Journal: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Effects of inhibition of AGEs on coronary artery tensions and BK channel densities and protein expression (a) Representative tracings for 60 mmol/L KCl and 100 nmol/L IBTX induced vascular tension alterations of coronary arterial rings from C+V, DM+V, C+A and DM+A groups. (b) Graph data showing the vascular tension alterations induced by KCl. (c) Graph data showing the vascular tension alterations (IBTX/KCl). (d and e) Whole-cell potassium currents before and after application of 100 nmol/L IBTX, and the I-V relationship of IBTX-sensitive currents of control and AGEs-cultured freshly isolated rat coronary arterial SMCs ( n = 3∼6 per group). (f) The representative tracings of baseline potassium currents and potassium currents after application of 100 nM IBTX in rat coronary arterial SMCs of the C+V, DM+V, C+A and DM+A groups, respectively ( n = 3∼5 per group). (g) Graph data showing IBTX-sensitive current densities at the testing potential of +100 mV in rat coronary arterial SMCs of the four groups. (h–j) The protein expressions of BK-α and BK-β1 in human coronary arterial SMCs in the BSA and BSA-AGEs groups ( n = 6∼9 per group). Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels. (k-l) The mRNA expression of BK-α and BK-β1 in rat coronary arteries of the C+V, DM+V, C+A and DM+A groups. β-actin was used as an internal control to normalize differences in the amount of total RNA in each rat sample ( n = 4 per group). (m and n) The mRNA expression of BK-α and BK-β1 in human coronary arterial SMCs of the NG, HG, NG+A, HG+A groups. GAPDH was used as an internal control to normalize differences in the amount of total RNA in each cell sample ( n = 4∼5 per group). (o–q) Protein expressions of BK-α and BK-β1 in rat coronary arteries of the C+V, DM+V, C+A and DM+A groups. Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels ( n = 5 per group). (r–t) Protein expressions of BK-α and BK-β1 in human coronary arterial SMCs of the NG, HG, NG+A, HG+A groups. Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels ( n = 5∼9 per group). (C+V: Control + Vehicle; C+A: Control + aminoguanidine; DM+V: DM + Vehicle; DM+A: DM + aminoguanidine. NG: normal glucose; HG: high glucose; NG+A: normal glucose + aminoguanidine; HG+A: high glucose + aminoguanidine).

Article Snippet: Human coronary arterial SMCs (ATCC, #PCS-100-021) and the culture medium (ATCC, #PCS-100-042 and #PCS-100-030) were purchased from ATCC.

Techniques: Inhibition, Expressing, Control, Cell Culture, Isolation

Journal: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Regulation of Akt in AGEs-mediated FBXO32-induced BK-β1 degradation (a and b) Protein expression of FBXO32 in rat coronary arteries of four groups ( n = 5 per group). (c and d) Protein expression of FBXO32 in human coronary arterial SMCs of four cell groups. Quantitative analysis of FBXO32 was normalized to GAPDH protein expression levels. (e–g) Phosphorylation levels of Akt and total Akt in rat coronary arteries of four groups ( n = 8 per group). (h–j) Phosphorylation levels of Akt and total Akt in human coronary arterial SMCs of four groups ( n = 3 per group). The phosphorylation level of Akt (k and n) and the protein expressions of FBXO32 (l and o) and BK-β1 (m and p) were measured after human coronary arterial SMCs were incubated for 96 h in DMEM containing 25.5 mmol/L glucose, or 25.5 mmol/L glucose with aminoguanidine in the absence or presence of MK2206 (0.3 μM) ( n = 5∼10 per group). MK2206 was added at the beginning and remained for 6 h (C+V: Control + Vehicle; C+A: Control + aminoguanidine; DM+V: DM + Vehicle; DM+A: DM + aminoguanidine. NG: normal glucose; HG: high glucose; NG+A: normal glucose + aminoguanidine; HG+A: high glucose + aminoguanidine.)

Article Snippet: Human coronary arterial SMCs (ATCC, #PCS-100-021) and the culture medium (ATCC, #PCS-100-042 and #PCS-100-030) were purchased from ATCC.

Techniques: Expressing, Phospho-proteomics, Incubation, Control

Journal: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Regulation of AMPK in Akt-mediated FBXO32-induced BK-β1 degradation by AGEs (a–c) Protein expression of p-AMPK and AMPK in rat coronary arteries from the four groups ( n = 8 per group). (d–f) Protein expression of p-AMPK and AMPK in human coronary arterial SMCs from the four groups ( n = 9 per group). Quantitative analysis of p-AMPK and AMPK was normalized to GAPDH protein expression levels. (g) Human coronary arterial SMCs were incubated for 96 h in DMEM containing 25.5 mmol/L glucose, or 25.5 mmol/L glucose and aminoguanidine in the absence or presence of Compound C (CC, 1 μM). Subsequently, the phosphorylation level of AMPK (h and i), AKT (j and k), and the protein expressions of FBXO32 (l) and BK-β1 (m) were measured ( n = 8 and 9 per group). Quantitative analysis of FBXO32 and BK-β1 was normalized to GAPDH protein expression levels.

Article Snippet: Human coronary arterial SMCs (ATCC, #PCS-100-021) and the culture medium (ATCC, #PCS-100-042 and #PCS-100-030) were purchased from ATCC.

Techniques: Expressing, Incubation, Phospho-proteomics

Journal: Quantitative Imaging in Medicine and Surgery

Article Title: Responsive alginate-cisplatin nanogels for selective imaging and combined chemo/radio therapy of proliferating macrophages

doi: 10.21037/qims.2018.09.01

Figure Lengend Snippet: Viability of J774A.1 cells treated with (A) TANgel or (B) free Cis at various Cis equivalent concentrations, with or without radiotherapy (RT, 3 Gy). **, P<0.01, ***, P<0.001. (C) Viability of normal cell lines (HDMVECn, HDF, and HCASMC) treated with TANgel at various Cis equivalent concentrations. TANgel, theranostic alginate-based nanogels.

Article Snippet: HDMVECn and HCASMC cells were maintained in vascular cell basal medium plus microvascular endothelial growth kit (ATCC).

Techniques:

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:

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