Journal: Molecules

Article Title: Anthocyanins Recovered from Agri-Food By-Products Using Innovative Processes: Trends, Challenges, and Perspectives for Their Application in Food Systems

doi: 10.3390/molecules26092632

Figure Lengend Snippet: A summary of human trials showing the potential health beneficial effects of anthocyanins.

Article Snippet: MEDOX ® (Purified anthocyanins) a , 320 mg/day , 16 weeks , 27 individuals with MCI ( n = 8) or stable non-obstructive coronary artery disease ( n = 19) (55–70 years old; 18 males and 9 females) , Open-label study , - ↓ RANTES; - Improved verbal memory function (learning, recall, and recognition) and cognitive speed. , [ ] .

Techniques: Purification, Control, Activity Assay, Clinical Proteomics, Activation Assay, Expressing, Modification, Bacteria, Comparison, Muscles

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: The CSPG4 gene is activated by Myocardin-Related Transcription Factors (MRTFs). To examine the transcriptional control of NG2/ CSPG4 we correlated the CSPG4 mRNA versus all other mRNAs ( www.GTExPortal.org ) and calculated the sum of correlation coefficients (R sum ) for all transcripts across 20 human tissues. Panel ( A ) shows the positive extreme of the resulting R sum distribution which has a theoretical maximum of 20 (seen only for CSPG4 itself, not included). Among the 600 (≈1%) most tightly correlating mRNAs we found classical smooth muscle cell (SMC) markers ( TAGLN , MYH11 ) and transcription factors ( SRF , MYOCD , MKL1 , all indicated by different green symbols). Two cell lineage markers ( MCAM and AOC3 , red symbols) were among the mRNAs in the absolute extreme (top 25). Examples of correlations between SRF , MYOCD and MKL1 versus CSPG4 in the transverse colon (N = 274) are shown in panels ( B – D ). P-values and Spearman Rho-coefficients are given in the respective panels. In panel ( E ), adenoviruses were used for overexpression of MRTFs (MYOCD, MRTF-A/ MKL1 , and MRTF-B/ MKL2 ) in primary human coronary artery smooth muscle cells in vitro. The CSPG4 mRNA level was determined by RT-qPCR and compared to that in cells treated with empty virus (ANOVA, followed by Dunnett's Multiple Comparison Test versus Null, N = 6 for all treatments). In this and the following figures showing RT-qPCR data, the relative mRNA level is represented by the official gene symbol in italics. Transcript levels were normalized to 18S as housekeeping gene (Pfaffl equation) and are given as fold change (FC). All statistical comparisons in panel ( E ) of figures 1 through 3 are versus Null as indicated by brackets. Panel ( F ) shows a western blot for NG2/CSPG4 following treatment with control (Null) virus and viruses encoding MRTFs. Membranes were cut horizontally in this, and the following, figures to allow for detection of multiple proteins on the same membrane. Full length blots (as full as possible) are found in Fig. . Panel ( G ) shows summarized data for the western blot experiments (N = 3). The bands migrating at ≥ 250 kDa were included in the analysis. In panel ( H ), cells were transduced with tagged MRTF-A/MKL1 (blue), fixed at 96 h, and stained for NG2/CSPG4 (red) and the intermediate filament synemin/SYNM (green), followed by confocal imaging. Red and green labels are shown in black and white below the colored panels for clarity. Summarized data from two independent experiments with three independent replicates each time (N = 6) is shown in panel ( I ). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, all versus Null.

Article Snippet: Human coronary artery smooth muscle cells (hCASMCs, Female 32 years, C-017-5C, Gibco) were purchased from Thermo Fisher and cultured in Medium-231 (M231500, Thermo Fisher) with smooth muscle growth supplement (SMGS, S00725) and antibiotics (50U/ml penicillin and 50 μg/ml streptomycin, Biochrom, A 2212) in a standard cell culture incubator (5% CO 2 ).

Techniques: Control, Over Expression, In Vitro, Quantitative RT-PCR, Virus, Comparison, Western Blot, Membrane, Transduction, Staining, Imaging

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: CD146/ MCAM correlates with CSPG4 across human tissues and is increased by MRTFs. Panels ( A – D ) show examples of correlations between CSPG4 and MCAM in four human tissues (stomach, ovary, prostate, and coronary artery). N-values, P-values, and Spearman Rho-coefficients are given in the graphs. In panel ( E ), MRTFs were overexpressed using adenoviruses and MCAM levels were determined by RT-qPCR (N = 6 throughout). Statistical comparisons are versus Null (here and in panel G ). Panel ( F ) shows a western blot for CD146/ MCAM and panel ( G ) shows summarized western blot data (N = 6). Panel ( H ) shows confocal imaging of CD146/ MCAM (green) following overexpression of MRTF-A/ MKL1 . Smooth muscle α-actin ( ACTA2 ) is shown in red, and blue represents MRTF-A/ MKL1 . Panel ( I ) shows summarized data on MCAM fluorescence in control cells (Null) and after overexpression of MRTF-A/ MKL1 (N = 6). ***P < 0.001, ****P < 0.0001 versus null.

Article Snippet: Human coronary artery smooth muscle cells (hCASMCs, Female 32 years, C-017-5C, Gibco) were purchased from Thermo Fisher and cultured in Medium-231 (M231500, Thermo Fisher) with smooth muscle growth supplement (SMGS, S00725) and antibiotics (50U/ml penicillin and 50 μg/ml streptomycin, Biochrom, A 2212) in a standard cell culture incubator (5% CO 2 ).

