Journal: Journal of molecular and cellular cardiology

Article Title: Inducing I to,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene.

doi: 10.1016/j.yjmcc.2021.11.004

Figure Lengend Snippet: Fig. 1. A. Plasmid vector for bicistronic for Kv4.3 and KChIP2.1 with amino acids shown in black (linkers in brown). A CMV or cardiac-specific troponin T promoter (cTnT) was used in different cell types. B. Western blot of KChIP2.1 expression by the bicistronic transgene in HEK293 cells. A single band corresponding to the estimated molecular weight of KChIP2.1 or KChIP2.1-Amcyan was detected in lysates from bicistronic transgene transfected cells but not from cells transfected with Kv4.3-mCherry alone. C Expression of Kv4.3-mCherry upstream of the P2A sequence was detected at an apparent molecular weight of ~80 kDa while expression of Kv4.3 alone was detected at ~60 kDa. These data are consistent with the expected weights of Kv4.3 (71 kDa) and mCherry (20 kDa), given a − 20% gel shift for K channel protein [61]. Additional higher molecular weight bands possibly reflect multimeric forms of Kv4.3. Lysates of cells transfected with untagged Kv4.3 and Amcyan-P2A-mCherry were used as positive and negative controls respectively and SYPRO® staining was used as internal loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 ◦C (see Fig. S2A,B).

Techniques: Plasmid Preparation, Western Blot, Expressing, Molecular Weight, Transfection, Sequencing, Gel Shift, Staining, Control

Journal: Journal of molecular and cellular cardiology

Article Title: Inducing I to,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene.

doi: 10.1016/j.yjmcc.2021.11.004

Figure Lengend Snippet: Fig. 2. Biophysical properties of Ito,f currents produced by Kv4.3 alone (-KChIP2.1) and with KChIP2.1 added via the bicistronic vector in HEK293 cells. A. Exemplar whole-cell currents elicited by 500 ms-depolarization pulses from −60 mV to +40 mV in 10 mV increments from a holding potential of −80 mV. B. The peak current at all potentials was increased by bicistronic addition of KChIP2.1 and the current activated at ~ −30 mV as expected for Kv4.3 in the presence of KChIP2.1. C. The fast time constant of Ito,f inactivation as a function of test potential (Kv4.3 alone: 7.66 ± 1.5 ms, n = 5 vs. Bicistronic Kv4.3/KChIP2.1: 21.2 ± 1.6 ms, n = 18 at +40 mV; p < 0.002 Students t-test). D. Time-dependent recovery from inactivation at −80 mV. To calculate the recovery rate from inactivation, normalised tail peak current amplitudes were recorded at +40 mV and plotted as a function of the inter-pulse interval (Δt) (protocol illustrated in the inset). Error bars show mean ± s. e.m.

Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 ◦C (see Fig. S2A,B).

Techniques: Produced, Plasmid Preparation

Journal: Journal of molecular and cellular cardiology

Article Title: Inducing I to,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene.

doi: 10.1016/j.yjmcc.2021.11.004

Figure Lengend Snippet: Fig. 3. Subcellular colocalization of Kv4.3 and KChIP2.1. A. Exemplar confocal images of living HEK293 cells expressing bicistronic Kv4.3-mCherry and KChIP2.1-AmCyan. B. Representative confocal images of a trans duced rabbit ventricular cardiomyocytes expressing Kv4.3-mCherry and KChIP2 (see also Fig. S2A). Surface membranes were labelled with an anti-Cav3 antibody (top). C shows an enlarged view of the boxed regions in B and the fluorescence intensity profile from each label at bottom. D Co-localization between Kv4.3 and KChIP2.1 in different subcellular compartments (sarcolemma -SL; t-tubules TT; and remaining cytoplasm -Cyt.) measured by unbiased Pearson's correlation coefficient (n/N = 8/3). Error bars show s.e. m.

Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 ◦C (see Fig. S2A,B).

