Journal: Redox Report : Communications in Free Radical Research

Article Title: ROS-Drp1-mitophagy feedback loop regulates myogenic differentiation via actin cytoskeleton remodeling-mediated MRTF-A/SRF axis

doi: 10.1080/13510002.2025.2536400

Figure Lengend Snippet: ROS-Drp1-mitophagy feedback loop controls myogenic differentiation by actin skeleton remodeling mediated MRTF-A/SRF activity. (A-B) Western blot analysis for F-actin, cofilin and p-cofilin was performed at 1 d of differentiation after C2C12 cells were treated with the combination of siDrp1, OE-Pink1 and 5 μM Antimycin A (AA). Mouse Gapdh was used to normalize protein expression. n = 3. (C-D) Western blot analysis for nuclear MRTF-A (n-MRTF-A) expression was performed at 1 d of differentiation after C2C12 cells were treated with the combination of siDrp1, OE-Pink1 and 5 μM AA. Histone H3 was used to normalize protein expression. n = 3. (E-F) Co-immunoprecipitation analysis was performed at 1 d of differentiation to determine the combination of MRTF-A with SRF after C2C12 cells were treated with the combination of siDrp1, OE-Pink1 and 5 μM AA. n = 3. (G-H) Western blot analysis for MyHC was performed at 3 d of differentiation after C2C12 cells were treated with the combination of siDrp1, OE-Pink1 and 5μM AA. Mouse Gapdh was used to normalize protein expression; n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies against Tom70 (14528-1-AP), SRF (66742-1-Ig), and MRTF-A for WB/IF were purchased from the Proteintech Group (Wuhan, China).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation

Journal: Redox Report : Communications in Free Radical Research

Article Title: ROS-Drp1-mitophagy feedback loop regulates myogenic differentiation via actin cytoskeleton remodeling-mediated MRTF-A/SRF axis

doi: 10.1080/13510002.2025.2536400

Figure Lengend Snippet: AAV9-shDrp1 injection blocks skeletal muscle regeneration by regulating actin/MRTF-A/SRF axis. (A) The Drp1 protein expression was detected at 4 w after AAV9-shNC or AAV9-shDrp1 injection. Mouse Gapdh was used to normalize protein expression. n = 3. (B) The weight of TA muscle at 11 d after CTX injection was examined in the AAV9-shNC and AAV9-shDrp1 group. n = 3. (C) H&E staining of TA muscle at 11 d after CTX injection in the AAV9-shNC and AAV9-shDrp1 group. Red arrows indicate the cell nuclei located in the center of muscle fibers. Scale bar, 100 μm. (D) IF staining of dystrophin at 11 d after CTX injection in AAV9-shNC and AAV9-shDrp1 group. White arrows indicate the cell nuclei located in the center of muscle fibers. Scale bar, 100 μm. (E-F) Western blot analysis for MyHC at 3, 5, 7, 9 and 11 d after CTX injection in the AAV9-shNC and AAV9-shDrp1 group. Mouse Gapdh was used to normalize protein expression. n = 3 at each time point. (G-I) Western blot analysis for F-actin, p-cofilin and cofilin was performed at 3 and 5 d after CTX injection in the AAV9-shNC and AAV9-shDrp1 group. Mouse Gapdh was used to normalize protein expression; n = 3. (J-K) Co-immunoprecipitation of MRTF-A and SRF in TA muscle at 5 d after CTX injection in the AAV9-shNC and AAV9-shDrp1 group. n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies against Tom70 (14528-1-AP), SRF (66742-1-Ig), and MRTF-A for WB/IF were purchased from the Proteintech Group (Wuhan, China).

Techniques: Injection, Expressing, Staining, Western Blot, Immunoprecipitation

Journal: Redox Report : Communications in Free Radical Research

Article Title: ROS-Drp1-mitophagy feedback loop regulates myogenic differentiation via actin cytoskeleton remodeling-mediated MRTF-A/SRF axis

doi: 10.1080/13510002.2025.2536400

Figure Lengend Snippet: Schematic model of ROS-Drp1-mitophagy feedback loop promotes myogenesis by actin cytoskeleton remodeling-mediated MRTF-A/SRF axis. At the beginning of myogenic differentiation, mitochondrial damage and reactive oxygen species (ROS) levels were elevated. Elevated ROS levels promote Drp1 expression to activate mitophagy and thereby form a ROS-Drp1-mitophagy feedback loop for balancing ROS levels. The ROS-Drp1-mitophagy axis regulates actin skeleton remodeling by promoting p-cofilin expression, allowing MRTF-A to translocate into nuclei and combine with SRF, thereby promoting skeletal muscle differentiation.

