Journal: Stem cells and development

Article Title: Efficient Differentiation of TBX18 + /WT1 + Epicardial-Like Cells from Human Pluripotent Stem Cells Using Small Molecular Compounds.

doi: 10.1089/scd.2016.0208

Figure Lengend Snippet: FIG. 5. Functional assay of EPLC-derived SMCs (EPL-SMCs). (A) Time-course of change in relative mean fluorescence intensity of Fluo-4-preloaded EPLC-derived cells, determined by flow cytometry after carbachol addition. Error bars represent SEM; n = 3/group; *p < 0.05, **p < 0.01 versus untreated cultures, analyzed by the Student’s t-test. (B) Total proportion of functional EPLC-SMCs displaying calcium transients after phenylephrine addition in D15 + 8 + 6 NT and TGFb1+ bFGF+TGFb1-induced cultures. Error bars represent SEM; n = 3/group; **p < 0.01 versus untreated cultures, analyzed by the Student’s t-test. (C) Calcium imaging examining the functional response of EPLC-SMCs and HCASMCs to phenylephrine. Average fluorescence intensity normalized to baseline (F/Fo) of five random cells exhibiting calcium transients was measured. Peaks correspond to active calcium cycles in response to 15mM phenylephrine added at 100 s, eliciting different responses. (D) Amplitude of calcium transients after phenylephrine addition. Error bars represent SEM; n = 5 cells; **p < 0.01 versus HCASMCs, analyzed by the Student’s t-test. (E) Kinetic parameters of calcium transients after phenylephrine addition. Error bars represent SEM; n = 5 cells; **p < 0.01 versus HCASMCs, analyzed by the Student’s t-test. (F) Fluo-4 AM-loaded EPLC- SMCs and HCASMCs displayed a change in cell surface area following phenylephrine stimulation. Scale bars, 100 mm. (G) Percentage change in cell surface area. Error bars represent SEM; n = 12 cells; **p < 0.01 versus D15 + 8 + 6 cells that did not exhibit calcium transients, analyzed by the Student’s t-test. HCASMCs, human coronary artery smooth muscle cells; NS, no significant difference. Color images available online at www.liebertpub.com/scd

Article Snippet: Primary human coronary artery SMCs (HCASMCs, ATCC) and EPL-SMCs were preloaded with 2.5 mM Fluo-4 AM in Tyrode’s solution at 37 C for 30 min.

Techniques: Functional Assay, Derivative Assay, Cytometry, Imaging

Journal: Scientific Reports

Article Title: Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

doi: 10.1038/s41598-024-66860-x

Figure Lengend Snippet: Morphology and SMC markers in human ITA SMC and ACA SMC. Human SMC derived from 3 distinct ACA donors and 3 distinct ITA donors were plated in T75 flasks with growth medium. Schwann cells and HeLa cells were used as controls. Representative photomicrographs of ACA SMC ( A ) and ITA SMC ( B ) taken at subconfluence are shown. Expression of α-actin ( C ), myh-11 ( D ), calponin ( E ), Sox-10 ( F ) and S100B ( G ) were determined as described in Methods and a representative blot from 3 independent experiments for each protein is shown. Expression of the senescence biomarkers p16INK4A and p21Cip1 were determined by Western blotting as described in Methods for growth arrested ACA SMC and ITA SMC. Expression was quantified and normalized to β-actin expression. Results from 3 independent experiments are shown in H.

Article Snippet: Cell lines of human ACA SMC from 3 distinct donors (Cat# 350q-05a) and cell lines of ITA SMC from 3 distinct donors (Cat# 358-05a) were obtained from Cell Applications (San Diego, CA).

Techniques: Derivative Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

doi: 10.1038/s41598-024-66860-x

Figure Lengend Snippet: Proliferative responses of human ITA SMC and ACA SMC to FBS. Human SMC derived from 3 distinct ACA donors and 3 distinct ITA donors were plated at equal seeding density in T25 flasks, grown to subconfluence, growth arrested and then exposed to 10% FBS. Cell counts were performed at 48 h, 72 h, 96 h, 120 h, 144 h, 168 h and 192 h. Results of combining data from the 3 ACA SMC lines and the 3 ITA SMC lines are shown in ( A ) and results from 6 to 10 independent experiments with each cell line are shown in ( B ). [SEM is presented in ( A ) and STD is presented in ( B ); ^— p < 0.05 vs control; *— p < 0.001 vs control; “— p < 0.05 vs ITA SMC].

