Journal: bioRxiv

Article Title: Neuregulin-1 protects against respiratory viral induced mortality

doi: 10.1101/2023.05.10.540232

Figure Lengend Snippet: ( A ) CD11c + cells are increased in the lungs of atopic mice. Flow cytometry of lung cell suspension showing frequency (left) and cell number (right) at day 3 post inoculation (PI) high dose SeV ( B ) Adoptive transfer of CD11c + cells isolated from NA or atopic mice into naïve mice 24 h before inoculation with high dose SeV delays but does not prevent mortality; n=4 per group. ( C ) Transcriptomic (RNAseq) comparison between FACS isolated lung CD11c + cells from atopic and NA mice identifies several disparately expressed gene products, including Nrg1 (n=4 per group). Selected gene products shown. ( D ) NRG1 is markedly increased in atopic mouse lung (“tissue”), BAL, and supernatant from 1 x10 6 atopic lung CD11c + cells (“CD11c sup”) cultured for 24 h. ( E ) The scRNA (10x Platform) tSNE coordinates show CD14 + monocytes and dendritic cell (“DC”) sub-populations expressing NRG1 in peripheral blood cells of healthy human donors.*p<0.05, **p<0.01, ****p<0.0001

Article Snippet: Cell culture basal media was supplemented with recombinant human NRG1 alpha (cat#: NBP2-35093, Novus Bio) for hBEC or with mouse NRG1 for mTEC on day five and three before and at the time of infection with 4000 pfu of rgRSV or SeV-GFP.

Techniques: Flow Cytometry, Suspension, Adoptive Transfer Assay, Isolation, Comparison, Cell Culture, Expressing

Journal: bioRxiv

Article Title: Neuregulin-1 protects against respiratory viral induced mortality

doi: 10.1101/2023.05.10.540232

Figure Lengend Snippet: ( A ) NRG1(1ng to 1000ng) i.n. (in 30µL) given daily to naïve mice for 5d before inoculation with high dose SeV reduces viral mortality; n=4 per group (1ng, 10ng 1000ng & PBS), n=8 per group (100 ng and 500 ng). ( B ) Ratio of EBD in the BAL to that in the lung shows reduced EBD in NRG1 treated mice on day 8 PI SeV. n≥8 per group, median ± IQR shown, Mann-Whitney U test.

Article Snippet: Cell culture basal media was supplemented with recombinant human NRG1 alpha (cat#: NBP2-35093, Novus Bio) for hBEC or with mouse NRG1 for mTEC on day five and three before and at the time of infection with 4000 pfu of rgRSV or SeV-GFP.

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Neuregulin-1 protects against respiratory viral induced mortality

doi: 10.1101/2023.05.10.540232

Figure Lengend Snippet: ( A ) Adding NRG1 to hBEC inoculated with rgRSV (left) and mTEC inoculated with GFP-SeV (right) reduces spread of infection. Representative images shown. ( B ) Quantification of (A) for rgRSV and hBEC and ( C ) for GFP-SeV and mTEC. GFP positive cells quantified by ImageJ. Representative images from ≥3 separate experiments. *p<0.05, **p<0.01. ( D ) Transcriptomic analysis of hBEC cultures treated with NRG1 (100 ng) on the basolateral side of the Transwell for 5 days and inoculated with RSV (4000 pfu). RNA was isolated 48 h PI RSV and qRT-PCR performed using a custom Prime PCR array plate: (i) Transcripts in which NRG1 treatment reduced gene expression from that seen in RSV infected cells. (ii) Transcripts with low level expression that show small but significant change in expression relative to naïve control with NRG1 alone or genes significantly increased with RSV but whose expression levels were not affected by NRG1. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3.

Article Snippet: Cell culture basal media was supplemented with recombinant human NRG1 alpha (cat#: NBP2-35093, Novus Bio) for hBEC or with mouse NRG1 for mTEC on day five and three before and at the time of infection with 4000 pfu of rgRSV or SeV-GFP.

