Journal: bioRxiv

Article Title: Effects of Hydroxychloroquine and Azithromycin on iPSC-derived Cardiomyocytes: Considerations for the Treatment of COVID-19 Patients

doi: 10.1101/2021.08.19.456950

Figure Lengend Snippet: A, Two representative western blots showing the expression of Nav1.5 and Cx43 in iPSC-CMs under different drug treatment conditions. B, Quantitation of protein expression levels of Nav1.5 in iPSC-CMs under different conditions; N = 6 independent differentiations. C, Quantitation of protein expression of Cx43 in iPSC-CMs under different drug treatment conditions; N = 6 independent differentiations. D, Representative images showing immunostaining for Nav1.5 (green) and Cx43 (red) in iPSC-CMs under different drug treatment conditions. Cell nuclei are shown in blue (Hoechst). Statistical evaluation was performed using one-way ANOVA with Tukey’s multiple comparison test (** p < 0.01, and *** p < 0.001).

Article Snippet: For staining, cells were incubated with the following primary antibodies: anti-α-actinin, clone EA-53 (1:500; mouse monoclonal, IgG1, Sigma-Aldrich, 7811), anti-Nav1.5 (1:200; rabbit polyclonal, Alomone Labs, ASC-005), and anti-Cx43, clone 2 (1:1000; mouse monoclonal, IgG1, BD Biosciences, 610061) at 4°C overnight.

Techniques: Western Blot, Expressing, Quantitation Assay, Immunostaining

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Schematics of full length Cx43 and αCT1 peptide. B) αCT1 interaction with ZO-1 PDZ domains as indicated by EDC zero-length cross-linking to GST fusion PDZ1, PDZ2 and PDZ3 polypeptides and neutravidin labeling of biotin-tagged peptide at concentrations of 5, 25 and 50 μM. The deletion of the CT Isoleucine (I) in αCT1-I renders this peptide incompetent to interact with the ZO-1 PDZ2 domain. C) Coomassie blue gel of EDC cross-linked products of kinase reaction mixtures containing GST-Cx43 CT and PKC-ε, with (αCT1) and without (Vehicle) αCT1. The fainter band above GST-Cx43 bands (indicated by lines) in the αCT1 lanes were cut from gels and analyzed by Tandem Mass Spectrometry (MS/MS). The boxes to right of gel show Cx43 CT peptides identified by MS/MS as being cross-linked to αCT1. D) Tandem mass spectrum of a quintuply charged crosslinked peptide (m/z: 674.1) between Cx43 345-366 (a-chain) and αCT1 peptide through Cx43 K346 and E8 in αCT1 (b-chain). Only the b-and y-sequence specific ions are labeled. Arrow indicates ion (b a5 2+ ) consistent with cross-linkage between Cx43 CT lysine K346 and the glutamic acid (E) residue of αCT1 at position −1.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Labeling, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Blots of EDC cross-linked products of kinase reaction mixtures containing GST-Cx43 CT, GST-Cx43 CT QQ/KK in which the lysine (K) residues were mutated to neutral glutamines (Q), PKC-ε and αCT1 (at 5, 10 and 25 μM) and a scrambled αCT1 (M4 scr) variant at the same concentrations. Only αCT1 is seen to be covalently linked by EDC to Cx43 CT in a concentration-dependent manner.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Variant Assay, Concentration Assay

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Schematics of Cx43 and the secondary structure of Cx43 CT from amino acid residues Glycine252 (G252) through to Isoleucine 382 (I382). The depiction of secondary structure in 2A has been modified from a diagram originally provided by Sosinsky and co-workers . B) ZDOCK and C) Schrodinger molecular modeling software analysis of the structure of a proposed αCT1-Cx43 CT complex. The protonated structure of αCT1 peptide and Cx43 CT (PDB:1r5s), constrained by a salt-bridge interaction between K346 in the Cx43 CT and the glutamic acid (E) at position −1 of αCT1. The αCT1-Cx43 interaction shown represents that based on the lowest energy minimization score determined in the model. D) Schrodinger molecular modeling software, a 2D map of αCT1-Cx43 CT in anti-parallel orientation showing location of amino acids predicted to bond to each other and the type of bond that is predicted to occur.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Modification, Software

