Journal: Cancers

Article Title: Serum Response Factor (SRF) Drives the Transcriptional Upregulation of the MDM4 Oncogene in HCC

doi: 10.3390/cancers13020199

Figure Lengend Snippet: ELK1 and ELK4 are essential co-factors for SRF-mediated transcriptional regulation of MDM4 in HCC. Luciferase activity of a MDM4 promoter reporter upon siRNA-mediated knockdown of ( A ) SRF, ( B ) ELK1, and ( C ) ELK4 in HepG2 and HLE cells compared to controls. ( D ) MDM4 mRNA levels after co-transfection of an ELK1 cDNA with siNS or siSRF. Transfection efficacy was confirmed by detection of ELK1 and SRF mRNA levels. ( E ) MDM4 mRNA levels following transfection of the indicated cDNA plasmids. ELK1 S383A represents an inactive variant, which cannot be activated by phosphorylation of S383 and is thus unable to initiate target gene transcription. Transfection efficacy was confirmed by detection of ELK1 and SRF mRNA levels. Data are presented as mean ± SEM. Mann-Whitney U test: * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: siNS, scrambled, nonsense; siSRF_1/_2, siELK1_1/_2 siELK4_1/_2, siRNA 1 and 2 specifically targeting SRF, ELK1 and ELK4, respectively; ELK1, ELK1 cDNA; ELK1 S383A, ELK1 S383A cDNA; GLuc, Gaussia luciferase; SEAP, Secreted Alkaline Phosphatase; norm., normalized against control.

Article Snippet: HLE cells were transiently transfected with a pCMV6-AC-GFP vector containing either a full-length human SRF cDNA (RG208596 from OriGene Technologies, Rockville, MD, USA), or a pCGN vector containing a full-length human ELK1 cDNA, an inactive ELK1 variant (ELK1 S383A cDNA), or a pCS2plus vector containing a SRF-VP16 cDNA [ ] following the manufacturer’s protocol, using Lipofectamine 3000 (Invitrogen, Karlsruhe, Germany). pCGN-ELK-1 and pCGN-ELK-1 S383A were a gift from Ron Prywes Lab (Addgene plasmids #27156 and #27160) [ ].

Techniques: Luciferase, Activity Assay, Knockdown, Cotransfection, Transfection, Variant Assay, Phospho-proteomics, MANN-WHITNEY, Control

Journal: PLoS ONE

Article Title: Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes

doi: 10.1371/journal.pone.0045514

Figure Lengend Snippet: Oligonucleotides and plasmids.

Article Snippet: pCGN-SRF , SRF expression plasmid , Addgene 11977, .

Techniques: Luciferase, Plasmid Preparation, Expressing, Control, Dominant Negative Mutation

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: Cell metabolism

Article Title: Pseudotemporal ordering of single cells reveals metabolic control of postnatal beta-cell proliferation

doi: 10.1016/j.cmet.2017.04.014

Figure Lengend Snippet: A. Heatmap showing Pearson correlation of gene expression profiles in all 387 beta-cells comparing proliferation genes with top pseudotemporally-regulated oxidative phosphorylation genes and TFs. Proliferation genes are depicted in red, oxidative phosphorylation genes in orange and TFs in black. B. Overview of RNA-seq analysis of lentiviral Srf overexpression in islets at P28. C. GSEA plots showing enrichment of proliferation genes regulated during pseudotime (left) and genes down-regulated during pseudotime (right) as an effect of Srf overexpression. RNA-seq data are from three independent transduction experiments. D. RPKM values of TFs Fos, Junb and Egr1 (top panel) and proliferation genes Mki67, Pcna and Ccne1 (bottom panel) in RNA-seq data from control and Srf-overexpressing islets. Data shown as mean ± SEM. E. Summary of metabolic regulators and effector TFs driving early neonatal beta-cell proliferation as revealed by reconstructing a pseudotemporal time course of beta-cell maturation, experimental validation, and prior literature. ** P < 0.01, *** P < 0.001. See also Figure S7 and Table S7.

Article Snippet: Lentivirus production and transduction GFP-tagged lentiviral plasmid (Origene PS100071) or GFP-tagged Srf lentiviral plasmid (Origene MR208120L2) was transfected with pCMV-R8.74 (Addgene 22036) and pMD2.G expression plasmid into HEK293T cells.

Techniques: Expressing, RNA Sequencing Assay, Over Expression, Transduction

Journal: Cell metabolism

Article Title: Pseudotemporal ordering of single cells reveals metabolic control of postnatal beta-cell proliferation

doi: 10.1016/j.cmet.2017.04.014

Figure Lengend Snippet: Key Resources Tables

Article Snippet: Lentivirus production and transduction GFP-tagged lentiviral plasmid (Origene PS100071) or GFP-tagged Srf lentiviral plasmid (Origene MR208120L2) was transfected with pCMV-R8.74 (Addgene 22036) and pMD2.G expression plasmid into HEK293T cells.