Techniques: Infection, Isolation, Quantitative RT-PCR, Expressing, Control

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: Disulfide-linked complexes isolated from human platelet-rich plasma following mechanism-based kinetic trapping using variants of PDI ( a ) or ERp57 ( b ) were subjected to immunoblotting using anti-FLAG (Green) and anti-HRG antibody (Red) under non-reducing condition. The representative blots from 3 independent experiments with similar results were shown. c The redox state of Cys was measured via differential alkylation using 12 C-IPA and 13 C-IPA (2-iodo-N-phenylacetamide) followed by chymotrypsin digestion and mass spectrometry analysis through a procedure as illustrated. d Redox state of Cys pairs was measured in purified human HRG incubated without (Nil) or with recombinant human PDI (2- or 10-fold molar excess). The number of peptides ( n value) analyzed in each group was indicated above the bars. e The predicted disulfide bonds were experimentally mapped using a method with disulfide-linked peptides and their relative abundance to the C6–C486 disulfide calculated. The cysteine pairing of the identified disulfide bonds in HRG was confirmed using label-free disulfide-linked peptides by mass spectrometry analysis. Except for C60–C71 and C87–C108 which crosslinked into a tripeptide with a molecular mass exceeding the limit of detection by the mass spectrometer, all disulfide bonds were mapped with relative abundance at a ratio of 0.3~8 as compared to the peptide containing C6–C486 bond. The data confirmed the identity of 3 disulfide bonds targeted by PDI. n.d., no peptide detected. f The disulfide pairs were located and mapped on the domain structure of human HRG with red color indicating the targets of PDI. Data are presented as mean values ± SEM. Source data are provided as a Source Data file.

Article Snippet: The samples were diluted 1:7 and incubated on pre-coated microtiter plates with or without 1 μM Zn 2+ at 37 °C for 1 h. To determine the effect of cations on HRG binding, purified human HRG (10 nM) was incubated on microtiter plates pre-coated with heparin (625 U/mL) in the presence of gradient concentrations of Ca 2+ (0.3, 1, 3 mM) or Zn 2+ (1, 3, 10, 30 μM) for 2 h. In all experiments HRP-conjugated mouse-anti human HRG (Angio-Proteomie, 1:10000) was added into washed plates and incubated for 1 h. After washing the TMB substrate was added and the plate was measured at OD 450 nm to quantitate the bound HRG.

Techniques: Isolation, Clinical Proteomics, Western Blot, Mass Spectrometry, Purification, Incubation, Recombinant

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: a Purified human HRG was pre-treated with vehicle or recombinant human PDI variants at indicated concentrations and incubated on heparin-coated surface with or without Zn 2+ (1 μM). The binding of HRG was determined by ELISA ( n = 3 independent samples). b Human plasma pre-treated with recombinant human PDI variants was incubated on HUVECs with Zn 2+ (10 μM). The binding of HRG and antithrombin on the cell surface were determined by immunofluorescence (scale bar: 20 μm). Human plasma pre-treated with recombinant human PDI variants was incubated on HUVECs with or without Zn 2+ (10 μM). The binding of HRG ( c ) and antithrombin ( d ) on the cell surface were determined by cell-based ELISA ( n = 6 independent samples). e Purified human HRG was pre-treated with vehicle or recombinant human PDI variants and incubated on FXIIa-coated surface with or without Zn 2+ (1 μM). The binding of HRG was determined by ELISA ( n = 6 independent samples). f Purified human HRG was pre-treated with vehicle or recombinant human PDI variants, and mixed with FXIIa (10 nM) with or without Zn 2+ (1 μM). The activity of FXIIa was determined using chromogenic substrate S2302 ( n = 6 independent samples). g Purified human HRG was pre-treated with vehicle or recombinant human PDI variants, mixed with FXIIa, and then added into DKO mouse plasma to initiate thrombin generation (TGA). The lag time and peak time ( h ), and the peak height and area under the curve (AUC) ( i ) from individual TGA curve were calculated ( n = 6 independent samples). j Recombinant mouse HRG was pre-treated with vehicle or recombinant mouse PDI variants, and then added into Hrg −/− mouse plasma. TGA was initiated by addition of aPTT reagent. The lag time and peak time ( k ), and the peak height and AUC ( l ) from individual TGA curve were calculated ( n = 6 independent samples). Veh vehicle. Data are presented as mean values ± SEM and analyzed by Welch’s ANOVA test ( c – f , h , I , k and l ). Source data are provided as a Source Data file.