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: SPR was used to analyze interactions of biotin-αCT1 and biotin-αCT1 variant peptides, immobilized to streptavidin-coated chips, with the Cx43 CT (Cx43-CT: amino acids 255 to 382) and Cx43 CT-KK/QQ as analytes, respectively. The mean of three runs is plotted for each analyte concentration. The exposure of the sensor chip to the specific analyte is indicated by the gray area. Sensorgrams obtained for: A) Cx43 CT and biotin-αCT1. B) Cx43 CT-KK/QQ and biotin-αCT1. C) Cx43 CT and biotin-M1 AALAI. D) Cx43 CT-KK/QQ and biotin-M1 AALAI. E) Cx43 CT and biotin-M3 DDLAI. F) Cx43 CT-KK/QQ and biotin-M3 DDLAI.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Variant Assay, Concentration Assay

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Melt curves (top) and first derivative of melt curves (bottom) for ZO-1 PDZ2 at 500 μg/mL in combination αCT1 at concentrations of 25, 50 and 100 μM. B) Temperature maxima (Tm) from Boltzman curves from left-to-right of Cx43 CT (Cx43-CT: amino acids 255 to 382) alone, Cx43 CT in combination with αCT1, and the αCT1 variants including: M1 AALAI, M2 AALEI, M3 DDLAI, M4 scrambled, αCT-I and αCT11. αCT1, αCT1-I and αCT11 show similar abilities to destabilize (i.e., significantly decrease the Tm of) Cx43 CT. **p<0.01, *** p<0.002, N=6. C) Temperature maxima (Tm) from Boltzman curves from left-to-right of PDZ2 alone, and PDZ2 in combination with αCT1 and αCT1variants including αCT1 variants including: M1 AALAI, M2 AALEI, M3 DDLAI, M4 scrambled, αCT-I and αCT11. M3 DDLAI, αCT1, and αCT11 show similar abilities to stabilize (i.e., significantly increase the Tm of) PDZ2. **p<0.01, ***p<0.002, N=6

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques:

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Blots of Cx43-pS368 (top) and total Cx43 (bottom) in kinase reactions mixtures including no-kinase controls with substrate (Cx43-CT: amino acids 255 to 382), but no PKC-ε (PKC-minus); Cx43-CT substrate with PKC-ε (PKC-plus); and mixtures containing PKC-ε, Cx43 CT, and biotin-tagged αCT1, biotin-tagged αCT1 mutant peptides with alanine substitutions (M1 AALAI, M2 AALEI, M3 DDLAI) and biotin-tagged M4 scrambled. Peptides are at 20 μM. B) Blots of Cx43-pS368 (top) and total Cx43 (bottom) in kinase reactions mixtures including no-kinase controls with Cx43 CT substrate, but no PKC-ε (PKC-minus); Cx43-CT substrate with PKC-ε (PKC-plus); and mixtures containing PKC-ε, Cx43 CT, and biotin-αCT1, biotin-αCT1-I or biotin-αCT11 (RPRPDDLEI with no antennapedia sequence at peptide NT) and biotin-M4 scrambled peptide. Peptides are at 20 μM. C) Chart showing that the ability of unmodified αCT1 and the Cx43 CT interaction-competent peptides biotin-αCT1-I or biotin-αCT11 to induce S368 phosphorylation was 3-5 fold greater than that of non-Cx43 CT interacting peptides. * p<0.05, ** p<0.01, *** p<0.002, N=5 αCT1 and M4, other peptides N=3.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Langendorff ischemia-reperfusion (I/R) injury protocols were performed on adult mouse hearts instrumented to monitor LV contractility (protocol in ). LV Systolic responses are shown in 7A-C : (A) Plots of left ventricular (LV) systolic developed pressure against balloon volume ; (B) LV maximal rate of tension development (+dP/dt) against balloon volume; (C) Maximal systolic elastance (E max ) – i.e., the slope from (A) ; (D) Plots of LV end diastolic pressure (EDP) against balloon volume; (E) Maximal rate of relaxation (-dP/dt) against balloon volume; (F) Stiffness, the reciprocal of the slope from (D) ; ( G) Percentage of LV contractile function recovery post-ischemia relative to baseline level. Data shown are mean ± S.E. N=4-8. *p<0.05, ***p<0.001, N=4-8 hearts/group. H) Blots of Cx43-pS368 (top) and total Cx43 (bottom) of LV samples infused with peptide for 20 minutes according to the protocol in . For hearts used in Western blots, the protocol did not proceed to the ischemia and reperfusion phases, being terminated after the peptide infusion step. Only those peptides competent to interact with Cx43 CT increase pS368 levels relative to total Cx43 above vehicle control.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Langendorff I/R protocols were performed on adult mouse hearts instrumented to monitor LV contractility. Protocol in , except that a 20-minute peptide infusion was begun after ischemic injury at the initiation of reperfusion. (A) Plots of left ventricular (LV) developed pressure against balloon volume; (B) Maximal systolic elastance (E max ), the slope from (A); (C) Maximal rate of tension development (+dP/dt) against balloon volume; (D) Plots of end diastolic pressure (EDP) against balloon volume; (E) Stiffness, the reciprocal of the slope from (D); (F) Maximal rate of relaxation (-dP/dt) against balloon volume. * p<0.05, *** p<0.001, N=4-8. G) Laser scanning confocal microscopic fields from sections of Vehicle control, αCT1, and αCT11 group hearts stained for Cx43 (green), nuclei (DAPI-blue), and Alexa647-conjugated streptavidin (red). H) Average intensities of biotinylated peptide (indicated by streptavidin Alexa647 fluorescence intensity level relative to background) in Vehicle control, αCT1, and αCT11 groups. ** p<0.05; not significant (ns) N=5 hearts/group. Scale bar = 5 μm.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Staining, Fluorescence

