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: 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

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: Pharmacological research

Article Title: A proteome-wide screen identifies the calcium binding proteins, S100A8/S100A9, as clinically relevant therapeutic targets in aortic dissection.

doi: 10.1016/j.phrs.2023.107029

Figure Lengend Snippet: Fig. 8. S100A8/A9 activates NF-κB and inhibits SRF transcriptional activity in VSMCs. (A-B) HASMCs were treated with recombinant human protein S100A8/ A9 heterodimer (rh-S100A8/A9, 1 μg/mL) for 48 h. The control group was treated with the same concentration of bovine serum albumin (BSA). Expression of genes related to phenotypic switch of VSMCs and inflammation were examined by qRT-PCR, n = 3. (C-D) RASMCs were treated as above and the expression related to VSMC phenotypic switch and inflammation were examined by qRT-PCR, n = 3. (E) The protein expression of SMMHC and OPN were determined by western blot, n = 3. (F) RASMCs were infected with NF-κB (titer: 2.5 ×106pfu/mL) adenoviral luciferase reporter vector. After treatment with rh-S100A8/A9 or TNFα (10 ng/mL) for 6 h, dual luciferase reporter assay was performed, n = 3. (G) 293 T cells were transfected with SRF luciferase reporter plasmid. The cells were treated with rh- S100A8/A9 at different time points. The relative fluorescence intensity was presented, n = 3. (H) RASMCs were treated with recombinant S100A8/A9 and BSA before ChIP-PCR was performed. The effect of rh-S100A8/A9 on SRF binding to Myh11 and Sm22α gene promoters was calculated, n = 3. IgG was used as the negative control. * P < 0.05, * * P < 0.01, * ** P < 0.001.

Article Snippet: For other antibodies, SMMHC (21404–1-AP), SRF (16821–1-AP), GAPDH (60004–1-Ig), and β-actin (81115–1-RR) were obtained from Proteintech (Wuhan, China), and Sigma-Aldrich (St. Louis, MO) provided myocardin (SAB4200539).

Techniques: Activity Assay, Recombinant, Control, Concentration Assay, Expressing, Quantitative RT-PCR, Western Blot, Infection, Luciferase, Plasmid Preparation, Reporter Assay, Transfection, Fluorescence, Binding Assay, Negative Control