Article Snippet: The samples were diluted 1:7 and incubated on pre-coated microtiter plates with or without 1 μM Zn 2+ at 37 °C for 1 h. To determine the effect of cations on HRG binding, purified human HRG (10 nM) was incubated on microtiter plates pre-coated with heparin (625 U/mL) in the presence of gradient concentrations of Ca 2+ (0.3, 1, 3 mM) or Zn 2+ (1, 3, 10, 30 μM) for 2 h. In all experiments HRP-conjugated mouse-anti human HRG (Angio-Proteomie, 1:10000) was added into washed plates and incubated for 1 h. After washing the TMB substrate was added and the plate was measured at OD 450 nm to quantitate the bound HRG.

Techniques: Purification, Recombinant, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Immunofluorescence, In-Cell ELISA, Activity Assay

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: a The accumulation of platelets and HRG during laser injury-induced thrombus formation in murine cremaster arterioles were visualized using Dylight-649-congugated anti-CD42c (Red) and Alexa-488-conjugated anti-HRG (Green), respectively, with irrelevant IgG as background control (488-IgG) (scale bar: 30 μm) (Supplementary Movie ). The median fluorescence intensity of HRG ( b ) and platelets ( c ) were calculated and plotted over time from mice treated or untreated with eptifibatide (10 μg/g body weight, repeated every 15~20 min). The area under the curve (AUC) for platelets ( d ) and HRG ( e ) were analyzed from each individual thrombus in different treatment groups as indicated. The number of thrombi ( n value) analyzed in each group was indicated above the bars. f A representative focal plane of HRG accumulation and vascular endothelium, as detected by Alexa-488-conjugated anti-HRG (Green) and Alexa-647-conjugated anti-CD31 (Red), respectively, approximately 5 min after laser-induced vessel injury, was extracted from in vivo two-photon scanning of the cremaster arterioles. The areas of the cremaster arteriole (ROI1, cyan) and the thrombus (ROI2, magenta) were manually defined (scale bar: 20 μm). g The intensities of green (HRG) and red (CD31) fluorescence were plotted spatially along the white solid line as indicated in f . h The scatter plot of pixel intensities of green (HRG) and red (CD31) fluorescence in ROI1 and ROI2 as indicated in f were presented. The Pearson’s correlation coefficients (r) for pixels with intensity above the threshold (indicated by the dashed lines) in the thrombus (ROI2) and in the area without the thrombus (ROI1-ROI2) were inserted. Veh vehicle, Epti eptifibatide. Data are presented as mean values ± SEM and analyzed by Kruskal–Wallis test with Dunn’s multiple comparisons ( d and e ). Source data are provided as a Source Data file.

Article Snippet: The samples were diluted 1:7 and incubated on pre-coated microtiter plates with or without 1 μM Zn 2+ at 37 °C for 1 h. To determine the effect of cations on HRG binding, purified human HRG (10 nM) was incubated on microtiter plates pre-coated with heparin (625 U/mL) in the presence of gradient concentrations of Ca 2+ (0.3, 1, 3 mM) or Zn 2+ (1, 3, 10, 30 μM) for 2 h. In all experiments HRP-conjugated mouse-anti human HRG (Angio-Proteomie, 1:10000) was added into washed plates and incubated for 1 h. After washing the TMB substrate was added and the plate was measured at OD 450 nm to quantitate the bound HRG.