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: EP gating strategy and in vitro results. (A) Transmission electron microscopy images of EP. (B) The EP gate was defined using 0.5 µm and 1.53 µm calibration beads. EP population shift was observed after staining with ASGR1 antibodies. (C) In vitro , TNF-α induced human hepatocyte–derived EP. Primary human hepatocytes following liver resection were isolated, cultured overnight, and stimulated with 10 and 20 ng/ml TNF-α. EP surface antigens were stained, and absolute AnnV + , CD130 + , Cx43 + , ASGR1 + EP were analyzed. The ordinary one-way ANOVA with Tukey’s post hoc test was used. The plots are indicated by the mean, and all error bars indicate the SD.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: In Vitro, Transmission Assay, Electron Microscopy, Staining, Derivative Assay, Isolation, Cell Culture

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: Time course of EP after LT (n = 11). (A) Total EP (/µl) (B) AnnV + , CD130 + , CD31 + EP (/µl) (C) Cx43 + , ASGR1 + , MDR3 + EP (/µl) were stained and analyzed. The Wilcoxon matched-pairs signed ranked test was used. The plots are indicated by the median, and all error bars indicate the interquartile range (IQR). A single asterisk indicates significance at p < 0.05 (D) Patients at risk for acute rejection. EP dynamics from preoperative state to POD 1 of the total, AnnV + , CD130 + , Cx43 + , ASGR1 + , MDR3 + , CD31 + EP (/µl) and their fold change were analyzed. Absolute EP were non-normally distributed; the two-tailed Mann-Whitney U test was used. The plots are indicated by the mean, and all error bars indicate the SD.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: EP profiles during histology-proven ACR. EP were determined in the blood plasma of patients with concomitant LBs. The patients were then classified as histology-proven ACR or non-ACR. EP surface antigens (A) AnnV + , (B) CD130 + , (C) Cx43 + , (D) ASGR1 + , (E) MDR3 + , (F) CD31 + EP were stained, and relative EP (%) were analyzed. (G) Example FSC-A (forward scatter) histograms of MDR3 + EP (%) (H) demonstrating the highest receiver operating characteristic (ROC) of all single antigens. One-way analysis of variance followed by Tukey’s post hoc test was used. The plots are indicated by the median, and all error bars indicate the IQR.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Clinical Proteomics, Staining