Article Snippet: Antibodies against Tom70 (14528-1-AP), SRF (66742-1-Ig), and MRTF-A for WB/IF were purchased from the Proteintech Group (Wuhan, China).

Techniques: Expressing

Journal: Journal of Personalized Medicine

Article Title: Exosomes from Microvascular Endothelial Cells under Mechanical Unloading Inhibit Osteogenic Differentiation via miR-92b-3p/ELK4 Axis

doi: 10.3390/jpm12122030

Figure Lengend Snippet: ELK4 is a direct target of miR-92b-3p and is responsible for miR-92b-3p-mediated suppression of osteogenic differentiation in MC3T3-E1 cells. ( A ) qRT–PCR analysis of ELK4 mRNA expression in MC3T3-E1 cells after transfection of mimic-92b-3p, inhibitor-92b-3p, or the corresponding control ( n = 3). ( B ) Western blotting analysis of the protein expression of ELK4 in MC3T3-E1 cells ( n = 3). ( C ) The relative luciferase activities of the ELK4 WT and MUT reporters were assessed after 293T cells were treated for 48 h with mimic-92b-3p and the equivalent controls ( n = 3). ( D ) Schematic representation of the luciferase reporters containing ELK4 3′-UTR WT or MUT sequences. ( E ) mRNA levels of ELK4 analyzed by qRT–PCR in MC3T3-E1 cells treated with Con Exos/Clino Exos (200 μg/mL) ( n = 3). ( F ) Protein levels of ELK4 analyzed by Western blotting ( n = 3). ( G ) qRT–PCR analysis of ALP, Osx, Runx2, and Ocn in MC3T3-E1 cells after the co-transfection of inhibitor-92b-3p, si-ELK4 and their negative controls in MC3T3-E1 cells ( n = 3). ( H ) Western blotting analysis of Osx, Runx2, and Ocn expression in MC3T3-E1 cells ( n = 3). ( I ) ALP activity analysis in MC3T3-E1 cells ( n = 3). ( J ) Representative images of ALP staining in MC3T3-E1 cells ( n = 3). * p < 0.05, ** p < 0.01 vs. control.

Article Snippet: After incubation with 5% skim milk (5% w / v ) for 2 h at room temperature, the membranes were co-incubated overnight at 4 °C with the following primary antibodies specific for GAPDH (1:1000; Cell Signaling Technology, USA), Runx2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), Osx (1:1000; Abcam, Cambridge, UK), Ocn (1:2000; Abcam, UK), ELK4 (1:1000; Proteintech, Rosemont, IL, USA), GM130 (1:1000; Proteintech, USA), CD9 (1:1000; Proteintech, USA), and TSG101 (1:1000; Proteintech, USA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Luciferase, Cotransfection, Activity Assay, Staining

Journal: Journal of Personalized Medicine

Article Title: Exosomes from Microvascular Endothelial Cells under Mechanical Unloading Inhibit Osteogenic Differentiation via miR-92b-3p/ELK4 Axis

doi: 10.3390/jpm12122030

Figure Lengend Snippet: A schematic diagram illustrating the molecular mechanisms of which exosomes derived from MVECs cultured under mechanical unloading regulate osteogenic differentiation. miR-92b-3p expression was increased in MVEC-secreted exosomes after mechanical unloading, resulting in the upregulation of miR-92b-3p expression in MC3T3-E1 cells cocultured with Clino Exos. ELK4, the direct target of miR-92b-3p, is decreased in MC3T3-E1 cells treated with Clino Exos, thus inhibiting osteogenic differentiation. The blue arrow represents inhibition, and the red arrow represents promotion.

Article Snippet: After incubation with 5% skim milk (5% w / v ) for 2 h at room temperature, the membranes were co-incubated overnight at 4 °C with the following primary antibodies specific for GAPDH (1:1000; Cell Signaling Technology, USA), Runx2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), Osx (1:1000; Abcam, Cambridge, UK), Ocn (1:2000; Abcam, UK), ELK4 (1:1000; Proteintech, Rosemont, IL, USA), GM130 (1:1000; Proteintech, USA), CD9 (1:1000; Proteintech, USA), and TSG101 (1:1000; Proteintech, USA).

Techniques: Derivative Assay, Cell Culture, Expressing, Inhibition

Journal: PeerJ

Article Title: Identification and validation of immune related core transcription factors GTF2I in NAFLD

doi: 10.7717/peerj.13735

Figure Lengend Snippet: The primer sequences.