Techniques: Control, Fluorescence, In Vivo

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: a Mouse plasma pre-treated with recombinant mouse PDI-CCCC or PDI-AAAA was incubated on mouse cerebral microvascular endothelial cells (bEnd.3) in the presence of Zn 2+ (100 μM). The binding of HRG on the cell surface were determined by immunofluorescence using Alexa-488-conjugated antibodies (scale bar: 20 μm). b Representative images of immunohistochemistry of HRG from cross sections of thrombus induced by FeCl in the carotid artery in mice treated with vehicle or Rutin (scale bar: 250 μm). c Representative images of HRG incorporation (Green), visualized using Alexa-488-conjugated anti-HRG, at indicated time points following laser injury in the cremaster arterioles in mice treated with vehicle or Rutin (5 μg/g body weight) (scale bar: 30 μm) (Supplementary Movie 2). The median fluorescence intensity ( d ) and the area under the curve (AUC) ( e ) of HRG were analyzed from each individual thrombus in the two groups. The number of thrombi (n value) analyzed in each group was indicated above the bars. f Mouse plasma pre-treated with recombinant mouse PDI-CCCC or PDI-AAAA was incubated on bEnd.3 cells in the presence of Zn 2+ (100 μM). The binding of antithrombin on the cell surface were determined by immunofluorescence using Alexa-488-conjugated antibodies (scale bar: 20 μm). g Representative images of immunohistochemistry of antithrombin from cross sections of thrombus induced by FeCl in the carotid artery in mice treated with vehicle or Rutin (scale bar: 250 μm). h Representative images of antithrombin incorporation (Green), visualized using Alexa-488-conjugated anti-antithrombin, at indicated time points following laser injury in the cremaster arterioles in mice treated with vehicle or Rutin (5 μg/g body weight) (scale bar: 30 μm) (Supplementary Movie ). The median fluorescence intensity ( i ) and AUC ( j ) of antithrombin were analyzed from each individual thrombus in the two groups. The number of thrombi (n value) analyzed in each group was indicated above the bars. Veh vehicle, Rutin quercetin-3-rutinoside. Data are presented as mean values ± SEM and analyzed by two-tailed Mann–Whitney U -test ( e and j ). Source data are provided as a Source Data file.

Article Snippet: The samples were diluted 1:7 and incubated on pre-coated microtiter plates with or without 1 μM Zn 2+ at 37 °C for 1 h. To determine the effect of cations on HRG binding, purified human HRG (10 nM) was incubated on microtiter plates pre-coated with heparin (625 U/mL) in the presence of gradient concentrations of Ca 2+ (0.3, 1, 3 mM) or Zn 2+ (1, 3, 10, 30 μM) for 2 h. In all experiments HRP-conjugated mouse-anti human HRG (Angio-Proteomie, 1:10000) was added into washed plates and incubated for 1 h. After washing the TMB substrate was added and the plate was measured at OD 450 nm to quantitate the bound HRG.

Techniques: Clinical Proteomics, Recombinant, Incubation, Binding Assay, Immunofluorescence, Immunohistochemistry, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: a The activity of antithrombin was measured in WT and Hrg −/− plasma on endothelial cell surface, or in the presence or absence of heparin. The number of independent samples (n value) analyzed in each group was indicated above the bars. b Immunoblotting of HRG antigen in equal amounts of plasma from F12 −/− mice treated with control or vivo-siRNA to HRG, with transferrin as the loading control. c Representative images of platelet accumulation (Red) and fibrin generation (Green), as visualized by Dylight-649-congugated anti-CD42c and Alexa-488-conjugated 59D8 antibody, respectively, at indicated time points following laser injury in the cremaster arterioles in F12 −/− mice treated with control or vivo-siRNA to HRG (scale bar: 30 μm) (Supplementary Movie ). The median fluorescence intensity of platelets ( d ) and fibrin ( e ) were calculated and plotted over time. The area under the curve (AUC) for platelets ( f ) and fibrin ( g ) were analyzed from each individual thrombus in the two groups. The number of thrombi ( n value) analyzed in each group was indicated above the bars. Ctrl, control. Data are presented as mean values ± SEM and analyzed by two-tailed Welch’s t -test ( a ) or two-tailed Mann–Whitney U -test ( f and g ). Source data are provided as a Source Data file.

