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: Oncogene

Article Title: Mitochondrial dysfunction is a key determinant of the rare disease lymphangioleiomyomatosis and provides a novel therapeutic target

doi: 10.1038/s41388-018-0625-1

Figure Lengend Snippet: Morphological and molecular characterization of bronchial SMC controls and LAM cell lines. a Study rationale and cellular morphology. Normal bronchial SMC controls ( n = 2) and patient-derived LAM cell lines ( n = 4) were stained for hematoxylin eosin (magnification ×10, size-bar 200 μm) and for alpha-smooth muscle actin (ASMA) (ASMA green, DAPI blue, magnification ×40, size bar 40 μm). Electron microscopy of mitochondria in LAM cells and normal bronchial SMC controls (magnification 200 nm, the scale bar 500 nm). b Nuclear receptor TaqMan arrays n = 2 (data were generated from pooled samples of normal bronchial SMC controls n = 2, or patient derived LAM cell lines n = 4, respectively). Heat map of LogRQ values are shown. Nuclear receptor TaqMan data presented as LogRQ ± technical error of the replicates. ANN analysis of the nuclear receptor arrays was performed to demonstrate hidden interactions among different nuclear receptors. c Deregulation of VEGF expression in LAM samples. qRT-PCR analysis of genes affecting angiogenesis were performed and beta-actin was used as inner control. Data are presented as mean of log RQ ± SEM. Significant changes are marked as asterisk ( P < 0.05); d Heat map of angiogenesis protein array. The figure presents mean of pixel intensity. e Angiogenesis array results of LAM cell lines n = 3 and normal SMC control n = 2 presented as mean of pixel intensity ± SEM. Significant changes are marked as asterisk ( P < 0.05). ANN analysis of angiogenesis protein interaction hierarchy. f Analysis of 798 miRNA absolute copy numbers by Nanostring. miRNA copy numbers detected by Nanostring in pooled LAM ( n = 4) and pooled, normal SMC ( n = 2) samples. The heat map represents the most deregulated 141 miRNA in LAM samples compared to normal SMC controls. Copy number differences of specific miRNAs that are involved in mitochondrial biogenesis detected after Nsolver analysis were further analyzed in individual cell lines (normal SMCs ( n = 2) and LAM ( n = 4)). Data are presented as average copy number ± SEM, significant changes are marked as asterisk ( P < 0.05)

Article Snippet: Controls were primary, normal human bronchial SMCs (Lonza, Basel, Switzerland).

Techniques: Derivative Assay, Staining, Electron Microscopy, Generated, Expressing, Quantitative RT-PCR, Control, Protein Array

Journal: Oncogene

Article Title: Mitochondrial dysfunction is a key determinant of the rare disease lymphangioleiomyomatosis and provides a novel therapeutic target

doi: 10.1038/s41388-018-0625-1

Figure Lengend Snippet: Proxison normalizes mitochondrial morphology and function in LAM cells. a Proxison (3 µM, 1 h)-treated normal SMC and LAM cells were incubated with 2.5 µM RH-123 and then fluorescence microscopy was used to analyze fluorescence intensity (magnification ×20, scale bar 50 µm). Quantification of fluorescence intensity in living cells was performed using ImageJ software. Data are presented as pixel intensity ± SEM; significant changes are marked as asterisk ( P < 0.05). b Proxison (3 µM, 1 h)-treated normal SMC and LAM cells were incubated with 2.5 µM RH-123 and then fluorescence was analyzed by flow cytometry. Data are presented as mean of RFU ± SEM; significant changes marked as * P < 0.05. c Representative morphological changes in the mitochondria of LAM cell lines following Proxison treatment. Electron microscopy of mitochondria of untreated and Proxison (3 µM, 1 h)-treated LAM cells and normal SMC control cells (scale bars are 500 and 200 nm, respectively). d qRT-PCR analysis of mitochondrial gene expression in untreated and Proxison (3 µM, 1 h) treated LAM cell lines ( n = 4) compared to normal SMC controls ( n = 2); Data are presented as mean log RQ ± SEM and significant changes are marked as asterisk, solid circle, solid rhombus, and solid square ( P < 0.05). e TrxR activity of Proxison (3 µM, 1h)-treated LAM cell lines ( n = 4) compared to normal SMC controls ( n = 2). TrxR activity is presented as mean ± SEM and significant changes are marked as asterisk ( P < 0.05). f qRT-PCR analysis of angiogenesis related gene expression in untreated and Proxison (3 µM, 1 h)-treated cell cultures (LAM n = 4, normal bronchial SMC n = 2). Data are presented as mean RQ ± SEM and significant changes are marked as asterisk, solid circle, and solid triangle (in all results significance was P < 0.05). g Proliferation capacity following Proxison treatment ( n = 3 technical repeats). Representative pictures of scratch assays in untreated and Proxison (3 µM, 1 h)-treated LAM cell lines ( n = 4) compared to normal SMC controls ( n = 2) after 12 h incubation. Data are presented as mean of cell growth (gap) area nm 2 ± SEM; significant changes are marked as asterisk ( P < 0.05). h Migration capacity of LAM cell lines. LAM cell lines ( n = 2) and normal SMCs ( n = 2) were treated with Rapamycin (20 nM, 24 h), Proxison (3 µM, 24 h), Rapamycin (20 nM, 24 h)+Proxison(3 µM, 24 h), and finally cells were pre-treated with Rapamycin for 48 h (20 nM/24 h) and then incubated with Proxison (3 µM, 24 h). Images are presented as the number of cells migrated through the membrane to the lower side of the chamber and were stained with DAPI. Data are presented as the percentage of migrated LAM cells compared to normal SMC ± SEM and significant changes are marked as ( 1 ), ( 2 ), ( 3 ), ( 4 ) and ( 5 ) (in all results significance was P < 0.05)

Article Snippet: Controls were primary, normal human bronchial SMCs (Lonza, Basel, Switzerland).