Article Snippet: Cell lines of human ACA SMC from 3 distinct donors (Cat# 350q-05a) and cell lines of ITA SMC from 3 distinct donors (Cat# 358-05a) were obtained from Cell Applications (San Diego, CA).

Techniques: Derivative Assay, Control

Journal: Scientific Reports

Article Title: Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

doi: 10.1038/s41598-024-66860-x

Figure Lengend Snippet: PDGF-BB induced proliferation is inhibited by anti-TSP1 neutralizing antibody A4.1 and by MiR21-3p inhibitors and MiR21-5p inhibitors in ACA SMC but not in ITA SMC. Human SMC derived from 3 distinct ACA donors ( A , C and D ) and 3 distinct ITA donors ( B , C and E ) were growth arrested as described in Methods. ( A ) and ( B ): The indicated groups were pretreated with vehicle, anti-TSP-1 antibody A4.1 (10 µg/ml), or a non-specific IgG (10 µg/ml) for 30 min prior to treatment with vehicle or PDGF-BB (20 ng/ml). Cell counts were determined 96 h later. [n ≥ 8 independent experiments; #— p < 0.05 compared to Cnt; *— p < 0.05 compared to PDGF-BB + IgG]. ( C ): Total RNA was extracted and quantitative RT-PCR performed in triplicate as described in Methods. ( D ) and ( E ): The indicated groups were pretreated with vehicle, 50 nM miR21-3p inhibitor, 50 nM miR21-5p inhibitor or 50 nM MiRCnt for 24 h prior to treatment with vehicle or PDGF-BB (20 ng/ml). Cell counts were determined 96 h later. [n ≥ 8 independent experiments; *— p < 0.05 compared to cnt; ^— p < 0.05 compared to TSP + MiR cnt].

Article Snippet: Cell lines of human ACA SMC from 3 distinct donors (Cat# 350q-05a) and cell lines of ITA SMC from 3 distinct donors (Cat# 358-05a) were obtained from Cell Applications (San Diego, CA).

Techniques: Derivative Assay, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

doi: 10.1038/s41598-024-66860-x

Figure Lengend Snippet: The effects of inhibitors on exogenous TSP1-induced proliferation of ACA SMC and ITA SMC. Human SMC derived from 3 distinct ACA donors and 3 distinct ITA donors were growth arrested and were treated with either vehicle (Cnt) or TSP1 (various concentrations). The results from the different ACA cell lines and different ITA cell lines were consistent and thus the data is presented as an aggregate of the ACA SMC and ITA SMC lines ( A ). In panels ( B ) and ( C ), growth arrested SMC were pretreated with vehicle, anti-TSP-1 antibody A4.1 (10 µg/ml), a non-specific IgG (10 µg/ml), 50 nM miR21-3p inhibitor, 50 nM miR21-5p inhibitor or 50 nM MiRCnt for 30 min prior to treatment with vehicle or TSP1 (12.5 µg/ml). [*— p < 0.05 compared to TSP1 + IgG and #— p < 0.05 compared to TSP1 + MiR cnt].

Article Snippet: Cell lines of human ACA SMC from 3 distinct donors (Cat# 350q-05a) and cell lines of ITA SMC from 3 distinct donors (Cat# 358-05a) were obtained from Cell Applications (San Diego, CA).

Techniques: Derivative Assay

Journal: Scientific Reports

Article Title: Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

doi: 10.1038/s41598-024-66860-x

Figure Lengend Snippet: Expression and secretion of TSP1 in PDGF-BB-treated ACA SMC and ITA SMC. Human SMC derived from 3 distinct ACA donors ( A ) and 3 distinct ITA donors ( B ) were growth arrested as described in Methods and then treated with either vehicle or PDGF-BB (20 ng/ml) for various time periods. TSP1 expression and β-actin expression were determined as described in Methods. Panel A shows a representative blot of lysate from ACA SMC treated with PDGF-BB for 24 h and lysate from ITA SMC treated with PDGF-BB for 24 h run on the same gel. A time course of TSP1 expression is shown in panels B–E. SMC isolated from ACA ( B and C ) and SMC isolated from ITA ( D and E ) were treated with PDGF-BB (20 ng/ml) for various times and representative blots are shown in B and D with results of 6 independent experiments shown in C and E. [*— p < 0.05 compared to basal levels]. In panels F and G, SMC were treated with PDGF-BB (20 ng/ml) for the indicated time points, and then conditioned medium was harvested and concentrated. This was followed by Western blot analysis with anti-TSP1 antibody as described in Methods. There were no statistically significant differences, compared to t = 0, for any of the time points in F or G.

