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: 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

Journal: Stem Cells International

Article Title: Resveratrol Enhances Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells through Inhibiting Canonical WNT Signal Pathway and Enhancing Serum Response Factor-miR-1 Axis

doi: 10.1155/2016/2524092

Figure Lengend Snippet: Serum response factor- (SRF-) miRNA-1 axis also involves the CMs differentiation enhanced by RSV. (a) Transcription levels of SRF in cultured EBs treated with RSV (50 μ M) were detected by qRT-PCR on day 4. (b, c) Western Blot was further carried out to determine the protein levels of RSV (50 μ M) treated EBs after 4 days. (d) miRNA-1 expression level was also detected using qRT-PCR 4 days after RSV (50 μ M) administration. (e, f) Knockdown of SRF using produced Lentivirus expressing shRNA targeting SRF was confirmed by Western Blot analysis. (g) SRF-miRNA-1 signal axis was confirmed in EBs treated with RSV (50 μ M) on day 4. (h) Inhibition of endogenous miRNA-1 in floating cultured EBs 4 days after transfection of Lentivirus within anti-miRNA-1. (i) The effects of modulation of SRF or/and miRNA-1 on the ratio of beating EBs were evaluated on day 24 ( n = 100). Data represent the mean ± s.d. of three biological replicates. ∗ P < 0.05 compared with control; # P < 0.05 compared with RSV alone.

Article Snippet: Human SRF shRNA lentiviral particles and the control shRNA lentiviral particles were purchased from OriGene (TL320530).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Produced, shRNA, Inhibition, Transfection, Control

Journal: Journal of molecular neuroscience : MN

Article Title: Interactions of Antibodies to the Gram-Negative Gastric Bacterium Helicobacter pylori with the Synaptic Calcium Sensor Synaptotagmin 5, Correlate to Impaired Vesicle Recycling in SiMa Human Neuroblastoma Cells.

doi: 10.1007/s12031-020-01670-0

Figure Lengend Snippet: Fig. 3 Western blot analysis of the cross-reactivity of antibodies directed to Helicobacter pylori (α-HPy) and Campylobacter jejuni (α-CJe) with different protein samples as provided by commercial HEK-293 overexpression lysates. a Cross-reactivity of α-HPy as revealed by a distinct immunopositive band can be observed for Syt5 and Vglut1 (Slc17a7), whereas Stmn4 and Ncan reveal no such band. Also a control lysate of non-transfected HEK293 cells, as well as an overexpression lysate of Srf is negative. b For α-CJe cross-reactivity as revealed by a distinct immunopositive band can be observed for Syt5 and Vglut1, whereas also in this case Stmn4 and Ncan reveal no such band. Again a control lysate of non-transfected HEK293 cells, as well as an overexpression lysate of Srf is negative. A corresponding negative control incubated with secondary antibodies only is shown in Supplementary Fig. 2

Article Snippet: Human SRFtransfected HEK293 overexpression lysate (Origene, cat. no. LY418874). hEXselect Multiprotein Array Analysis A high-density multiprotein array (MPA) (hEXselect, Engine, Berlin, Germany, Order No. 1003) derived from a cDNAbank of two first trimester human fetal brain samples, containing 23,806 E. coli expression clones representing a total of 10,000 human proteins (Büssow et al. 1998; Horn et al. 2006), was incubated with either α-HPy or α-CJe according to the manufacturer’s protocol.

Techniques: Western Blot, Over Expression, Control, Transfection, Negative Control, Incubation

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