7628, Cell Signaling); rabbit anti‐Atrogin‐1 (1:1000, cat#AP2041, ECM Biosciences); rabbit anti‐MuRF1 (1:1000, cat#MP3401, ECM Biosciences).

Techniques: Over Expression, Electrotransfer, Western Blot

Journal: Acta Physiologica (Oxford, England)

Article Title: miR‐29c improves skeletal muscle mass and function throughout myocyte proliferation and differentiation and by repressing atrophy‐related genes

doi: 10.1111/apha.13278

Figure Lengend Snippet: miR‐29c overexpression downregulates the mRNAs related to muscle atrophy by directly binding to the MuRF1 3′ UTR. C2C12 cells were incubated with a miR‐29c or miR‐29b mimics 24 hours before the onset of differentiation for 1, 3 or 5 days. A scrambled sequence was used as a control. The mRNA levels of (A) FoxO1, (B) FoxO3, (C) MuRF1 and (D) Atrogin‐1 were determined by qPCR. Data are expressed in arbitrary units (au) as mean and SEM (n = 4) and normalized to endogenous 18s ribosomal RNA levels. (E) C2C12 cells were treated with a miR‐29c mimic at increasing concentrations (10, 20, 40 and 80 ŋM), and MuRF1 mRNA levels were determined by qPCR. * P ≤ 0.05 vs scrambled group. Statistical analysis included one‐way ANOVA followed by a Tukey's posttest. * P ≤ 0.05 vs scrambled group. (F) Diagram depicting the miR‐29c sequence and the 3′ untranslated region (3′UTR) of MuRF1. Seed sequences are highlighted in grey, and the mutations are indicated on the top in red. Luciferase reporter activity driven by the wild‐type MuRF1 3′UTR (WT‐pmiRGlo3′UTRMuRF1) or a mutated version (MUT‐pmiRGlo3′UTRMuRF1) was determined in C2C12 (G) and HEK293 (H) cells. A scrambled sequence was used as a control, and the cells were treated with a miR‐29c mimic. Renilla coexpression was used to normalize the transfection. Data were expressed in arbitrary units (au) as mean and SEM; n = 3. * P ≤ 0.05 vs the scrambled group (blue bars) and & P ≤ 0.05 vs vector only group (white bars)

Article Snippet: The following primary antibodies were used: rabbit anti‐HDAC4 (1:1000, cat#7628, Cell Signaling); rabbit anti‐Atrogin‐1 (1:1000, cat#AP2041, ECM Biosciences); rabbit anti‐MuRF1 (1:1000, cat#MP3401, ECM Biosciences).

Techniques: Over Expression, Binding Assay, Incubation, Sequencing, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Cancer medicine

Article Title: TRIM55 inhibits colorectal cancer development via enhancing protein degradation of c-Myc.

doi: 10.1002/cam4.6020

Figure Lengend Snippet: FIGURE 1 TRIM55 expression is downregulated in CRC. (A,B) The mRNA (A) and (B) protein levels of TRIM55 were analyzed in CRC cell lines (DLD1, HCT116, SW620, and SW480) and fetal colon cell line FHC. (C) The protein expression of TRIM55 was analyzed in CRC tissues and paired-normal tissues from 14 CRC patients. (D) IHC staining of TRIM55 was conducted in CRC tissues and paired-normal tissues from 22 CRC patients. (E,F) Tissue microarray (TMA) was performed and the prognosis of patients with high or low expression of TRIM55 was analysis in CRC TMA cohort. (G) Kaplan–Meier analysis was performed using TCGA dataset colon adenocarcinoma (COAD) cohort based on TRIM55 expression levels. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: SDS- PAGE and western blot were conducted using the following antibodies: anti- GAPDH (BM3874, Boster), anti- TRIM55 (A15917, Abclonal), anti- MMP2 (orb12416, Biorbyt), anti- MMP9 (27306- 1- AP, Proteintech), anti- NCadherin (22018- 1- AP, Proteintech), anti- Bak (A0498, Abclonal), anti- Bax (sc- 7480), anti- Bcl2 (ET7110- 51, HuaBio), anti- Caspase 3 (ET1602- 39, HuaBio), anti- c- Myc (AF0358, Affinity), anti- p21/Cip1 (27296- 1- AP, Proteintech), anti- E- Cadherin (20874- 1- AP, Proteintech), anti- P27/Kip1 (NBP1- 32213, Novus), anti- survivin (NBP2- 48494, Novus), mouse anti- HA- Tag mAb (AE065, Abclonal), and mouse anti His- Tag mAb (AE003, Abclonal).