Article Snippet: The samples were diluted 1:7 and incubated on pre-coated microtiter plates with or without 1 μM Zn 2+ at 37 °C for 1 h. To determine the effect of cations on HRG binding, purified human HRG (10 nM) was incubated on microtiter plates pre-coated with heparin (625 U/mL) in the presence of gradient concentrations of Ca 2+ (0.3, 1, 3 mM) or Zn 2+ (1, 3, 10, 30 μM) for 2 h. In all experiments HRP-conjugated mouse-anti human HRG (Angio-Proteomie, 1:10000) was added into washed plates and incubated for 1 h. After washing the TMB substrate was added and the plate was measured at OD 450 nm to quantitate the bound HRG.

Techniques: Activity Assay, Clinical Proteomics, Western Blot, Control, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: a Immunoblotting of HRG and FXII antigens in equal amounts of plasma from 4 groups of mice as indicated: WT , Hrg −/− , F12 −/− and DKO , with transferrin as the loading control. b Tissue factor-induced thrombin generation (TGA) in the plasma from 4 groups of mice was evaluated in the presence of endothelial cells. The peak height ( c ) and area under the curve (AUC) ( d ) from individual TGA curve were calculated in these groups as indicated. The number of animals (n value) analyzed in each group was indicated above the bars. e The time to complete arterial occlusion following FeCl 3 -induced carotid injury were recorded in 4 groups of mice as indicated. The number of animals (n value) analyzed in each group was indicated above the bars. f Representative images of platelet accumulation (Red) and fibrin generation (Green), visualized by Dylight-649-congugated anti-CD42c and Alexa-488-conjugated 59D8 antibody, respectively, at indicated time points following laser injury in the cremaster arterioles in 4 groups of mice as indicated (scale bar: 30 μm) (Supplementary Movie ). The median fluorescence intensity of platelets ( g ) and fibrin ( h ) were calculated and plotted over time. The AUC for platelets ( i ) and fibrin ( j ) were analyzed from each individual thrombus in different groups. The number of thrombi ( n value) analyzed in each group was indicated above the bars. Data are presented as mean values ± SEM and analyzed by Welch’s ANOVA test ( c – e ) or Kruskal–Wallis test with Dunn’s multiple comparisons ( i and j ). Source data are provided as a Source Data file.

Article Snippet: The samples were diluted 1:7 and incubated on pre-coated microtiter plates with or without 1 μM Zn 2+ at 37 °C for 1 h. To determine the effect of cations on HRG binding, purified human HRG (10 nM) was incubated on microtiter plates pre-coated with heparin (625 U/mL) in the presence of gradient concentrations of Ca 2+ (0.3, 1, 3 mM) or Zn 2+ (1, 3, 10, 30 μM) for 2 h. In all experiments HRP-conjugated mouse-anti human HRG (Angio-Proteomie, 1:10000) was added into washed plates and incubated for 1 h. After washing the TMB substrate was added and the plate was measured at OD 450 nm to quantitate the bound HRG.

Techniques: Western Blot, Clinical Proteomics, Control, Fluorescence

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: a The disulfide pairs were predicted on the domain structure of mouse HRG based on that of human HRG, with red color indicating the targets of PDI. The gain-of-function mutant of mouse HRG ( gof-HRG) was generated by replacing 6 Cys of the 3 target disulfide bonds of PDI: C246-C261, C270–C467 and C315–C320 to Ala, which locked the variant in a reduced form. The binding of wt-HRG and gof-HRG on heparin ( b ) ( n = 12 independent samples) and FXIIa ( c ) ( n = 9 independent samples) was determined by ELISA. d , e The accumulation of HRG following laser injury in the cremaster arterioles of Hrg −/− mice infused with different HRG variants were visualized using Alexa-488-conjugated anti-HRG. The median fluorescence intensity of HRG ( d ) was plotted over time. The area under the curve (AUC) for HRG ( e ) was analyzed from each individual thrombus in different groups. The number of thrombi (n value) analyzed in each group was indicated above the bars. f – i Platelet accumulation and fibrin generation following laser injury in the cremaster arterioles of Hrg −/− mice infused with different HRG variants were visualized by Dylight-649-congugated anti-CD42c and Alexa-488-conjugated 59D8 antibody, respectively. The median fluorescence intensity of platelets ( f ) and fibrin ( g ) were calculated and plotted over time. The AUC for platelets ( h ) and fibrin ( i ) were analyzed from each individual thrombus in different groups. The number of thrombi (n value) analyzed in each group was indicated above the bars. Data are presented as mean values ± SEM and analyzed by two-tailed Welch’s t -test ( b and c ) or two-tailed Mann–Whitney U -test ( e , h and i ). Source data are provided as a Source Data file.