Article Snippet: Cell lines of human ACA SMC from 3 distinct donors (Cat# 350q-05a) and cell lines of ITA SMC from 3 distinct donors (Cat# 358-05a) were obtained from Cell Applications (San Diego, CA).

Techniques: Expressing, Derivative Assay, Isolation, Western Blot

Journal: Oncotarget

Article Title: TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells

doi: 10.18632/oncotarget.10853

Figure Lengend Snippet: A. Cell viability was determined with MTT assay in human coronary artery SMCs incubated with 10, 30, 60, and 90 μmol/L lysoPC for 72 h. Data are mean ± SEM (* P < 0.05, ** P < 0.01 vs . control, 0 μmol/L lysoPC). B. Cell viability was determined in human coronary artery SMCs incubated with 10, 30, and 60 μmol/L lysoPC for 24-72 h. Data are mean ± SEM ( n = 3 individual experiments, * P < 0.05, ** P < 0.01 vs . 0 day).

Article Snippet: The primary human coronary artery SMCs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in α-MEM/F12 (Invitrogen, Hong Kong, China).

Techniques: MTT Assay, Incubation, Control

Journal: Oncotarget

Article Title: TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells

doi: 10.18632/oncotarget.10853

Figure Lengend Snippet: A. Cell cycling progression was not affected by 30 or 60 μmol/L lysoPC (24 h) in human coronary artery SMCs. B. Flow cytometry analysis was made in cells treated with 30 or 60 μmol/L (24 h) and co-stained with PI and Annexin V-FITC (V, viability; D, death; LA, late apoptosis; EA, early apoptosis) C. Percentage of cells that show viability, early apoptosis, late apoptosis and death after treatment with 30 or 60 μmol/L lysoPC ( n = 3, * P < 0.05, ** P < 0.01 vs . vehicle control). D. Representative microphotographs of TUNEL staining in human coronary artery SMCs treated with 30 or 60 μmol/L lysoPC (24 h). TUNEL labelling is stained in green and nuclei are labelled by DAPI staining in blue. Scale bar = 50 μm. E. The percentage of TUNEL-positive cells in total cells. The DAPI staining cells were countered from five randomly picked regions (** P < 0.01 vs . vehicle control). F. Detection of the caspase-3 activity in human coronary artery SMCs treated with 30 or 60 μmol/L lysoPC for 24 h. ( n = 3 individual experiments, * P < 0.05, ** P < 0.01 vs . 0, i.e. vehicle control).

Article Snippet: The primary human coronary artery SMCs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in α-MEM/F12 (Invitrogen, Hong Kong, China).

Techniques: Flow Cytometry, Staining, Control, TUNEL Assay, Activity Assay

Journal: Oncotarget

Article Title: TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells

doi: 10.18632/oncotarget.10853

Figure Lengend Snippet: A. Cytosolic free Ca 2+ was determined in Tyrode's solution containing 5% FBS with a confocal microscopy technique in human coronary artery SMCs preloaded with Fluo-3 AM (data are mean ± SEM). The pseudoratio dF/F was applied to express intracellular free Ca 2+ level, where dF is the measured fluorescence intensity of fluo 3, and F is the initial level of fluorescence intensity. LysoPC 30 μmol/L ( n = 27) or 60 μmol/L ( n = 27, ** P < 0.01 vs . 10 μmol/L lysoPC), but not 10 μmol/L lysoPC ( n = 25), induced a significant sustained Ca 2+ influx. B. LysoPC (30 μmol/L) induced sustained Ca 2+ increase (data are mean ± SEM) in the presence of 1.8 mmol/L Ca 2+ in bath medium ( n = 28), but not in the medium with 0 Ca 2+ and 1 mmol/L EGTA ( n = 26, ** P < 0.01 vs . 1.8 mmol/L Ca 2+ ). C. Cell apoptosis determined in 1.8 and 0.9 mm/L Ca 2+ medium in human coronary artery SMCs by co-staining with PI and Annexin V-FITC (V, viability; D, death; LA, late apoptosis; EA, early apoptosis.) after treatment with 60 μmol/L lysoPC for 24 h. D. Percent values of viability, early apoptosis, late apoptosis and death after treatment with 60 μmol/L lysoPC. ( n = 3, * P < 0.05, ** P < 0.01 vs . vehicle control; # P < 0.05, ## P < 0.01 vs . 1.8 mmol/L Ca 2+ ).