Techniques: Expressing, Fluorescence

Journal: Journal of molecular and cellular cardiology

Article Title: Inducing I to,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene.

doi: 10.1016/j.yjmcc.2021.11.004

Figure Lengend Snippet: Fig. 4. Characterization of Ito,f currents encoded by the bicistronic Kv4.3/KChIP2.1construct in rabbit ventricular cardiomycytes. A. Typical patch-clamp recordings of Ito currents from freshly isolated cardiomyocytes and 40 h after transduction. Currents were evoked by step depolarizations to potentials between −30 and + 40 mV (in 10 mV increments). B. The left panel shows current-voltage relationships of intrinsic Ito (n/N = 7/3) and exogenous Ito,f (n/N = 16/8) and the fast time constant of Ito,f inactivation (right hand panel). C. Concentration-dependence of the Ito,f block by 4-AP block (n/N = 7/4). Exemplar recordings of the Ito,f currents at +30 mV before and after application of 1 mmol/l of 4-AP are shown in the inset. D. Exemplar Ito,f traces recorded from a transduced myocyte under basal conditions and in the presence of NS5806 (5 μmol/l). The right hand panel shows the augmentation of the mean peak Ito,f current-voltage relationship by 5 μmol/l NS5806 (p < 0.001, n/N = 7/3, matched pair t-test). The cells used for these experiments were cultured for 48 h (40 h in A,B) to further increase peak Ito,f to minimise the contribution from NS5806-augmented native Ito (typically 3 pA/pF [21]). Error bars show mean ± s.e.m.

Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 ◦C (see Fig. S2A,B).

Techniques: Patch Clamp, Isolation, Transduction, Concentration Assay, Blocking Assay, Cell Culture

Journal: Diagnostics

Article Title: A Pilot Study of Exploring miRNA–Protein Interaction Networks in Pancreatic Ductal Adenocarcinoma Patients: Implications for Diagnosis and Prognosis

doi: 10.3390/diagnostics15192479

Figure Lengend Snippet: The distribution of levels of the target proteins between PDAC patients and healthy individuals. ( a ) ESR1, ( b ) HCFC1, ( c ) KCNA1, ( d ) CACNG3, and ( e ) EPC1. Differences between the groups were analyzed using the Mann–Whitney U test. Bars represent mean protein concentration, and error bars indicate standard deviation (mean ± SD). * p < 0.05 was considered statistically significant.

Article Snippet: Quantitative analysis of ESR1 (Human Estrogen Receptor Alpha, ESR1 ELISA Kit, E7871Hu), HCFC1 (Human Host Cell Factor C1, HCFC1 ELISA Kit, E0307Hu), KCNA1 (Human Voltage-Gated Potassium Channel Subunit Alpha-1, KCNA1 ELISA Kit, E1757Hu), EPC1 (Human Enhancer of Polycomb Homolog 1, EPC1 ELISA Kit, E1137529Hu), and CACNG3 (Human Voltage-Dependent Calcium Channel Subunit Gamma-3, CACNG3 ELISA Kit, E4572Hu) proteins in the serum samples of PDAC patients was performed using sandwich-format ELISA kits (BT Lab, Bioassay Technology Laboratory, Shanghai, China).

Techniques: MANN-WHITNEY, Protein Concentration, Standard Deviation

Journal: Diagnostics

Article Title: A Pilot Study of Exploring miRNA–Protein Interaction Networks in Pancreatic Ductal Adenocarcinoma Patients: Implications for Diagnosis and Prognosis

doi: 10.3390/diagnostics15192479

Figure Lengend Snippet: Scatter plot matrix demonstrating the pairwise correlations among protein serum concentration levels of ESR1, HCFC1, KCNA1, CACNG3, and EPC1. The upper triangle of the matrix displays Pearson correlation coefficients (r) with corresponding p -values. Significant positive correlations were observed for each miRNA. * p < 0.05 and ** p < 0.001 were considered statistically significant.

Article Snippet: Quantitative analysis of ESR1 (Human Estrogen Receptor Alpha, ESR1 ELISA Kit, E7871Hu), HCFC1 (Human Host Cell Factor C1, HCFC1 ELISA Kit, E0307Hu), KCNA1 (Human Voltage-Gated Potassium Channel Subunit Alpha-1, KCNA1 ELISA Kit, E1757Hu), EPC1 (Human Enhancer of Polycomb Homolog 1, EPC1 ELISA Kit, E1137529Hu), and CACNG3 (Human Voltage-Dependent Calcium Channel Subunit Gamma-3, CACNG3 ELISA Kit, E4572Hu) proteins in the serum samples of PDAC patients was performed using sandwich-format ELISA kits (BT Lab, Bioassay Technology Laboratory, Shanghai, China).