Article Snippet: The samples were diluted 1:7 and incubated on pre-coated microtiter plates with or without 1 μM Zn 2+ at 37 °C for 1 h. To determine the effect of cations on HRG binding, purified human HRG (10 nM) was incubated on microtiter plates pre-coated with heparin (625 U/mL) in the presence of gradient concentrations of Ca 2+ (0.3, 1, 3 mM) or Zn 2+ (1, 3, 10, 30 μM) for 2 h. In all experiments HRP-conjugated mouse-anti human HRG (Angio-Proteomie, 1:10000) was added into washed plates and incubated for 1 h. After washing the TMB substrate was added and the plate was measured at OD 450 nm to quantitate the bound HRG.

Techniques: Mutagenesis, Generated, Variant Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Circulation

Article Title: Endothelial-Derived Neuregulin Protects the Heart against Ischemic Injury

doi: 10.1161/CIRCULATIONAHA.110.991125

Figure Lengend Snippet: Endothelial deletion of neuregulin (NRG) impairs post-ischemic recovery in Langendorff perfused hearts. A. Western blot of pooled (3-4 hearts/sample) isolated, purified cardiac endothelial cells from either vehicle or tamoxifen treated mice demonstrating a significant reduction of NRG expression after tamoxifen induction. A reprobe of the same blot using Actin as a loading control is shown in lower panel. B. Left ventricular developed pressures (LVDP) in perfused hearts from vehicle or tamoxifen treated mice. Baseline and ischemic LVDP were not significantly different between groups. Reperfusion (post-ischemic, taken at end of 30 min reperfusion) LVDP was significantly impaired in CFF NRG knockout animals (CFF tamoxifen) (*P<0.05 vs. all other groups, N=5/group). C. Rate-pressure products (RPP) in the same perfused hearts. Reperfusion RPP was also significantly impaired in the NRG knockout animals (*P<0.05 vs. all other groups).

Article Snippet: Following a short gene induction period (3 days tamoxifen injection), NRG repletion was achieved by injecting animals daily with 2.5 μg of recombinant human NRG (rNRG, R&D Systems) throughout the remainder of the tamoxifen induction period (total 9 days, as above).

Techniques: Western Blot, Isolation, Purification, Expressing, Control, Knock-Out

Journal: Circulation

Article Title: Endothelial-Derived Neuregulin Protects the Heart against Ischemic Injury

doi: 10.1161/CIRCULATIONAHA.110.991125

Figure Lengend Snippet: Endothelial deletion of neuregulin (NRG) increases infarct size and blunts erbB4 activation. Mice were pretreated with tamoxifen or vehicle (N=4-5/group) then subjected to left coronary artery ligation. A. Area at risk (AAR, % total ventricle) was not significantly different between the groups; however, the infarct sizes (expressed as % total ventricle or % AAR) were significantly larger in the NRG deleted (CFF tamoxifen) animals compared to tamoxifen treated (C++ tamoxifen) or vehicle treated (CFF vehicle) controls. Administration of exogenous recombinant NRG (rNRG) rescued the defect, leading to smaller infarct sizes in NRG deleted animals (CFF tam + rNRG) (*P<0.05, vs. C++ tamoxifen and CFF tam + rNRG.) B. Immunoprecipitation and Western blotting for phosphorylated erbB4 or total erbB4 in left ventricular lysates from non-infarcted controls or infarcted animals (*P<0.05 vs. CFF tamoxifen infarct)

Article Snippet: Following a short gene induction period (3 days tamoxifen injection), NRG repletion was achieved by injecting animals daily with 2.5 μg of recombinant human NRG (rNRG, R&D Systems) throughout the remainder of the tamoxifen induction period (total 9 days, as above).

