Journal: Journal of Natural Medicines

Article Title: Cellular mechanism for herbal medicine Junchoto to facilitate intestinal Cl − /water secretion that involves cAMP-dependent activation of CFTR

doi: 10.1007/s11418-018-1207-9

Figure Lengend Snippet: Junchoto (JCT)-induced CFTR currents in HEK293T cells transiently transfected with CFTR. a Representative records of whole-cell current activation in CFTR- ( left ) and vector-transfected ( right ) cells before and after application of 400 μg/mL JCT ( filled bars ), taken during the application of alternating pulses from 0 to ± 40 mV every 10 s. The asterisks denote times when step pulses were applied. The inset shows a membrane displaying immunoblot of CFTR protein from control (vector-transfected) and CFTR transfected HEK293T cells in the upper lane. Note that the lower band is only detected in CFTR-transfected HEK293T cells. Alpha-tubulin bands with molecular mass 50 kDa were detected at equal levels in the lower lane. b The current response to step pulses from −100 to +100 mV for CFTR ( top traces ) and the vector ( bottom trace ). c I – V relationships for the mean JCT-activated current densities for cells expressing CFTR ( open circles ; n = 6) and vector ( filled triangles ; n = 6)

Article Snippet: The blots were incubated with anti-CFTR antibody (1:1000 dilution, CUSABIO and CUSAb, MD, USA: CSB-PA001608) or monoclonal anti-α-tubulin (as an internal standard, 1:2000 dilution; Sigma-Aldrich: T6074), and stained using the enhanced chemiluminescence system (Thermo Scientific, Rockford, IL, USA).

Techniques: Transfection, Activation Assay, Plasmid Preparation, Membrane, Western Blot, Control, Expressing

Journal: Journal of Natural Medicines

Article Title: Cellular mechanism for herbal medicine Junchoto to facilitate intestinal Cl − /water secretion that involves cAMP-dependent activation of CFTR

doi: 10.1007/s11418-018-1207-9

Figure Lengend Snippet: Effects of small interfering RNA (siRNA) silencing of CFTR on JCT-induced anion currents in Caco-2 cells. a RT-PCR analysis of CFTR mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in control, mock-treated and CFTR siRNA-treated Caco-2 cells. The data represent 3 similar experiments. No PCR product was amplified when reverse transcriptase was omitted from the reaction in the RT(−) group. The nucleotide sequences of the PCR products obtained with CFTR-specific primers were completely identical to the corresponding sequences in human CFTR (4302-4722 Sequence ID: NM_000492.3). b Immunoblot of CFTR protein from control, mock-treated and CFTR siRNA-treated Caco-2 cells. Alpha-tubulin bands with molecular mass 50 kDa were detected at equal levels. c – h Representative time courses of the JCT-evoked whole-cell currents recorded at +100 and −100 mV under ramp clamp in mock siRNA-treated cells ( c ) and in CFTR siRNA-treated cells ( e ). Gray bar and solid bar show application of 400 μg/mL of JCT and 10 μM of FSK, respectively. Corresponding I – V relationships at time points a–c in d and a–c in f . g , h Averages of JCT-induced whole-cell current at +100 mV in mock and CFTR siRNA ( g ), and FSK-induced whole-cell current ( h ) ( n = 5–6). Data points show the mean ± SEM. * P < 0.05 compared to mock at +100 mV

Article Snippet: The blots were incubated with anti-CFTR antibody (1:1000 dilution, CUSABIO and CUSAb, MD, USA: CSB-PA001608) or monoclonal anti-α-tubulin (as an internal standard, 1:2000 dilution; Sigma-Aldrich: T6074), and stained using the enhanced chemiluminescence system (Thermo Scientific, Rockford, IL, USA).

Techniques: Small Interfering RNA, Reverse Transcription Polymerase Chain Reaction, Control, Amplification, Reverse Transcription, Sequencing, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: miR-200b downregulates CFTR during hypoxia in human lung epithelial cells

doi: 10.1186/s11658-017-0054-0

Figure Lengend Snippet: Regulation of CFTR during hypoxia in human lung epithelial cells, Calu3 cells (left panels) and 16HBE14o- cells (right panels). a CFTR mRNA is reduced during hypoxia. CFTR mRNA levels were monitored in qRT-PCR experiments. The results from 3 independent experiments ( n = 12) are plotted normalized to 18S rRNA levels and expressed as a fold-change over the normoxic control. b Hypoxia sequentially increases HIF-1α protein levels and reduces CFTR protein levels. Protein expression levels of were monitored with SDS-PAGE and Western Blot and normalized to β-actin levels. Two individual samples (4 μg of total protein per lane) were tested for each time point and the experiments were repeated twice. Error bars represent standard deviations. Significant changes ( P < 0.05) are marked with an asterisk

Article Snippet: The membranes were then blocked with BSA (Sigma-Aldrich) dissolved in PBS/Tween-20 (3% BSA, 0.5% Tween-20 for 1–2 h), followed by immunoblotting with the primary antibody specified for each experiment CFTR (Merck MM13–4 diluted at 1:1000), HIF-1α (Abcam ab16066, diluted at 1:1000); and beta ACTIN (Abcam ab1801, diluted at 1:1000).