Techniques: Recombinant, Staining, Sequencing, Imaging, In Situ, Enzyme-linked Immunosorbent Assay, Sample Prep, Real-time Polymerase Chain Reaction, Software

Journal: Nature Communications

Article Title: FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis

doi: 10.1038/s41467-021-22771-3

Figure Lengend Snippet: a HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h, then treated with actinomycin D for different periods of time. RT-qPCR assay analysis of vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels ( n = 5 independent experiments). b HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h, then transfected with various VEGFR2 promoter truncation constructs or a mutant (serum response factor [SRF] binding site) construct for 24 h. After this, luciferase activity was measured ( n = 5 independent experiments). c Chromatin immunoprecipitation assays using an anti-SRF antibody to amplify VEGFR2 promoter in HUVECs transfected with or without FUNDC1 siRNA ( n = 5 independent experiments). d HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h and then treated with VEGF for 30 min. Expression of SRF and phosphorylated SRF at Ser103 (pSRF) was determined by western blot assay ( n = 5 independent experiments). e Cytosolic Ca 2+ levels, as indicated by the fluorescent probe Fluo-4, AM ( n = 5 independent experiments). HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h and then exposed to VEGF (20 ng/mL) for the indicated times. f HUVECs were infected with adenovirus encoding control (Ad-Cont) or mitochondrial–endoplasmic reticulum (ER) linker (Ad-Linker) for 24 h and then treated with BAPTA-AM (10 µM) or control dimethylsulfoxide (vehicle) for 30 min. Levels of SRF and pSRF (Ser103) were detected by western blot assay ( n = 5 independent experiments). g HUVECs were pre-transfected with or without SRF siRNA for 24 h, then infected with adenovirus encoding control or mitochondria–ER linker for 24 h. VEGFR2 protein expression was detected by western blot assay ( n = 5 independent experiments). Statistical significance was assessed using two-tailed t -tests for two groups and one-way ANOVA with post hoc multiple comparisons test for multiple groups. *p < 0.05 vs Scr siRNA; Scr siRNA+VEGF; Ad-Cont+Vehicle or Ad-Cont+Scr siRNA. All values are mean ± S.D.

Article Snippet: SiRNA for FUNDC1, IP3R1, VEGFR2, serum response factor (SRF), and controls were obtained from Santa Cruz Biotechnology (sc-36563, sc-91118, sc-42475, and sc-29318; Dallas, Texas).

Techniques: Transfection, Quantitative RT-PCR, Construct, Mutagenesis, Binding Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Expressing, Western Blot, Infection, Control, Two Tailed Test

Journal: Nature Communications

Article Title: FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis

doi: 10.1038/s41467-021-22771-3

Figure Lengend Snippet: a Transduction efficiency of peptides 1, 2, and a control peptide in Human umbilical vein endothelial cells (HUVECs) ( n = 5 independent experiments). b HUVECs were treated with different peptides (20 μM) for 24 h, then subjected to immunoprecipitation with antibody against FUNDC1(HA) to quantify the interaction of FUNDC1 and IP3R1 ( n = 3 independent experiments). c HUVECs were treated with different peptides (20 μM) for 24 h, then cytosolic Ca 2+ was detected using the fluorescent probe Fluo-4, AM ( n = 5 independent experiments). d HUVECs were treated with different peptides. After 24 h, the cell lysates were collected and subjected to western blot assays to detect the expression of VEGFR2, IP3R1, SRF, and phosphorylated SRF at Ser103 (pSRF) ( n = 5 independent experiments). e Three-dimensional spheroids and representative images of spheroid-sprouting were analyzed ( n = 5 independent experiments). Scale bar, 100 µm. f Matrigel containing vascular endothelial growth factor (VEGF) was injected subcutaneously into 6-week-old wild-type mice, then the mice received an intravenous injection of the indicated peptides. After 10 days, matrigel plugs were removed for analysis of new vessel formation by histological and Hb assay ( n = 5 mice/group). Quantification of Hb extracted from matrigel plugs of different groups. g C57BL/6 mice with LLC tumors that were approximately 90 mm 3 in size were treated with intravenous injections of different peptides (10 mg/kg/3 days). After 28 days, the tumors were harvested and quantified. Representative images of tumors ( n = 5 mice/group). h Immunostaining of LLC tumor sections and quantification of relative CD31-positive area. ( n = 5 mice/group). Scale bar, 100 µm. Statistical significance was assessed using one-way ANOVA with post hoc multiple comparisons test for multiple groups. *p < 0.05 vs Ctrl peptide. All values are mean ± S.D.

Article Snippet: SiRNA for FUNDC1, IP3R1, VEGFR2, serum response factor (SRF), and controls were obtained from Santa Cruz Biotechnology (sc-36563, sc-91118, sc-42475, and sc-29318; Dallas, Texas).

Techniques: Transduction, Control, Immunoprecipitation, Western Blot, Expressing, Injection, Immunostaining