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: ( 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 PDGF-BB. Transfected SMCs exhibiting cytoplasmic GFP expression were scored as described in Methods. Data represent average per cent positive±s.e.m. N =3 independent experiments; * P <0.01 versus control 0.1% CS; ** P <0.01 versus control PDGF and WT PTEN PDGF; *** P <0.01 versus control PDGF. ( f ) Non-transfected (Ctrl) and HA–PTEN-transfected SMCs were co-transfected with an Acta2 promoter-Luciferase reporter construct. SMCs were serum-restricted for 48 h followed by stimulation with vehicle control or 20 ng ml −1 PDGF-BB for an additional 24 h. Luciferase activity normalized to β-galactosidase was determined; shown are fold changes from Ctrl serum-restricted (0.1% CS) SMCs. Data represent average fold changes±s.e.m. N =3 independent experiments; * P <0.01 versus Ctrl 0.1% CS; ** P <0.01 versus Ctrl PDGF. Molecular weight markers were cropped out for final SRF blot; please see . Scale bars for all images, 20 μm.

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

Techniques: Staining, Confocal Microscopy, Immunoprecipitation, Western Blot, Transfection, Construct, Expressing, Luciferase, Activity Assay, Molecular Weight

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: 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, Produced, shRNA, Inhibition, Transfection

Journal: Journal of Molecular Neuroscience

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: 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.

Article Snippet: Human SRF -transfected HEK293 overexpression lysate (Origene, cat. no. LY418874).

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

Journal: Cell Death & Disease

Article Title: Both direct and indirect suppression of MCL1 synergizes with BCLXL inhibition in preclinical models of gastric cancer

doi: 10.1038/s41419-025-07481-8

Figure Lengend Snippet: A , B Role of ELK1, STAT3 and SRF in regulating the transcriptional activity of MCL1 under basal conditions and in response to HER2-targeting drugs. SNU-216 cells expressing empty vector or the indicated transcriptional factors were left untreated or treated with 100 ng/ml trastuzumab ( A ) or 200 nM lapatinib ( B ) for 72 h or 48 h, respectively. The levels of MCL1 mRNA were determined using RT-qPCR. C Effect of HER2-targeting drugs on STAT3 and SRF expression. SNU-216, NCI-N87 and GCIY were treated with 100 ng/ml trastuzumab or 200 nM lapatinib for 72 h or 48 h, respectively. The protein levels of p-STAT3, total STAT3, and SRF were determined by Western blotting. D , E Dual luciferase reporter assays to identify the binding site(s) of STAT3 and SRF in the MCL1 promoter. Left: diagram depicting the putative binding sites of STAT3 ( D ) or SRF ( E ) in the MCL1 promoter predicted by the JASPAR database. Right: Dual luciferase reporter assays conducted in HEK293T. F Reduced binding of STATS and SRF in the MCL1 promoter upon HER2-targeting drug treatments. SNU-216 cells were left untreated or treated with 100 ng/ml trastuzumab or 200 nM lapatinib for 72 h or 48 h, respectively. ChIP was conducted using specific antibodies against STAT3, SRF, and an IgG isotype control. G Co-IP analysis of the interaction of endogenous STAT3 and SRF in SNU-216 cells. H Direct interaction of exogenous STAT3 and SRF. HEK293T cells were transiently transfected with HA-tagged STAT3, Flag-tagged SRF, either individually or both. Equivalent lysates were immunoprecipitated with anti-HA or anti-Flag antibody and immunoblotted with the indicated antibodies. I Effect of overexpressing or deleting STAT3 on SRF expression. The mRNA (left) and protein (right) levels of SRF in SNU-216 cells with STAT3 overexpression or depletion were determined. J Similar experiments to those in panel I were conducted to detect the effect of manipulating SRF on STAT3 expression in SNU-216 cells. K Reduced binding of STAT3 in the SRF promoter upon HER2-targeting drug treatments. Similar experiments to those in ( F ) were conducted. L Depletion of STAT3 attenuated the binding of SRF in the MCL1 promoter. Similar experiments to those in ( F ) were performed in SNU-216 cells engineered to express sg STAT3 . ChIP was conducted at 72 h after addition of DOX to induce the deletion of STAT3 . 2 sgRNAs were tested. M , N Deleting STAT3 enhanced the sensitivity of GC lines to BCLXL inhibition. SNU-216 ( M ) and GCIY ( N ) cells inducibly expressing sg STAT3 or the sgRNA empty vector were treated with DOX alone, or in combination with indicated concentrations of BCLXLi. Cell viability was determined 48 h later. Two-way ANOVA was used for statistical significance. O STAT3 inhibitor synergized with BCLXLi in GC cell lines, regardless of their p-STAT3 levels. GC cell lines with high p-STAT3 levels (HGC-27, MKN45 and AGS) and the ones with low p-STAT3 levels (SNU-216, NCI-N87, GCIY) were treated with indicated concentrations of BCLXLi and STAT3 inhibitor and cell viability was determined 48 h later. Data in ( A ), ( B ), ( D ), ( E ), ( I , left ), ( J , left ), ( M – O ) represent the means ± SD of ≥3 independent experiments; data in panel ( C ), ( F – H ), ( I , right ), ( J , right ), ( K ) and ( L ) are representatives of 2 independent experiments.

Article Snippet: The GV492 lentiviral expression constructs of human ELK1, ELK3, ELK4, STAT3, SRF, as well as the GV366 (C-terminally HA-tagged) and GV657 (C-terminally Flag-tagged) constructs for transient expression of human STAT3 and SRF were purchased from Shanghai Genechem Co., Ltd.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Luciferase, Binding Assay, Control, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Over Expression, Inhibition