Techniques: Quantitative RT-PCR, Western Blot, Imaging, Over Expression, Fluorescence, Control

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: The MCAM and AOC3 gene loci have SRF binding motifs that confer responsiveness to MRTF-SRF signaling. Panel ( A ) shows graphical representations of the CSPG4 , MCAM and AOC3 gene loci with known (red arrows, ENCODE data accessible via the UCSC genome browser) and predicted (lighter red/pink arrows) binding sites for MRTF-SRF . Gene loci and binding sites are not drawn to scale. Panel ( B ) shows effects of SRF silencing (by 43 ± 4%, P < 0.0001, N = 6), using a short hairpin construct, on the indicated mRNA levels measured by RT-qPCR. Panel ( C ) shows reporter assays for MCAM (S1-S3 plasmid and S2-S4 plasmid, see panel A ) and AOC3 . We used HEK 293 cells for transfection of the reporter plasmids in ( C ), because these cells were more readily transfected compared to human coronary artery smooth muscle cells used elsewhere. Panels ( D ) and ( E ) show time-courses of mRNA induction following overexpression of myocardin (N = 3 for all time points). CSPG4 , MCAM and AOC3 were all increased at least as fast as the direct target gene ACTA2 . Panel ( F ) shows that MRTF-A/MKL1 increases the mRNA level of KDM3A, and that short hairpin silencing of KDM3A (shKDM3A) antagonizes this effect (N = 5–6). Panel ( G ) shows RT-qPCR for MCAM , AOC3 , and CSPG4 in MRTF-A/MKL1-transduced cells in the absence and presence of shKDM3A (N = 5–6). Panel ( H ) shows western blots for cells transduced with null, MKL1, and MKL1 plus shKDM3A viruses. The bar graph at the bottom shows the quantitative analysis for MCAM (vs. HSP90). Quantitative analysis of the KDM3A protein level similarly showed it to be significantly increased by MRTF-A transduction and reduced by KDM3A silencing (not shown). Panel ( I ) shows the effect of MRTF-A/MKL1 on transcript levels of three transcription factors ( SOX10 , ASCL1 , and OLIG2 ) that control CSPG4 expression in brain glial cells. None of these transcription factors were significantly increased, while the positive controls ( ACTA2 , CSPG4 ) were increased in the same samples (N = 6).

Article Snippet: Human coronary artery smooth muscle cells (hCASMCs, Female 32 years, C-017-5C, Gibco) were purchased from Thermo Fisher and cultured in Medium-231 (M231500, Thermo Fisher) with smooth muscle growth supplement (SMGS, S00725) and antibiotics (50U/ml penicillin and 50 μg/ml streptomycin, Biochrom, A 2212) in a standard cell culture incubator (5% CO 2 ).

Techniques: Binding Assay, Construct, Quantitative RT-PCR, Plasmid Preparation, Transfection, Over Expression, Western Blot, Transduction, Control, Expressing

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: SMC differentiation, actin dynamics, and loss of Ternary Complex Factors (TCFs) all affect AOC3 , MCAM, and CSPG4 expression. In panel ( A ), human coronary artery smooth muscle cells were incubated with either growth supplement (GS) or differentiation supplement (DS) for 72 h and the mRNA levels of AOC3 , MCAM , and CSPG4 were determined by RT-qPCR (N = 6). In panels ( B ) and ( C ), the effects of Latrunculin B (LatB), which depolymerizes actin, were tested using two different protocols ( B 24 h static, and C 20 h with drug + 4 h washout in cycles for 96 h, N > 6). Transcript levels were assayed using RT-qPCR. All cells in ( B ) and ( C ) were transduced with MRTF-A/ MKL1 . Panel ( D ) (72 h treatment) and panel ( E ) (time-course) show effects of the MRTF-SRF inhibitor CCG-1423. Panel ( F ) compares transcript levels in freshly isolated mouse caudal arteries with mouse caudal arteries that were organ cultured in the presence of vehicle (DMSO) or CCG-1423 (96 h). In panel ( G ), the levels of Aoc3 , Mcam , and Cspg4 were determined by RT-qPCR in mouse embryonic fibroblasts (MEFs) from wild type (WT) mice, and in MEFs from mice that lack three Ternary Complex Factors (Elk1, Elk3, and Elk4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the respective controls.

Article Snippet: Human coronary artery smooth muscle cells (hCASMCs, Female 32 years, C-017-5C, Gibco) were purchased from Thermo Fisher and cultured in Medium-231 (M231500, Thermo Fisher) with smooth muscle growth supplement (SMGS, S00725) and antibiotics (50U/ml penicillin and 50 μg/ml streptomycin, Biochrom, A 2212) in a standard cell culture incubator (5% CO 2 ).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Transduction, Isolation, Cell Culture

Journal:

Article Title: Resveratrol, a Red Wine Constituent, Blocks the Antimitogenic Effects of Estradiol on Human Female Coronary Artery Smooth Muscle Cells

doi: 10.1210/jc.2010-0460

Figure Lengend Snippet: Expression of ER-α and ER-β in coronary artery smooth muscle cells (A) and attenuation by resveratrol (Resv; 5 μmol/liter) of the concentration-dependent inhibitory effects of estradiol (1–100 nmol/liter) on 2.5% serum (FCS)-induced DNA synthesis (B), [3H]proline incorporation (C), and cell proliferation (D) in human VSMCs. Values represent mean ± sem from at least four independent experiments, each conducted in triplicate. *, P < 0.05 vs. control cells treated with FCS alone; §, significant reversal of the inhibitory effects of estradiol.

Article Snippet: Cell culture Female human coronary artery VSMCs were obtained from Cascade Biologics (Portland, OR) and cultured as described previously ( 13 ) .

Techniques: Expressing, Concentration Assay, DNA Synthesis