Article Snippet: The primary human coronary artery SMCs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in α-MEM/F12 (Invitrogen, Hong Kong, China).

Techniques: Confocal Microscopy, Fluorescence, Staining, Control

Journal: Oncotarget

Article Title: TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells

doi: 10.18632/oncotarget.10853

Figure Lengend Snippet: A. RT-PCR (left) and Western blots (right) of TRPC isoforms in human coronary artery SMCs (passages 3, 4, and 6). B. Western blots and ratio of protein levels of TRPC1, TRPC3 or TRPC4 channels in human coronary artery SMCs transfected with 20 or 50 nmol/L siRNAs targeting TRPC1, TRPC3 or TRPC4 channels (relative to b-actin and lipofectamine 2000, n = 3 individual experiments, * P < 0.05, ** P < 0.01 vs . control siRNA or lipofectamine). C. Ca 2+ influx (data are mean ± SEM) induced by 30 μmol/L lysoPC in cells transfected with 50 nmol/L control siRNA ( n = 27), TRPC1 siRNA ( n = 27, ** P < 0.01 vs . control siRNA), TRPC3 siRNA ( n = 28, ** P < 0.01 vs . control siRNA), or TRPC4 siRNA ( n = 28). D. Co-immunoprecipitation showing the interaction between TRPC1 and TRPC3 proteins in cells treated without or with 60 μmol/L lysoPC. IP indicates the antibody used to pull down the interacting proteins. Ig G represents the negative control.

Article Snippet: The primary human coronary artery SMCs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in α-MEM/F12 (Invitrogen, Hong Kong, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Control, Immunoprecipitation, Negative Control

Journal: Oncotarget

Article Title: TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells

doi: 10.18632/oncotarget.10853

Figure Lengend Snippet: A. Western blots of Bax, Bcl-2, caspase-3, and p-Akt(S473) in human coronary artery SMCs treated with 60 μmol/L lysoPC for 3 h or 6 h incubation with medium containing 1.8 or 0.9 mmol/L Ca 2+ . B. Relative ratio of Bax, Bcl-2, caspase-3, or p-Akt ( n = 3, * P < 0.05, ** P < 0.01 vs . vehicle; # P < 0.05, ## P < 0.01 vs .1.8 mmol/L Ca 2+ ).

Article Snippet: The primary human coronary artery SMCs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in α-MEM/F12 (Invitrogen, Hong Kong, China).

Techniques: Western Blot, Incubation

Journal: Oncotarget

Article Title: TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells

doi: 10.18632/oncotarget.10853

Figure Lengend Snippet: A. Western blots of Bax, Bcl-2, caspase-3, and p-Akt in human coronary artery SMCs transfected with 50 nM control siRNA, TRPC1 siRNA, TRPC3 siRNA or TRPC4 siRNA for 72 h, and then treated with vehicle (V) or 60 μmol/L lysoPC for 3 and 6 h. B. Relative mean levels of Bax, Bcl-2, caspase-3, and p-Akt in human coronary artery SMCs with the same treatment as in A ( n = 3, * P < 0.05, ** P < 0.01 vs . vehicle, # P < 0.05, ## P < 0.01 vs . control siRNA).

Article Snippet: The primary human coronary artery SMCs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in α-MEM/F12 (Invitrogen, Hong Kong, China).

Techniques: Western Blot, Transfection, Control

Journal: Cell Biology International Reports

Article Title: Microarray analysis of ox-LDL (oxidized low-density lipoprotein)-regulated genes in human coronary artery smooth muscle cells

doi: 10.1042/CBR20100006

Figure Lengend Snippet: Effect of n (normal) and ox (oxidized) LDL (2 μg/ml) on the proliferation of human coronary artery SMCs

Article Snippet: Human coronary artery SMCs were purchased from Clonetics and cultured in SmBM (SMC basal medium) containing SmBM-3 growth supplements [FBS (fetal bovine serum) (5%), bovine insulin (50 ng/ml), hr (human recombinant)-EGF (epidermal growth factor) (5.0 ng/ml), hr-FGF-B (20 ng/ml) and GA-1000 (Gentamicin, Amphotericin B)] supplied by BioWhittaker Inc.

Techniques: