Journal: Nature genetics

Article Title: Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease

doi: 10.1038/s41588-024-01669-y

Figure Lengend Snippet: a . The CHD MPRA library included 6590 REF-ALT pairs. After pooled library synthesis of barcoded oligos, the oligos were PCR amplified and cloned into lentivirus genome backbone. A minimal promoter (miniP)-GFP cassette was then inserted into the cloned oligo library. b . Summary of activity of CHD MPRA library. Plot on bottom indicates the occurrence of the indicated annotation with a vertical line. Enrichment score represents enrichment of the indicated set of annotations at either end of the list of all regions, ranked by activity. Enrichment p-value was determined by 1-sided permutation test, with Bonferroni correction. Active enhancers had barcodes overrepresented in RNA compared to DNA (DESeq2 P adj < 0.05). c . Pearson correlation (PCC) between regions shared between the Mutagenesis MPRA and the CHD MPRA. The same genomic sequences had different barcodes in the two assays. d . Validation of the effect of variants on transcription factor binding. EMSA assay was used to test the binding of SRF or TBX20 to REF or ALT variant sequences. For the GLB1L3 CRE, ALT disrupted the SRF motif and reduced SRF binding in the EMSA assay. For the PIP4K2A CRE, ALT generated a TBX20 motif and increased TBX20 binding in the EMSA assay. Representative of three independent experiments. Two-tailed t-test. n = 3 per group. Graph shows mean ± SD.

Article Snippet: Recombinant human proteins used in this study included SMAD2 (Abcam, ab85329), SRF (OriGene, TP308596), TBX20 (OriGene, TP762422), HIC2 (OriGene, TP760963), SOX9 (OriGene, TP308944) and GATA4 (OriGene, TP310945).

Techniques: Amplification, Clone Assay, Activity Assay, Mutagenesis, Genomic Sequencing, Biomarker Discovery, Binding Assay, Variant Assay, Generated, Two Tailed Test

Journal: Nature genetics

Article Title: Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease

doi: 10.1038/s41588-024-01669-y

Figure Lengend Snippet: a . BCOR downregulation in SMAD2 Het and KO iPSC-CMs. Gene expression was measured by RNA-seq. One-way ANOVA with Dunnett’s multiple comparison test versus WT. n = 3. b . Effect of ncDNVs on binding of transcription factors to CREs near CHD genes. 39 bp duplexes centered on ncDNVs neighboring 4 CHD genes were synthesized. Binding of purified, recombinant proteins to the REF or ALT sequence was measured by electrophoretic mobility shift assay (EMSA). SMAD2 and HIC2 bound CREs near BCOR and ACVRL1 more strongly for REF compared to ALT. In contrast, SRF and TBX20 bound CREs near ADAMTS6 and MYOCD more strongly for ALT compared to REF. Note lower free probe in MYOCD -ALT compared to REF. Results are representative of at least three independent experiments. Quantification of TBX20 EMSA: mean ± SD; n = 3; two-sided t-test. Graphs in a and b show mean ± SD.

Article Snippet: Recombinant human proteins used in this study included SMAD2 (Abcam, ab85329), SRF (OriGene, TP308596), TBX20 (OriGene, TP762422), HIC2 (OriGene, TP760963), SOX9 (OriGene, TP308944) and GATA4 (OriGene, TP310945).

Techniques: Gene Expression, RNA Sequencing, Comparison, Binding Assay, Synthesized, Purification, Recombinant, Sequencing, Electrophoretic Mobility Shift Assay

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: Nature communications

Article Title: Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation.

doi: 10.1038/ncomms10830

Figure Lengend Snippet: Figure 7 | Nuclear PTEN blocks PDGF-mediated repression of SM gene transcription. (a,b) SMCs were serum-restricted for 48 h followed by stimulation with vehicle control or 20 ng ml 1 PDGF-BB for 24 h (a) or 48 h (b). (a) SMCs were fixed, immunofluorescently stained for PTEN (green) and analysed for PTEN localization using confocal microscopy; nuclei were stained for DAPI (blue). (b) PTEN was immunoprecipitated (IP) from cytoplasmic (cyto) and nuclear (nuc) fractions of vehicle- or PDGF-stimulated SMCs. Co-immunoprecipitating SRF was detected by immunoblotting (IB). Representative western blot from three separate experiments. (c) SMCs were transfected with a construct expressing SRF–GFP, maintained in serum-restricted conditions or stimulated with 20 ng ml 1 PDGF-BB, fixed and analysed for GFP localization; nuclei were stained for DAPI (blue). Shown are representative images (two serum-restricted and four PDGF-stimulated cells are shown); arrows indicate cytoplasmic localized SRF–GFP; nuclei are outlined with white lines. (d) SMCs were transfected with HA-tagged wild-type PTEN (WT), nuclear localized PTEN (NLS) or nuclear excluded PTEN (NES). SMCs were maintained in serum-restricted conditions or stimulated with 20 ng ml 1 PDGF-BB, fixed, immunofluorescently stained for HA (red) and analysed for PTEN localization; nuclei were stained for DAPI (blue). Arrowheads, HA–PTEN-transfected SMCs. (e) SMCs were transfected with GFP–SRF (ctrl) or co-transfected with GFP–SRF and WT PTEN or nuclear localized PTEN (NLS) then maintained in serum-restricted conditions or stimulated with 20 ng ml 1

Article Snippet: Plasmid-encoding human SRF tagged with GFP (RG208596) was obtained from OriGene.

Techniques: Control, Staining, Confocal Microscopy, Immunoprecipitation, Western Blot, Transfection, Construct, Expressing