Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: A & B, illustration of a HCN4 -subunit with the cytoplasmic NH2- and COOH-terminus, six transmembrane segments (S1–S6), including the S4 voltage sensor (‘+’ sign denotes amino acid residues with positive charge), the pore loop between S5 and S6, and the C-linker (CL) with the cyclic-nucleotide binding domain (CNBD). The red (seven) and blue (three) circles denote the location of the novel variants identified in the early-onset AF cases and referents, respectively.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Binding Assay

Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: A, current recordings of wild type HCN4, p.Lys189Arg and p.Gly1077Ser channels (see methods). B, plots of activation curves for wild type HCN4, p.Lys189Arg and p.Gly1077Ser. C & D, plots of V1/2 and k for wild type HCN4 and the seven variants. The dashed black line through p.Pro257Ser denotes the lack of measureable current (see figure 3). The V1/2 and k for the six variants expressing current were not significantly different from wild type (t-test, p> 0.05). The numbers in the parentheses represent the number of cells.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Activation Assay, Expressing

Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: Electrophysiology properties of wild type and variant HCN4 channels.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Variant Assay, Expressing

Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: A, Currents were elicited from a holding current of -35 mV to a test pulse of -150 mV (fully-activated voltage) for 4 seconds and returned back to the holding current. The wild type HCN4 channel produced a current and the p.Pro257Ser mutant did not. B, confocal micrographs of wild type HCN4 and p.Pro257Ser channels expressed in CHO cells. The cells were stained with rabbit anti-HCN4 antibody (green) and DAPI (blue). The wild type HCN4 channel is expressed on the cell membrane and in the cytoplasm, whereas the p.Pro257Ser mutant channel is restricted to the cytoplasm. The scale bar denotes 50μm.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Produced, Mutagenesis, Staining

Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: A, current recordings of wild type HCN4, p.Asn688Ser and p.Arg1068His channels. B, plots of activation curves for wild type HCN4, p.Asn688Ser and p.Arg1068His channels. C & D, plots of V1/2 and k for wild type HCN4 and the three mutants. The V1/2 and k for the three mutants were not significantly different from wild type (t-test, p> 0.05). The numbers in parentheses represent the number of analyzed cells.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Activation Assay

Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: A, current recordings of wild type HCN4 (2 μg) and wild type HCN4 (1 μg)+p.Pro257Ser (1 μg). B, plot of current density (pA/pF) measured at -150 mV for wild type HCN4 and wild type HCN4+p.Pro257Ser. C, plots of activation curves for wild type HCN4 and wild type HCN4+p.Pro257Ser. The number in parentheses represents the number of cells. D, confocal micrographs of co-expressed wild type HCN4 and p.Pro257Ser constructs tagged with unique C-terminal epitopes in CHO cells (see methods); i) wild type HCN4-myc (green)+ wild type HCN4-V5 (red) and ii) wild type HCN4-myc (green)+p.Pro257Ser-V5(red). Co-expressed wild type HCN4-myc and wild type HCN4-V5 channels both traffick and are distributed together on cell membrane. ii) wild type HCN4-myc +p.Pro257Ser-V5 images show the wild type HCN4-myc channel expressed on the cell membrane and the p.Pro257Ser-V5 channel distributed in the cytoplasm and not on the cell membrane. Cells were also stained with DAPI (blue) to visualize the nucleus which is shown in the merged images. The scale bar denotes 50μm.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Activation Assay, Construct, Staining

Journal: BMC Gastroenterology

Article Title: Inhibition of TLR4 enhances oxaliplatin chemotherapy sensitivity in esophageal squamous cell carcinoma by suppressing inflammation and glycolysis

doi: 10.1186/s12876-026-04663-2

Figure Lengend Snippet: OXA activated TLR4 signaling in ESCC cells. The cells were treated by OXA (25 µM) for 24 h. A , B . The mRNA expressions of TLR4 and MYD88 in ESCC cell lines and normal esophageal cells were detected by qRT-PCR. C . ICC showed the immunocytochemical activity of NF-κB p65, p-NF-κB p65, COX-2, and MYD88. Scale bar: 50 μm. D . qRT-PCR analysis of mRNA levels of IL-1β, IL-6, COX-2, CXCL5, and CXCL8. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control group

Article Snippet: Serum concentrations of IL-6 (JL20268, Jonin) and IL-1β (KE10003, Proteintech) were quantified using commercial ELISA kits according to manufacturers’ protocols.

Techniques: Quantitative RT-PCR, Activity Assay, Control

Journal: BMC Gastroenterology

Article Title: Inhibition of TLR4 enhances oxaliplatin chemotherapy sensitivity in esophageal squamous cell carcinoma by suppressing inflammation and glycolysis

doi: 10.1186/s12876-026-04663-2

Figure Lengend Snippet: TLR4 knockout enhances the sensitivity of OXA chemotherapy in ESCC in vivo. A . Number of tumors. B . HE staining of esophageal epithelial tissues. Scale bar: 50 μm. C . Body weight of mice. D , E . the levels of serum IL-1β and IL-6 in mice from different groups were detected by ELISA. F . The immunohistochemical activities of PCNA, CK14, Cyclin D1, COX-2, S100A8 and S100A9 in the esophageal tissue were detected. Scale bar: 100 μm. G , H . The mRNA levels of inflammatory cytokines and glycolysis-related proteins in esophageal tissue were detected by qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. WT + 4NQO + OXA group

Article Snippet: Serum concentrations of IL-6 (JL20268, Jonin) and IL-1β (KE10003, Proteintech) were quantified using commercial ELISA kits according to manufacturers’ protocols.

Techniques: Knock-Out, In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Quantitative RT-PCR

Journal: BMC Gastroenterology

Article Title: Inhibition of TLR4 enhances oxaliplatin chemotherapy sensitivity in esophageal squamous cell carcinoma by suppressing inflammation and glycolysis

doi: 10.1186/s12876-026-04663-2

Figure Lengend Snippet: Schematic diagram illustrating the mechanism by which TLR4 inhibition enhances oxaliplatin (OXA) chemosensitivity in esophageal squamous cell carcinoma (ESCC). OXA treatment upregulates TLR4 and its downstream adaptor protein MYD88, which activates the phosphorylation of NF-κB p65. This activation drives two parallel pathways: (1) the inflammatory response, characterized by the upregulation of pro-inflammatory factors such as IL-6, COX-2, and CXCL5; (2) the glycolytic metabolic reprogramming, mediated by the HIF-1α/GLUT1 axis and enhanced expression of glycolytic enzymes including PFKM and LDHB. These two pathways synergistically promote ESCC cell proliferation, migration, and invasion, ultimately reducing OXA chemosensitivity. Inhibition of TLR4 (via genetic knockout, shRNA knockdown, or pharmacological inhibitor TAK-242) or its downstream mediator MYD88 (via shRNA knockdown or inhibitor ST2825) blocks NF-κB p65 phosphorylation, thereby suppressing both the inflammatory response and glycolytic activity. This dual inhibition disrupts the adaptive survival mechanisms of ESCC cells, potentiating the anti-tumor efficacy of OXA

Article Snippet: Serum concentrations of IL-6 (JL20268, Jonin) and IL-1β (KE10003, Proteintech) were quantified using commercial ELISA kits according to manufacturers’ protocols.

Techniques: Inhibition, Phospho-proteomics, Activation Assay, Expressing, Migration, Knock-Out, shRNA, Knockdown, Activity Assay