Article Snippet: After protein quantification using a microplate reader, an appropriate amount of loading buffer was added and subsequently changed by heating in 95 °C for 15 min. After polyacrylamide gel electrophoresis (30 g protein usage) and subsequent routine western blot experimental steps, GAPDH (diluted 1:25,000, 60004-1, Proteintech, USA), CHD2 (diluted 1:1,000, 12311-1-AP, Proteintech, USA) were respectively incubated overnight with the corresponding PVDF membrane region with the GTF2I (1:1,000, ab248269, abcam, USA) antibody at 4 °C overnight.

Techniques:

Journal: PeerJ

Article Title: Identification and validation of immune related core transcription factors GTF2I in NAFLD

doi: 10.7717/peerj.13735

Figure Lengend Snippet: (A–B) The mRNA and protein of GTF2I was high expression in the FFA group, while the difference in CHD2 was not obvious. (C) H & E staining was used as pathological evidence for the diagnosis of NAFLD. (D–E) RT-qPCR revealed the mRNA expression of both GTF2I and CHD2 were significantly higher in NAFLD patients than in normal participants. (F) WB showed that GTF2I but not CHD2 was significantly higher in NAFLD patients than in normal participants. (G) The immunohistochemical results of GTF2I in liver tissues of NAFLD and normal participants. Arrows served as a marker of positive areas. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After protein quantification using a microplate reader, an appropriate amount of loading buffer was added and subsequently changed by heating in 95 °C for 15 min. After polyacrylamide gel electrophoresis (30 g protein usage) and subsequent routine western blot experimental steps, GAPDH (diluted 1:25,000, 60004-1, Proteintech, USA), CHD2 (diluted 1:1,000, 12311-1-AP, Proteintech, USA) were respectively incubated overnight with the corresponding PVDF membrane region with the GTF2I (1:1,000, ab248269, abcam, USA) antibody at 4 °C overnight.

Techniques: Expressing, Staining, Biomarker Discovery, Quantitative RT-PCR, Immunohistochemical staining, Marker

Journal: eLife

Article Title: CCDC113 stabilizes sperm axoneme and head-tail coupling apparatus to ensure male fertility

doi: 10.7554/eLife.98016

Figure Lengend Snippet: ( A–C, F ) Head-tail coupling apparatus (HTCA)-associated proteins (SUN5, CENTLEIN, PMFBP1, SPATA6) were expressed alone or co-expressed with CCDC113 in HEK293T cells, and the interactions between CCDC113 and these HTCA-associated proteins were examined by co-immunoprecipitation. CCDC113 interacted with SUN5 and CENTLEIN, but did not interact with PMFBP1 and SPATA6. IB: immunoblotting; IP: immunoprecipitation. ( D ) SUN5 interacted with CCDC113. pECMV-FLAG- Ccdc113 and pEGFP-GFP- Sun5 were transfected into HEK293T cells. At 48 hr after transfection, the cells were collected for immunoprecipitation (IP) with anti-GFP antibody and analyzed with anti-FLAG and anti-GFP antibodies. ( E ) CENTLEIN interacted with CCDC113. pCDNA -FLAG- Centlein and pEGFP-GFP- Ccdc113 were transfected into HEK293T cells. At 48 hr after transfection, the cells were collected for IP with anti-FLAG antibody and analyzed with anti-FLAG and anti-GFP antibodies. The % Input is displayed below the corresponding figures for quantification. n=3 independent experiments. Data are presented as mean ± SD; *p<0.05, ns indicates no significant difference. ( G ) Immunofluorescence of CCDC113 (red) and SUN5 (green) in mature spermatozoa. Nuclei were stained with DAPI (blue). ( H ) Immunofluorescence of CCDC113 (red) and CENTLEIN (green) in testicular step 13–14 spermatid. Nuclei were stained with DAPI (blue). ( I ) Immunofluorescence analysis for SPATA6 (green) and α/β-tubulin (red) was performed in Ccdc113 +/+ and Ccdc113 –/– spermatozoa. Nuclei were stained with DAPI (blue). ( J ) Quantification ratio of SPATA6 on the detached sperm tail (n=3 independent experiments). At least 200 spermatozoa were analyzed for each mouse. ( K ) Quantification ratio of CCDC113 on the detached sperm tail (n=3 independent experiments). At least 200 spermatozoa were analyzed from each mouse. Data are presented as mean ± SD; ***p<0.001, ****p<0.0001. ( L ) Immunofluorescence analysis for CCDC113 (red) was performed in wild-type (WT), Sun5 –/– , Centlein –/– , and Pmfbp1 –/– spermatozoa. Nuclei were stained with DAPI (blue). Figure 7—source data 1. Original files for western blot in . Figure 7—source data 2. Labelled files for western blot in .

Article Snippet: Rabbit anti-SPATA6 antibody (11849-1 AP, Proteintech) was used at a 1:100 dilution for immunofluorescence.

Techniques: Immunoprecipitation, Western Blot, Transfection, Immunofluorescence, Staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Targeting AGGF 1 (angiogenic factor with G patch and FHA domains 1) for Blocking Neointimal Formation After Vascular Injury

doi: 10.1161/JAHA.117.005889

Figure Lengend Snippet: AGGF 1 regulates expression of phenotypic switching markers of vascular smooth muscle cells ( VSMC s). A, The platelet‐derived growth factor subunit B homodimer ( PDGF ‐ BB ) decreases the expression levels of α‐ SMA (α smooth muscle actin), SM 22 (smooth muscle protein 22‐α or transgelin), and MYH 11 (myosin heavy polypeptide 11, smooth muscle) at the protein level. PDGF ‐ BB does not affect the expression level of AGGF 1. B, AGGF 1 blocks PDGF ‐induced downregulation of contractile markers at the protein level. C, AGGF 1 blocks PDGF ‐induced downregulation of contractile markers at the mRNA level. NC indicates negative control. D, AGGF 1 increases the expression levels of α‐ SMA , SM 22, and MYH 11 in mouse VSMC line MOVAS ‐1 VSMC s. E, AGGF 1 increases the expression levels of α‐ SMA , SM 22, and MYH 11 in primary VSMC s isolated from mouse aortas. F, The expression levels of α‐ SMA , SM 22, and MYH 11 in MOVAS ‐1 VSMC s are significantly less than in primary mouse aortic VSMC s. G, Knockdown of SRF encoding the serum response factor by si RNA (si SRF ) abolishes the effect of AGGF 1 on PDGF at the protein level. H, Knockdown of SRF by si RNA (si SRF ) abolishes the effect of AGGF 1 on PDGF at the mRNA level. * P <0.05 (n=3/group). NS indicates not significant.

Article Snippet: The 6xHis‐tagged AGGF1 protein was purified as described by us previously., , Antibodies against AGGF1, SM22, α‐SMA, MYH11, serum response factor (SRF), myocardin, and Enhanced Green Fluorescent Protein (EGFP) were from Proteintech.

Techniques: Expressing, Derivative Assay, Negative Control, Isolation, Knockdown

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Targeting AGGF 1 (angiogenic factor with G patch and FHA domains 1) for Blocking Neointimal Formation After Vascular Injury

doi: 10.1161/JAHA.117.005889

Figure Lengend Snippet: AGGF 1 regulates transcriptional activation of vascular smooth muscle cells ( VSMC s) phenotypic switching markers. A, Luciferase assays showing that AGGF 1 increases transcriptional activation of VSMC s contractile marker genes encoding α‐ SMA (α smooth muscle actin), SM 22 (smooth muscle protein 22‐α or transgelin), and MYH 11 (myosin heavy polypeptide 11, smooth muscle) in the presence of SRF (serum response factor) ( SRF vs SRF +rh AGGF 1). NC indicates negative control. B, Luciferase assays showing that the platelet‐derived growth factor subunit B homodimer ( PDGF ‐ BB ) represses SRF ‐induced transcriptional activation of VSMC s contractile marker genes encoding α‐ SMA , SM 22, and MYH 11, but the effects are abolished by AGGF 1 protein. C, Chromatin immunoprecipitation assays to detect protein– DNA interaction between SRF and the CA rG elements at the promoter/regulatory regions of VSMC s contractile marker genes. PDGF reduces the SRF binding to CA rG elements, but the effects are abolished by AGGF 1 protein. D, Co‐immunoprecipitation assays showing that the AGGF 1 protein increases the interaction between SRF and myocardin in MOVAS ‐1 VSMC s with overexpression of both myocardin and SRF . An anti‐myocardin antibody was used for immunoprecipitation, and an anti‐ SRF antibody was used for immunoblotting. E, Co‐immunoprecipitation assays showing that PDGF reduced the interaction between SRF and myocardin, but the effect was reversed by AGGF 1. * P <0.05 and ** P <0.01 (n=3/group).

Article Snippet: The 6xHis‐tagged AGGF1 protein was purified as described by us previously., , Antibodies against AGGF1, SM22, α‐SMA, MYH11, serum response factor (SRF), myocardin, and Enhanced Green Fluorescent Protein (EGFP) were from Proteintech.

Techniques: Activation Assay, Luciferase, Marker, Negative Control, Derivative Assay, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Over Expression, Western Blot