Techniques: Quantitative RT-PCR, Expressing, SDS Page, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: miR-200b downregulates CFTR during hypoxia in human lung epithelial cells

doi: 10.1186/s11658-017-0054-0

Figure Lengend Snippet: Downregulation of CFTR expression during hypoxia is HIF-1 dependent and relies on both the 5′ and 3′ UTRs of CFTR mRNA. a Calu3, 16HBE14o- and HeLa WT CFTR cells were treated with hypoxia mimetics (500 μM DMOG for 12 h (light grey) and 200 μM CoCl 2 for 12 h (dark grey)) and the mRNA levels were monitored in qRT-PCR experiments. CFTR mRNA levels from 2 independent experiments ( n = 8) are plotted relative to 18S rRNA levels and expressed as a fold change over the untreated control. b Calu3 cells were transfected with 5′UTR CFTR luciferase reporter (white), 3′UTR CFTR luciferase reporter (light grey) and co-transfected with both 5′UTR and 3′UTR CFTR luciferase reporters (dark grey) and treated with hypoxia mimetics (500 μM DMOG or 200 μM CoCl 2 for 12 h) and the luciferase activity was monitored. These reporters were normalized to internal controls ( Renilla ) firefly luciferase activities from 2 independent experiments ( n = 6) and plotted and expressed as a fold-change over non-treated control. Error bars represent standard deviations. Significant changes ( P < 0.05) are marked with an asterisk

Article Snippet: The membranes were then blocked with BSA (Sigma-Aldrich) dissolved in PBS/Tween-20 (3% BSA, 0.5% Tween-20 for 1–2 h), followed by immunoblotting with the primary antibody specified for each experiment CFTR (Merck MM13–4 diluted at 1:1000), HIF-1α (Abcam ab16066, diluted at 1:1000); and beta ACTIN (Abcam ab1801, diluted at 1:1000).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay

Journal: Cellular & Molecular Biology Letters

Article Title: miR-200b downregulates CFTR during hypoxia in human lung epithelial cells

doi: 10.1186/s11658-017-0054-0

Figure Lengend Snippet: Hypoxia induces miR-200b in human airway epithelial cells in a HIF-1-dependent manner. a The predicted target site of miR-200b in CFTR 3′UTR is shown above. The miR-200b target site was predicted in human CFTR 3′UTR only, Hypoxia-induced changes in the expression profiles of miR-200b and miR-200c in Calu3 and 16HBE14o- cells are shown. The miRNA levels were monitored in qRT-PCR experiments. The results from 2 independent experiments ( n = 8) are plotted normalized to RNU48 and expressed as a fold-change over the normoxic control. b Calu3 and 16HBE14o- cells were treated with hypoxia mimetic (200 μM CoCl 2 for 12 h) and the miRNA levels were monitored in qRT-PCR experiments. miR-200b and miR-200c levels were measured in 3 independent experiments ( n = 10) and are plotted relative to RNU44 levels and expressed as a fold change over the untreated controls. Error bars represent standard deviations (SD). Significant changes ( P < 0.05) are marked with an asterisk

Article Snippet: The membranes were then blocked with BSA (Sigma-Aldrich) dissolved in PBS/Tween-20 (3% BSA, 0.5% Tween-20 for 1–2 h), followed by immunoblotting with the primary antibody specified for each experiment CFTR (Merck MM13–4 diluted at 1:1000), HIF-1α (Abcam ab16066, diluted at 1:1000); and beta ACTIN (Abcam ab1801, diluted at 1:1000).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cellular & Molecular Biology Letters

Article Title: miR-200b downregulates CFTR during hypoxia in human lung epithelial cells

doi: 10.1186/s11658-017-0054-0

Figure Lengend Snippet: Model for negative regulation of CFTR expression during hypoxia by HIF-1 and miR-200b. During hypoxia, HIF-1 activity is induced and HIF-1 binds to the hypoxia response element (HRE) sequence located in CFTR 5′UTR and has been reported to decrease CFTR expression (Zheng et al. ). Our studies show that HIF-1 induces miR-200b expression and binds to the target sequence (TS miR-200b) located at the 3′UTR of CFTR mRNA, which further decreases CFTR mRNA and protein expression

Article Snippet: The membranes were then blocked with BSA (Sigma-Aldrich) dissolved in PBS/Tween-20 (3% BSA, 0.5% Tween-20 for 1–2 h), followed by immunoblotting with the primary antibody specified for each experiment CFTR (Merck MM13–4 diluted at 1:1000), HIF-1α (Abcam ab16066, diluted at 1:1000); and beta ACTIN (Abcam ab1801, diluted at 1:1000).

Techniques: Expressing, Activity Assay, Sequencing