Techniques: Expressing, Immunohistochemistry, Microarray

Journal: Cancer medicine

Article Title: TRIM55 inhibits colorectal cancer development via enhancing protein degradation of c-Myc.

doi: 10.1002/cam4.6020

Figure Lengend Snippet: FIGURE 2 Overexpression of TRIM55 suppresses CRC cell growth. (A–E) SW620 and SW480 cells were transfected with empty vector or TRIM55 overexpression vector (TRIM55-OE). The relative mRNA (A) and protein (B) levels of TRIM55 were examined by qPCR and western blot 48 hlater. (C) Cell proliferation, (D) DNA incorporation, and (E) colony formation of SW620 and SW480 cells were analyzed by CCK-8 assay, EDU DNA incorporation assay, and colony formation assay respectively. (F–I) SW620 cells were transfected with empty vector or TRIM55-OE. 5 × 106 cells were inoculated into nude mice to establish the CRC xenograft tumor model. After 36 days, tumors were harvested (F) and weighted (G). Tumor growth was measured every 4 days and the growth curve was plotted (H). (I) H&E and IHC staining of KI-67 and PCNA were performed to evaluate the cell proliferation in tumor. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: SDS- PAGE and western blot were conducted using the following antibodies: anti- GAPDH (BM3874, Boster), anti- TRIM55 (A15917, Abclonal), anti- MMP2 (orb12416, Biorbyt), anti- MMP9 (27306- 1- AP, Proteintech), anti- NCadherin (22018- 1- AP, Proteintech), anti- Bak (A0498, Abclonal), anti- Bax (sc- 7480), anti- Bcl2 (ET7110- 51, HuaBio), anti- Caspase 3 (ET1602- 39, HuaBio), anti- c- Myc (AF0358, Affinity), anti- p21/Cip1 (27296- 1- AP, Proteintech), anti- E- Cadherin (20874- 1- AP, Proteintech), anti- P27/Kip1 (NBP1- 32213, Novus), anti- survivin (NBP2- 48494, Novus), mouse anti- HA- Tag mAb (AE065, Abclonal), and mouse anti His- Tag mAb (AE003, Abclonal).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, CCK-8 Assay, Colony Assay, Immunohistochemistry

Journal: Cancer medicine

Article Title: TRIM55 inhibits colorectal cancer development via enhancing protein degradation of c-Myc.

doi: 10.1002/cam4.6020

Figure Lengend Snippet: FIGURE 3 Overexpression of TRIM55 inhibits CRC cell migration and invasion. (A–C) SW620 and SW480 cells were transfected with empty vector or TRIM55-OE. (A) Cell migration was analyzed by wound-healing assay. (B) Cell invasion was analyzed by transwell assay. (C) The protein expression of TRIM55, MMP-2, MMP-9, E-CAD, N-CAD, and GAPDH were analyzed by western blot and quantified. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: SDS- PAGE and western blot were conducted using the following antibodies: anti- GAPDH (BM3874, Boster), anti- TRIM55 (A15917, Abclonal), anti- MMP2 (orb12416, Biorbyt), anti- MMP9 (27306- 1- AP, Proteintech), anti- NCadherin (22018- 1- AP, Proteintech), anti- Bak (A0498, Abclonal), anti- Bax (sc- 7480), anti- Bcl2 (ET7110- 51, HuaBio), anti- Caspase 3 (ET1602- 39, HuaBio), anti- c- Myc (AF0358, Affinity), anti- p21/Cip1 (27296- 1- AP, Proteintech), anti- E- Cadherin (20874- 1- AP, Proteintech), anti- P27/Kip1 (NBP1- 32213, Novus), anti- survivin (NBP2- 48494, Novus), mouse anti- HA- Tag mAb (AE065, Abclonal), and mouse anti His- Tag mAb (AE003, Abclonal).

Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Wound Healing Assay, Transwell Assay, Expressing, Western Blot

Journal: Cancer medicine

Article Title: TRIM55 inhibits colorectal cancer development via enhancing protein degradation of c-Myc.

doi: 10.1002/cam4.6020

Figure Lengend Snippet: FIGURE 5 TRIM55 directly interacts with c-Myc. (A,B) HEK-293 T cells were transfected with HA-vector, HA-TRIM55, or His-c-Myc. Co-IP assay was done using anti-HA (A) or anti-His (B) antibody. The immunoprecipitated protein was analyzed by western blot. (C,D) Co-IP assay was performed in SW620 or SW480 cell lysate using anti-TRIM55 or anti-c-Myc antibody. The immunoprecipitated protein was analyzed by western blot. (E) Immunofluorescence staining of c-Myc and TRIM55 was performed in SW620 and SW480 cells. DAPI staining was used to localize the nucleus.

Article Snippet: SDS- PAGE and western blot were conducted using the following antibodies: anti- GAPDH (BM3874, Boster), anti- TRIM55 (A15917, Abclonal), anti- MMP2 (orb12416, Biorbyt), anti- MMP9 (27306- 1- AP, Proteintech), anti- NCadherin (22018- 1- AP, Proteintech), anti- Bak (A0498, Abclonal), anti- Bax (sc- 7480), anti- Bcl2 (ET7110- 51, HuaBio), anti- Caspase 3 (ET1602- 39, HuaBio), anti- c- Myc (AF0358, Affinity), anti- p21/Cip1 (27296- 1- AP, Proteintech), anti- E- Cadherin (20874- 1- AP, Proteintech), anti- P27/Kip1 (NBP1- 32213, Novus), anti- survivin (NBP2- 48494, Novus), mouse anti- HA- Tag mAb (AE065, Abclonal), and mouse anti His- Tag mAb (AE003, Abclonal).

Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Immunofluorescence, Staining

Journal: Cancer medicine

Article Title: TRIM55 inhibits colorectal cancer development via enhancing protein degradation of c-Myc.

doi: 10.1002/cam4.6020

Figure Lengend Snippet: FIGURE 4 TRIM55 overexpression suppresses cyclin D1 and c-Myc. (A) Gene Ontology (GO) enrichment analysis of the top 800 genes with TRIM55-activated or TRIM55-Suppressed correlation coefficient. (B,C) Gene set enrichment analysis (GSEA) of the enriched signaling pathways with TRIM55-High or TRIM-55-low expression using Hallmark signaling dataset. (D) SW620 and SW480 cells were transfected with empty vector or TRIM55-OE. The protein expression levels of TRIM55, survivin, P27, p21/Cip1, Cyclin D1, c-Myc, cleaved-caspase 3, Bcl2, BAX, BAK, and GAPDH were examined by western blot. (E) IHC staining of c-Myc and cyclin D1 was performed using SW620 xenograft tumor tissues. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: SDS- PAGE and western blot were conducted using the following antibodies: anti- GAPDH (BM3874, Boster), anti- TRIM55 (A15917, Abclonal), anti- MMP2 (orb12416, Biorbyt), anti- MMP9 (27306- 1- AP, Proteintech), anti- NCadherin (22018- 1- AP, Proteintech), anti- Bak (A0498, Abclonal), anti- Bax (sc- 7480), anti- Bcl2 (ET7110- 51, HuaBio), anti- Caspase 3 (ET1602- 39, HuaBio), anti- c- Myc (AF0358, Affinity), anti- p21/Cip1 (27296- 1- AP, Proteintech), anti- E- Cadherin (20874- 1- AP, Proteintech), anti- P27/Kip1 (NBP1- 32213, Novus), anti- survivin (NBP2- 48494, Novus), mouse anti- HA- Tag mAb (AE065, Abclonal), and mouse anti His- Tag mAb (AE003, Abclonal).

