primary cultured pcasmcs  (Thermo Fisher)

Journal: Oncotarget

Article Title: Activation of PERK branch of ER stress mediates homocysteine-induced BK Ca channel dysfunction in coronary artery via FoxO3a-dependent regulation of atrogin-1

doi: 10.18632/oncotarget.17721

Figure Lengend Snippet: Treatment with ER stress inhibitors, either 4-PBA or TUDCA preserves the relaxant response in both homocysteine- ( A ) and tunicamycin- ( B ) exposed arteries. Homocysteine exposure lowers the protein level of BK Ca β1 subunit in PCASMCs (n=5) whereas shows no significant effect on the protein content of α subunit (n=8) ( C & D ). Inhibition of ER stress during homocysteine exposure restores BK Ca β1 protein level ( D ). Homocysteine does not alter mRNA expression levels of both α and β1 subunits of BK Ca ( E & F , n=7). N indicates the number of independent experiments, each obtained from vessels (a & b) or cell isolates (c-f) of different heart. *p<0.05, **p<0.01 vs. control; # p<0.05, ## p<0.01 vs. Hcy or TM. Hcy: homocysteine, TM: tunicamycin.

Article Snippet: Briefly, primary cultured PCASMCs were harvested with trypsin-EDTA (Thermo Fisher Scientific, USA) and centrifuged at 500×g for 5 min to collect the cell pellet.

Techniques: Inhibition, Expressing, Control

Journal: Oncotarget

Article Title: Activation of PERK branch of ER stress mediates homocysteine-induced BK Ca channel dysfunction in coronary artery via FoxO3a-dependent regulation of atrogin-1

doi: 10.18632/oncotarget.17721

Figure Lengend Snippet: While the overall level of FoxO3a expression remains unchanged ( A , n=7), nuclear to cytoplasmic expression ratio of FoxO3a significantly increases in homocysteine-exposed PCASMCs ( B , n=4). Inhibition of ER stress and selective blockade of the PERK branch of ER stress both effectively inhibit homocysteine-induced FoxO3a nuclear translocation ( B & C , n=4) and atrogin-1 upregulation ( D & E , n=4). *p<0.05, # p<0.05; **p<0.01, ## p<0.01. Hcy: homocysteine, TUDCA and 4-PBA: ER stress inhibitors, GSK: GSK2606414, PERK selective blocker.

Article Snippet: Briefly, primary cultured PCASMCs were harvested with trypsin-EDTA (Thermo Fisher Scientific, USA) and centrifuged at 500×g for 5 min to collect the cell pellet.

Techniques: Expressing, Inhibition, Translocation Assay

Journal: Oncotarget

Article Title: Activation of PERK branch of ER stress mediates homocysteine-induced BK Ca channel dysfunction in coronary artery via FoxO3a-dependent regulation of atrogin-1

doi: 10.18632/oncotarget.17721

Figure Lengend Snippet: Silencing of the FoxO3a gene by siRNA prevents atrogin-1 upregulation (A) and BK Ca β1 downregulation (B) in homocysteine-exposed PCASMCs. The role of atrogin-1 as a critical mediator of homocysteine-induced BK Ca β1 loss is evidenced by the preserved protein level of BK Ca β1 in homocysteine-exposed cells that are transfected with atrogin-1 siRNA (C) . *p<0.05, **p<0.01; # p<0.05, ## p<0.01. Silencing of the FoxO3a gene prevents homocysteine-induced inhibition of BK Ca channel currents ( D & E ). Original traces and I-V curves of whole-cell K + current under unstimulated condition (D - left panel) and in response to NS1619 (E - left panel) in cells from different treatment groups before and after application of the BK Ca channel blocker iberiotoxin. *p<0.05, **p<0.01, ***p<0.001 vs. basal, # p<0.05, ## p<0.01, ### p<0.001 NS1619+Ibtx vs. NS1619. Summarized data of BK Ca channel currents from 5 independent experiments under conditions without (D - right panel) or with NS1619 stimulation. (E - lower panel), each obtained from cell isolates of different heart. *p<0.05. Hcy: homocysteine, Ibtx: iberiotoxin.

Article Snippet: Briefly, primary cultured PCASMCs were harvested with trypsin-EDTA (Thermo Fisher Scientific, USA) and centrifuged at 500×g for 5 min to collect the cell pellet.

Techniques: Transfection, Inhibition