serum response factor  (Proteintech)

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, 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: Expressing, RNA Sequencing Assay, Comparison, Binding Assay, Synthesized, Purification, Recombinant, Sequencing, Electrophoretic Mobility Shift Assay

Journal: BMB Reports

Article Title: Quercetin induces dual specificity phosphatase 5 via serum response factor

doi: 10.5483/BMBRep.2023-0051

Figure Lengend Snippet: SRF transcription factor modulated the expression of DUSP5 gene. (A) A representative Western blot image demonstrates the protein expression levels of DUSP5 and p-ERK1. The transfection efficiency of SRF siRNA was assessed using an anti-SRF antibody. (B) HCT116 cells were transiently co-transfected with the human DUSP5 promoter region and siRNA. The induction of the DUSP5 promoter was quantified using a luciferase assay. The data presented are relative to the DMSO-treated sample, which is assigned a value of 1.0. (C) SRF knock-down HCT116 cells were treated with DMSO or quercetin. Cell viability was determined using an MTS assay. The DMSO-treated samples were designated as 100%. (D) A chromatin immunoprecipitation (ChIP) assay was performed to quantify the enhancement of the DUSP5 gene using quantitative real-time PCR (qRT-PCR). The fold enrichment of ChIP was calculated using the comparative Ct method and normalized to normal rabbit IgG. All bar graphs represent the mean data ± standard deviation (SD) from three independent experiments. Statistical significance was determined using *P < 0.05, **P < 0.01, and ***P < 0.001. (E) The molecular mechanism underlying the effects of quercetin on SRF and DUSP5 expression can be summarized by a schematic diagram. Quercetin induces the upregulation of DUSP5 through the activation of SRF. This increased expression of DUSP5 potentially affects the dephosphorylation of ERK. However, it should be noted that quercetin itself can mediate the phosphorylation of ERK, which ultimately contributes to the anticancer activity exhibited by quercetin.

Article Snippet: Serum response factor (SRF) small interfering RNA (siRNA) (sc-36563, Santa Cruz) or negative control siRNA (Bioneer, Daejeon, Korea) were transfected in HCT116 cells using PepMuteTM siRNA Transfection Reagent (SignaGen).

Techniques: Expressing, Western Blot, Transfection, Luciferase, Knockdown, MTS Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Activation Assay, De-Phosphorylation Assay, Phospho-proteomics, Activity Assay