Techniques: Concentration Assay

Journal: Diagnostics

Article Title: A Pilot Study of Exploring miRNA–Protein Interaction Networks in Pancreatic Ductal Adenocarcinoma Patients: Implications for Diagnosis and Prognosis

doi: 10.3390/diagnostics15192479

Figure Lengend Snippet: Kaplan–Meier survival curves showing overall survival durations of patients based on the expression levels of target proteins. ( a ) ESR1, ( b ) HCFC1, ( c ) KCNA1, ( d ) CACNG3, and ( e ) EPC1.

Article Snippet: Quantitative analysis of ESR1 (Human Estrogen Receptor Alpha, ESR1 ELISA Kit, E7871Hu), HCFC1 (Human Host Cell Factor C1, HCFC1 ELISA Kit, E0307Hu), KCNA1 (Human Voltage-Gated Potassium Channel Subunit Alpha-1, KCNA1 ELISA Kit, E1757Hu), EPC1 (Human Enhancer of Polycomb Homolog 1, EPC1 ELISA Kit, E1137529Hu), and CACNG3 (Human Voltage-Dependent Calcium Channel Subunit Gamma-3, CACNG3 ELISA Kit, E4572Hu) proteins in the serum samples of PDAC patients was performed using sandwich-format ELISA kits (BT Lab, Bioassay Technology Laboratory, Shanghai, China).

Techniques: Expressing

Journal: Acta pharmacologica Sinica

Article Title: Knockout of interleukin-17A diminishes ventricular arrhythmia susceptibility in diabetic mice via inhibiting NF-κB-mediated electrical remodeling.

doi: 10.1038/s41401-021-00659-8

Figure Lengend Snippet: Fig. 4 Effects of IL-17 knockout on the Ito and KChIP2 expression in the hearts of diabetic mice. a Representative traces of the Ito. b Current density-voltage (I–V) relationship of the Ito. n = 8–11 cells. c Current density-voltage (I–V) relationship of the Iss. n = 9–16 cells. d Mean membrane capacitance of the Ito and Iss n = 9–16 cells. e The protein and mRNA levels of Kv4.2. n = 7. f The protein and mRNA levels of Kv4.3. n = 7. g The protein and mRNA levels of KChIP2. n = 5. *P < 0.05 vs. WT mice; #P < 0.05 vs. WT+DM mice. WT wild-type, IL-17 KO IL-17 knockout, WT+DM wild-type+diabetes mellitus, IL-17 KO+DM IL-17 knockout+diabetes mellitus.

Article Snippet: The primary antibodies included rabbit anti-mouse Nav1.5 (Alomone Labs, Israel), Kv4.2 (Alomone Labs, Israel), Kv4.3 (Alomone Labs, Israel), KChIP2 (Boster, China), Cav1.2 (Alomone Labs, Israel), and NF-κB (CST, USA). β-Actin was used as an internal control.

Techniques: Knock-Out, Expressing, Membrane

Journal: Acta pharmacologica Sinica

Article Title: Knockout of interleukin-17A diminishes ventricular arrhythmia susceptibility in diabetic mice via inhibiting NF-κB-mediated electrical remodeling.

doi: 10.1038/s41401-021-00659-8

Figure Lengend Snippet: Fig. 8 Knockout of IL-17 protects against ventricular arrhythmias in STZ-induced diabetic mice. Knockout of IL-17 downregulates the expression of NF-κB, which suppresses the expression of KCNIP2, which encodes potassium voltage-gated channel interacting protein 2; CACNA1C, which encodes the pore-forming subunit of the voltage-gated L-type calcium channel Cav1.2; and SCN5A, which encodes the pore-forming subunit of the voltage-gated sodium channel Nav1.5. Decreased expression of KChIP2 and Nav1.5 prolonged the APD and slowed the conduction velocity, which increased susceptibility to ventricular arrhythmias. KCNIP2, potassium voltage-gated channel interacting protein 2; CACNA1C, calcium voltage-gated channel subunit alpha 1 C; KCND2, potassium voltage-gated channel subfamily D member 2; KCND3, potassium voltage-gated channel subfamily D member 3; SCN5A, sodium voltage-gated channel alpha subunit 5; WT+DM, wild-type+diabetes mellitus; IL-17 KO+DM, IL-17 knockout+diabetes mellitus.

Article Snippet: The primary antibodies included rabbit anti-mouse Nav1.5 (Alomone Labs, Israel), Kv4.2 (Alomone Labs, Israel), Kv4.3 (Alomone Labs, Israel), KChIP2 (Boster, China), Cav1.2 (Alomone Labs, Israel), and NF-κB (CST, USA). β-Actin was used as an internal control.

Techniques: Knock-Out, Expressing