Techniques: Incubation, Fluorescence, Microscopy, Software, Flow Cytometry, Electron Microscopy, Control, Quantitative RT-PCR, Gene Expression, Activity Assay, Migration, Membrane, Staining

Journal: ACS biomaterials science & engineering

Article Title: TiO 2 -Based Nanotopographical Cues Attenuate the Restenotic Phenotype in Primary Human Vascular Endothelial and Smooth Muscle Cells

doi: 10.1021/acsbiomaterials.9b01475

Figure Lengend Snippet: Representative HIM images of primary human coronary endothelial muscle cells on (A) flat (scale bar = 2 μm) and (B) NT90 surfaces (scale bar = 200 nm). Representative HIM images of primary human coronary SMCs on (C) flat (scale bar = 1 μm) and (D) NT90 surface (scale bar = 2 μm). Cells were fixed after 2 days of culture. Filopodia are indicated by white arrows.

Article Snippet: Cell Culture and Cell Proliferation Assays Primary human coronary artery ECs and primary human coronary artery SMCs were purchased from PromoCell (Heidelberg, Germany).

Techniques:

Journal: ACS biomaterials science & engineering

Article Title: TiO 2 -Based Nanotopographical Cues Attenuate the Restenotic Phenotype in Primary Human Vascular Endothelial and Smooth Muscle Cells

doi: 10.1021/acsbiomaterials.9b01475

Figure Lengend Snippet: EC and SMC cell coverage on flat and NT surfaces. (A) EC and (E) SMC cell numbers on flat and NT surfaces were measured using a CyQUANT assay, which quantifies DNA content in cell lysates. (B) EC and (F) SMC area were quantified using fluorescence microscopy and ImageJ. Representative images of (C,D) ECs and (G,H) SMCs cultured on flat and NT surfaces are shown. Green: phalloidin. Blue: DAPI. Scale bar: 150 μm.

Article Snippet: Cell Culture and Cell Proliferation Assays Primary human coronary artery ECs and primary human coronary artery SMCs were purchased from PromoCell (Heidelberg, Germany).

Techniques: CyQUANT Assay, Fluorescence, Microscopy, Cell Culture

Journal: ACS biomaterials science & engineering

Article Title: TiO 2 -Based Nanotopographical Cues Attenuate the Restenotic Phenotype in Primary Human Vascular Endothelial and Smooth Muscle Cells

doi: 10.1021/acsbiomaterials.9b01475

Figure Lengend Snippet: Total FAK and pFAK were quantified by ELISA using cell lysates from (A,B) ECs and (C,D) SMCs cultured on flat and NT90 surfaces. Total FAK concentration was normalized to the total protein content. The pFAK concentration was normalized to the total FAK concentration. Data are expressed as mean ± SD.

Article Snippet: Cell Culture and Cell Proliferation Assays Primary human coronary artery ECs and primary human coronary artery SMCs were purchased from PromoCell (Heidelberg, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay

Journal: ACS biomaterials science & engineering

Article Title: TiO 2 -Based Nanotopographical Cues Attenuate the Restenotic Phenotype in Primary Human Vascular Endothelial and Smooth Muscle Cells

doi: 10.1021/acsbiomaterials.9b01475

Figure Lengend Snippet: Effect of NT topography on inflammatory response. (A) VCAM-1 gene expression in ECs. (B) SMC cell numbers when cultured on flat or NT90 surfaces in control media or under stimulation with 2 ng/mL TNFα, an inflammatory cytokine and known mitogen for SMCs. (C) MCP-1 secretion by SMCs cultured on flat or NT surfaces was measured in conditioned media using ELISA. Data are expressed as mean ± SD.

Article Snippet: Cell Culture and Cell Proliferation Assays Primary human coronary artery ECs and primary human coronary artery SMCs were purchased from PromoCell (Heidelberg, Germany).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: ACS biomaterials science & engineering

Article Title: TiO 2 -Based Nanotopographical Cues Attenuate the Restenotic Phenotype in Primary Human Vascular Endothelial and Smooth Muscle Cells

doi: 10.1021/acsbiomaterials.9b01475

Figure Lengend Snippet: Effect of varying NT diameter on EC and SMC response. Cell growth rates on flat and NT surfaces using (A) ECs and (D) SMCs. Significant values are represented as follows: x NT30; # NT50; *NT90. One symbol: p < 0.05; two symbols: p < 0.01. (B) Relative VCAM-1 mRNA expression in ECs cultured on flat and NT surfaces. Values are normalized to mRNA expression on flat surfaces. (C) SMC cell numbers cultured on flat and NT surfaces in control media and in media containing 2 ng/mL TNFα. (E) MCP-1 secretion by SMCs cultured on flat and NT surfaces. Note: data shown here for flat and NT90 surfaces are identical as those in previous figures. They are shown here as a reference for comparison purposes.

Article Snippet: Cell Culture and Cell Proliferation Assays Primary human coronary artery ECs and primary human coronary artery SMCs were purchased from PromoCell (Heidelberg, Germany).

Techniques: Expressing, Cell Culture