Techniques: Over Expression, Protein-Protein interactions, Expressing, Transfection, Plasmid Preparation, Western Blot, Immunohistochemistry

Journal: Cancer medicine

Article Title: TRIM55 inhibits colorectal cancer development via enhancing protein degradation of c-Myc.

doi: 10.1002/cam4.6020

Figure Lengend Snippet: FIGURE 6 TRIM55 decreases the expression level of c-Myc via protein ubiquitination. (A) SW620 transfected with empty vector, TRIM55-WT, or TRIM55-CS was treated with cycloheximide (CHX). c-Myc, TRIM55, and GAPDH levels in SW620 cells were analyzed via western blot. (B) HCT116 cells transfected with si-NC, si-TRIM55#1, or si-TRIM55#2 were treated with CHX for indicated times. c-Myc, TRIM55, and GAPDH levels in SW620 cells were analyzed via western blot. (C) SW620 cells transfected with empty vector, TRIM55-WT, TRIM55-CS, were treated with or without MG132. c-Myc, TRIM55, and GAPDH levels in SW620 cells were analyzed via western blot. (D) HCT116 cells transfected with si-NC, si-TRIM55#1, si-TRIM55#2, were treated with or without MG132. c-Myc, TRIM55, and GAPDH in HCT116 cells were analyzed via western blot. (E) SW620 or SW480 cells were transfected with empty vector or TRIM55 overexpression vector. DLD1 or HCT116 cells were transfected with si-NC, si-TRIM55#1, or si-TRIM55#2. The c-Myc mRNA levels were analyzed by qPCR 48 h later. (F) SW620 cells were transfected with control vector, TRIM55 WT, or TRIM55 CS. After 48 h, cell lysates were performed co-IP using anti-c-Myc antibody. The precipitated proteins were probed with Poly-Ub, c-Myc, TRIM55, and GAPDH. (G) SW620 cells were transfected with empty vector, His-c-Myc, with or without HA-TRIM55. After 48 h, cell lysates were performed co-IP using anti-His antibody. The precipitated proteins were probed with Poly-Ub, His, HA, and GAPDH.

Article Snippet: SDS- PAGE and western blot were conducted using the following antibodies: anti- GAPDH (BM3874, Boster), anti- TRIM55 (A15917, Abclonal), anti- MMP2 (orb12416, Biorbyt), anti- MMP9 (27306- 1- AP, Proteintech), anti- NCadherin (22018- 1- AP, Proteintech), anti- Bak (A0498, Abclonal), anti- Bax (sc- 7480), anti- Bcl2 (ET7110- 51, HuaBio), anti- Caspase 3 (ET1602- 39, HuaBio), anti- c- Myc (AF0358, Affinity), anti- p21/Cip1 (27296- 1- AP, Proteintech), anti- E- Cadherin (20874- 1- AP, Proteintech), anti- P27/Kip1 (NBP1- 32213, Novus), anti- survivin (NBP2- 48494, Novus), mouse anti- HA- Tag mAb (AE065, Abclonal), and mouse anti His- Tag mAb (AE003, Abclonal).

Techniques: Expressing, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Western Blot, Over Expression, Control, Co-Immunoprecipitation Assay

Journal: Cancer medicine

Article Title: TRIM55 inhibits colorectal cancer development via enhancing protein degradation of c-Myc.

doi: 10.1002/cam4.6020

Figure Lengend Snippet: FIGURE 7 Overexpression of c-Myc partially antagonizes the function of TRIM55 overexpression. (A–E) SW620 or SW480 cells were transfected with vector, TRIM55 overexpressing vector, c-Myc overexpressing vector, or both TRIM55 and c-Myc overexpressing vector. (A) The expression levels of TRIM55, c-Myc and GAPDH were analyzed by western blot 48 h later. (B) Cell viability was assessed by CCK-8 kit. (D) Cell growth was assessed by colony formation assay. (E) Cell invasion was analyzed by transwell assay. (C) SW620 and S480 cells were transfected with control vector, TRIM55 WT or TRIM55 CS overexpressing vectors. Cell growth was evaluated by colony formation assay. (F) IHC staining of TRIM55 and c-Myc was conducted using CRC tissues and the IHC score of c-Myc in TRIM55-low or TRIM55-high tissues were analyzed. (G) The graph depicted the potential regulation of c-Myc degradation by TRIM55 in CRC. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: SDS- PAGE and western blot were conducted using the following antibodies: anti- GAPDH (BM3874, Boster), anti- TRIM55 (A15917, Abclonal), anti- MMP2 (orb12416, Biorbyt), anti- MMP9 (27306- 1- AP, Proteintech), anti- NCadherin (22018- 1- AP, Proteintech), anti- Bak (A0498, Abclonal), anti- Bax (sc- 7480), anti- Bcl2 (ET7110- 51, HuaBio), anti- Caspase 3 (ET1602- 39, HuaBio), anti- c- Myc (AF0358, Affinity), anti- p21/Cip1 (27296- 1- AP, Proteintech), anti- E- Cadherin (20874- 1- AP, Proteintech), anti- P27/Kip1 (NBP1- 32213, Novus), anti- survivin (NBP2- 48494, Novus), mouse anti- HA- Tag mAb (AE065, Abclonal), and mouse anti His- Tag mAb (AE003, Abclonal).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, CCK-8 Assay, Colony Assay, Transwell Assay, Control, Immunohistochemistry

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 1 MuRF1 and MuRF2 are highly induced during skeletal muscle regeneration. (a) MuRF1, MuRF2 and Mafbx/Atrogin‐1 immunoblots 3 and 10 days after TA CTX injury. Sarcomeric actin is a loading control. (b‐g) Immunolocalization of MuRF1 and (j‐o) MuRF2, 3 and 10 days after CTX injury in TA muscle. In h‐i and p‐q, primary antibodies were not used in the assay. Immunodetection of laminin (green) was used to outline muscle fibers. (h‐i and p‐q) No primary antibodies were added to the assay. Images were obtained with a fluorescence microscope and a 40x air medium objective. Bar, 50µm

Article Snippet: The primary antibodies used for immunofluorescence were: (1) MuRF1 and (2) MuRF2 [27, 32, 33]; (3) Laminin (Cat#L9393, Sigma); (4) eMHC (Cat# ab49461, Abcam); (5) MyoD (Cat#IMG132, Imgenex); (6) Pax7 (Cat# ab34360, Abcam); (7) β‐Catenin (Cat# 18‐272‐ 196288, Genway).

Techniques: Western Blot, Control, Immunodetection, Fluorescence, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 2 Combined deletion of MuRF1 and MuRF2 causes deficient skeletal muscle regeneration. TA muscles from WT (a and e), from MuRF1 KOs (b and f), MuRF2 KOs (c and g), or from MuRF1&2 dKOs (d and h) were CTX injured and histologically analyzed 3 or 10 days later, respectively. We also histologically analyzed WT and MuRF1&2 dKO mice at 28 days (i and j respectively) after CTX. TA from MuRF1&2 dKO mice presented indications of defective regeneration in all time points analyzed (d, h and j, see text for detailed description). Bar, 20µm. (k) Average density of necrotic fibers in WT and MuRF1&2 dKO 24h after CTX injury; error bars indicate standard deviations (N=3)

Article Snippet: The primary antibodies used for immunofluorescence were: (1) MuRF1 and (2) MuRF2 [27, 32, 33]; (3) Laminin (Cat#L9393, Sigma); (4) eMHC (Cat# ab49461, Abcam); (5) MyoD (Cat#IMG132, Imgenex); (6) Pax7 (Cat# ab34360, Abcam); (7) β‐Catenin (Cat# 18‐272‐ 196288, Genway).

Techniques: Muscles

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig.3 Simultaneous deletion of MuRF1 and MuRF2 decreases the number of cells positive to Pax7 and Myod. TA muscles from WT, or MuRF1&2 dKO mice were investigated by immunofluorescence 3 (a‐d and j‐m) or 10 days (e‐h and n‐q) after CTX injections. Immunolocalization and percentage of positive eMHC positive myofibers was also determined (u). DAPI (blue), Pax7 and Myod (red), laminin (green) and eMHC (green). Images were obtained with a fluorescence microscope and a 40x air medium objective. Large bar, 50 µm, small bar 10 µm

Article Snippet: The primary antibodies used for immunofluorescence were: (1) MuRF1 and (2) MuRF2 [27, 32, 33]; (3) Laminin (Cat#L9393, Sigma); (4) eMHC (Cat# ab49461, Abcam); (5) MyoD (Cat#IMG132, Imgenex); (6) Pax7 (Cat# ab34360, Abcam); (7) β‐Catenin (Cat# 18‐272‐ 196288, Genway).

Techniques: Muscles, Immunofluorescence, Fluorescence, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 4 Simultaneous deletion of MuRF1 and MuRF2 causes decreased protein expression of myogenic factors and increased apoptosis. (a) Western blots for Myogenin, Myf‐5, FHL2, MARP2 and GAPDH in TA of MuRF1&2 dKO animals 3 and 10 days after CTX injury and silver staining showing even protein loading. Fluorescence images showing apoptotic nuclei by TUNEL assay in WT (b‐d and h‐j) and MuRF1&2 dKO (e‐g and k‐m) muscles 3 (b‐g) and 10 (h‐ m) days after CTX injury. Images were obtained with a fluorescence microscope and a 40x air medium objective. Bar, 30 µm. (n) Percentage of apoptotic nuclei in WT and MuRF1&2 dKO muscles 3 and 10 days after CTX injury

Article Snippet: The primary antibodies used for immunofluorescence were: (1) MuRF1 and (2) MuRF2 [27, 32, 33]; (3) Laminin (Cat#L9393, Sigma); (4) eMHC (Cat# ab49461, Abcam); (5) MyoD (Cat#IMG132, Imgenex); (6) Pax7 (Cat# ab34360, Abcam); (7) β‐Catenin (Cat# 18‐272‐ 196288, Genway).

Techniques: Expressing, Western Blot, Silver Staining, Fluorescence, TUNEL Assay, Muscles, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 5 Simultaneous deletion of MuRF1 and MuRF2 time‐shifts the localization/expression of β‐catenin during regeneration. TA muscles from WT (a‐e and k‐o), or MuRF1&2 dKO mice (f‐j and p‐t) were studied by immunofluorescence 3 (a‐j) or 10 days (k‐t) after CTX injections. DAPI (blue), β‐catenin (red), MyoD (green) and laminin (white). Arrows indicate nuclei simultaneously positive to MyoD, B‐catenin and Dapi. Arrow heads indicate non‐nuclear β‐catenin labeling. Images were obtained with a fluorescence microscope and a 40x air medium objective. Bar, 20 µm. (u) Number of β‐catenin positive cells/mm 2 in WT and MuRF1&2 dKO TA muscles. ***p<0.001 vs WT

Article Snippet: The primary antibodies used for immunofluorescence were: (1) MuRF1 and (2) MuRF2 [27, 32, 33]; (3) Laminin (Cat#L9393, Sigma); (4) eMHC (Cat# ab49461, Abcam); (5) MyoD (Cat#IMG132, Imgenex); (6) Pax7 (Cat# ab34360, Abcam); (7) β‐Catenin (Cat# 18‐272‐ 196288, Genway).

Techniques: Expressing, Muscles, Immunofluorescence, Labeling, Fluorescence, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 6 siRNA knock down of MuRF1 and MuRF2 reduce myogenesis. (a‐d) Phase contrast micrographs of primary myoblast culture 2 days after induction of differentiation. Images were taken with a 4x objective. Cells were siRNA knocked down for MuRF1 (b), MuRF2 (c) and MuRF1 and MuRF2 combined (d). (a) Arrowheads indicate the normal differentiation pattern generating myotubes. (b‐d) Arrows indicate a severe differentiation deficit. (e) Fusion index of siRNA knocked down cells. (f) Myotube area of siRNA knocked down cells. (g) Number of myotube by field of siRNA knocked down cells. The fusion index was calculated as the ratio of the nuclei number in myotubes with two or more nuclei versus the total number of nuclei. Ten representative images per sample were scored for myotube number and area occupied by myotubes relative to the total area ( a p<0.05 vs Control). Fluorescence images showing reduced expression of eMHC after siRNA knock down for MuRF1 (k‐m) MuRF2 (n‐p) and MuRF1 and MuRF2 combined (q‐s) compared to control (h‐j). Images were obtained with a fluorescence microscope. Bars, 20 µm

Article Snippet: The primary antibodies used for immunofluorescence were: (1) MuRF1 and (2) MuRF2 [27, 32, 33]; (3) Laminin (Cat#L9393, Sigma); (4) eMHC (Cat# ab49461, Abcam); (5) MyoD (Cat#IMG132, Imgenex); (6) Pax7 (Cat# ab34360, Abcam); (7) β‐Catenin (Cat# 18‐272‐ 196288, Genway).

Techniques: Knockdown, Control, Fluorescence, Expressing, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 7 siRNA knock down of MuRF1 and MuRF2 reduce MyoD positive cell number. Cells were siRNA knocked down for MuRF1 (e‐h), MuRF2 (i‐l) and MuRF1 and MuRF2 combined (m‐p). Immunoflurescence for Pax7 (red) and MyoD (green) and dapi staining was performed. (q) Percentage of positive nuclei for MyoD. (r) Percentage of positive nuclei for Pax7. (s) Percentage of positive nuclei for Pax7 and MyoD. Ten representative images per group were scored for percentage of positive nuclei (*p<0.05 vs Control). Images were obtained with a fluorescence microscope and a 40x air medium objective. Bar, 30 µm

Article Snippet: The primary antibodies used for immunofluorescence were: (1) MuRF1 and (2) MuRF2 [27, 32, 33]; (3) Laminin (Cat#L9393, Sigma); (4) eMHC (Cat# ab49461, Abcam); (5) MyoD (Cat#IMG132, Imgenex); (6) Pax7 (Cat# ab34360, Abcam); (7) β‐Catenin (Cat# 18‐272‐ 196288, Genway).

Techniques: Knockdown, Staining, Control, Fluorescence, Microscopy

Journal: JCSM Rapid Communications

Article Title: MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

doi: 10.1002/j.2617-1619.2019.tb00010.x

Figure Lengend Snippet: Fig. 8 siRNA knock down of MuRF1 and MuRF2 induce accumulation of BAF57 in the nucleus of primary myoblasts culture 2 days after induction of differentiation. (a‐j) Immunolocalization of BAF57 in cells siRNA knocked down for MuRF1 (c and d), MuRF2 (e and f) and MuRF1 and MuRF2 combined (g and h). (i) Representative immunoblots for BAF57 and GAPDH in cytoplasmic fraction from cells siRNA knocked down for MuRF1 (MuRF1 RNAi), MuRF2 (MuRF2 RNAi) and MuRF1 and MuRF2 combined (MuRF1/2 RNAi). (j) Representative immunoblots for BAF57 and GAPDH in nuclear fraction from cells siRNA knocked down for MuRF1 (MuRF1 RNAi), MuRF2 (MuRF2 RNAi) and MuRF1 and MuRF2 combined (MuRF1/2 RNAi). Bar, 40 µm

Article Snippet: The primary antibodies used for immunofluorescence were: (1) MuRF1 and (2) MuRF2 [27, 32, 33]; (3) Laminin (Cat#L9393, Sigma); (4) eMHC (Cat# ab49461, Abcam); (5) MyoD (Cat#IMG132, Imgenex); (6) Pax7 (Cat# ab34360, Abcam); (7) β‐Catenin (Cat# 18‐272‐ 196288, Genway).

Techniques: Knockdown, Western Blot

Journal: The Journal of physiology

Article Title: Preferential skeletal muscle myosin loss in response to mechanical silencing in a novel rat intensive care unit model: underlying mechanisms.

doi: 10.1113/jphysiol.2010.202044

Figure Lengend Snippet: Figure 10. Cross-sections of rat gastrocnemius muscle Cross-sections of rat gastrocnemius muscle stained for the MuRF1 (red) or MuRF2 (green) and DAPI (blue). In control muscles, both MuRF1 and -2 are localised to the cytoplasm, and nuclear localisation is not prominent (arrowheads). ICU conditions induce strong nuclear translocation of both MuRFs that persists up to 9 days. At this stage, MuRFs are colocalising in the nucleus (arrowheads) but also accumulate in a perinuclear area in the cytoplasm (arrows). Scale bars, 10 μm. The overlay image for each row is shown at the left. All sections stained simultaneously and images recorded with identical gain settings. See Methods for details.

Article Snippet: First antibodies were: goat-anti MuRF1 or -2 (Abcam, UK), MuRF2-specific rabbit polyclonal antibodies (Pizon et al. 2002), mouse monoclonal p62 (Abcam, UK) and rabbit anti-SRF (Santa Cruz biotechnology Inc., CA, USA).

Techniques: Staining, Control, Muscles, Translocation Assay

Journal: Brazilian Journal of Medical and Biological Research

Article Title: The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts

doi: 10.1590/1414-431X20198551

Figure Lengend Snippet: MuRF2 is expressed in fibroblasts. Semi-quantitative PCR for MuRF1 ( A ) and MuRF2 ( B ), and cyclophilin as a housekeeping gene ( C ) in skeletal muscle fibroblasts, C2C12, lung, and pancreatic islets. Immunofluorescence for MuRF2 in skeletal muscle fibroblasts ( D and G ), cardiac fibroblasts ( E and H ), and mouse embryonic fibroblasts (MEFs) ( F and I ). J : Real-time PCR in skeletal muscle fibroblasts treated with scrambled (SCRBd) or MuRF2 siRNA. K : Representative immunoblots for MuRF2 and GAPDH in skeletal muscle fibroblasts treated with SCRBd or MuRF2 siRNA. The images belong to the same membrane. L : Relative band intensities were determined by densitometry. Scale bar, 50 µM ( D-I ). Data are reported as means±SE. *P<0.05 vs SCRBd (Student’s t -test).

Article Snippet: Primary antibodies used were: MuRF2 ( ); Akt (Cell Signaling #9272, USA); GAPDH (Cell Signaling #2118); pAkt Ser473 (Cell Signaling #4058); pAkt Thr308 (Cell Signaling #4056).

Techniques: Real-time Polymerase Chain Reaction, Immunofluorescence, Western Blot, Membrane

Journal: Brazilian Journal of Medical and Biological Research

Article Title: The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts

doi: 10.1590/1414-431X20198551

Figure Lengend Snippet: Knock-down of MuRF2 in skeletal muscle fibroblasts decreased lamellipodia formation. Staining for phalloidin and DAPI 24 h after the onset of wound healing assays at low ( A and C , 100×; bar, 100 µM) and high ( B and D , 400×; bar, 50 µM) magnifications; Skeletal muscle fibroblasts treated with scrambled (SCRBd) ( A and B ) and with MuRF2 siRNA ( C and D ). Arrowheads indicate actin stress fibers, asterisks indicate misfolded actin cytoskeleton, and arrows indicate cell migration direction. E : Quantification of lamellipodia-containing cells. F : Representative immunoblots of pAkt Ser473, pAkt Thr308, total Akt, and GAPDH in skeletal muscle fibroblasts. The images belong to the same membrane. G - J : Densitometry from western blots. K : Real-time PCR for collagen 1 (Col1) in skeletal muscle fibroblast. Data are reported means±SE. *P<0.05 vs SCRBd ( t -test).

Article Snippet: Primary antibodies used were: MuRF2 ( ); Akt (Cell Signaling #9272, USA); GAPDH (Cell Signaling #2118); pAkt Ser473 (Cell Signaling #4058); pAkt Thr308 (Cell Signaling #4056).

Techniques: Knockdown, Staining, Migration, Western Blot, Membrane, Real-time Polymerase Chain Reaction

Journal: Brazilian Journal of Medical and Biological Research

Article Title: The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts

doi: 10.1590/1414-431X20198551

Figure Lengend Snippet: Knock-down of MuRF2 in skeletal muscle fibroblasts decreased lamellipodia formation. Staining for phalloidin and DAPI 24 h after the onset of wound healing assays at low ( A and C , 100×; bar, 100 µM) and high ( B and D , 400×; bar, 50 µM) magnifications; Skeletal muscle fibroblasts treated with scrambled (SCRBd) ( A and B ) and with MuRF2 siRNA ( C and D ). Arrowheads indicate actin stress fibers, asterisks indicate misfolded actin cytoskeleton, and arrows indicate cell migration direction. E : Quantification of lamellipodia-containing cells. F : Representative immunoblots of pAkt Ser473, pAkt Thr308, total Akt, and GAPDH in skeletal muscle fibroblasts. The images belong to the same membrane. G - J : Densitometry from western blots. K : Real-time PCR for collagen 1 (Col1) in skeletal muscle fibroblast. Data are reported means±SE. *P<0.05 vs SCRBd ( t -test).

Article Snippet: Primary antibodies used were: MuRF2 ( ); Akt (Cell Signaling #9272, USA); GAPDH (Cell Signaling #2118); pAkt Ser473 (Cell Signaling #4058); pAkt Thr308 (Cell Signaling #4056).

Techniques: Knockdown, Staining, Migration, Western Blot, Membrane, Real-time Polymerase Chain Reaction