Techniques: Activation Assay, Ligation, Recombinant, Immunoprecipitation, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Biased activation of the receptor tyrosine kinase HER2

doi: 10.1007/s00018-023-04806-8

Figure Lengend Snippet: Single-particle tracking of HER2 in live HeLa cells treated with the ligands EREG and NRGβ1. A HER2 was targeted with a Cy3B-labeled anti-HER2 nanobody, and the mobility of HER2 was measured in the absence and presence of EREG and NRGβ1. B Distribution of diffusion coefficients for resting (gray), EREG- (purple), and NRGβ1-treated (lilac) HeLa cells at 22 °C. C Global diffusion coefficient per condition (violin plots with dotted lines marking the quartiles, dashed lines the median, and stars representing mean values). D Exemplary bright-field image of a living HeLa cell treated with NRGβ1, and single-molecule trajectories colored for their diffusion mode, immobile (blue), confined (green), free (orange). E Relative occurrences of immobile, confined, and freely diffusing HER2 receptors in live HeLa cells. F The diffusion coefficient for the individual diffusion modes immobile (i), confined (c), and free (f) (violin plots, dense dashed lines represent the quartiles, loosely dashed lines represent the median). The data shown was assembled from 160 cells. Error bars are defined by SEMs; p > 0.05 no significant difference (no label), p < 0.05 significant difference (*), p < 0.01 very significant difference (**), p < 0.001 highly significant difference (***)

Article Snippet: For stimulated cells, 20 nM epidermal growth factor (EGF) (#AF-100-15), transforming growth factor alpha (TGFα) (#100-16A), neuregulin beta 1 (NRGβ1) (#100-03), or epiregulin (EREG) (#100-04) (all from PeproTech, Waltham, MA, USA) were added 5 min after measurement start.

Techniques: Single-particle Tracking, Labeling, Diffusion-based Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Biased activation of the receptor tyrosine kinase HER2

doi: 10.1007/s00018-023-04806-8

Figure Lengend Snippet: Temporal response of HER2 activation in living HeLa cells following treatment with EGF, TGFα, EREG, or NRGβ1. A Schematic representation of the time-course SPT experiment. Cells were seeded sparsely and imaged sequentially. After imaging 5 cells in resting condition, the respective ligand was added to the cell dish and the measurement continued. Diffusion mode and coefficients were calculated per cell and pooled into 5 min time intervals. Diamonds represent mean diffusion coefficients per segment (colored) or cell (grey; mean values are colored in black with error bars representing the SEM). B Relative change in the fraction of immobile particles (dot plots) plotted against time. Bars show HER2 phosphorylation obtained from western blots ( N = 3). C Relative change in the diffusion coefficient of freely diffusing particles over time. Relative changes were calculated from mean values of 40 cells per interval. Receptor models indicate the expected ligand-orchestrated interactions between HER2 and other receptors of the family. The dotted lines represent mean values of the relative change over the time of ligand stimulation. Error bars in dot plots represent the standard error of the difference (SED); error bars in bar plots show the standard error of the mean (SEM). Significance was tested for stimulated cells vs. untreated cells from the same sample before calculating the relative change; p > 0.05 no significant difference (no label), p < 0.05 significant difference (*), p < 0.01 very significant difference (**), p < 0.001 highly significant difference (***)

Article Snippet: For stimulated cells, 20 nM epidermal growth factor (EGF) (#AF-100-15), transforming growth factor alpha (TGFα) (#100-16A), neuregulin beta 1 (NRGβ1) (#100-03), or epiregulin (EREG) (#100-04) (all from PeproTech, Waltham, MA, USA) were added 5 min after measurement start.

Techniques: Activation Assay, Imaging, Diffusion-based Assay, Phospho-proteomics, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Biased activation of the receptor tyrosine kinase HER2

doi: 10.1007/s00018-023-04806-8

Figure Lengend Snippet: Proposed model for ligand-induced activation of HER2 derived from single-particle tracking data shown in a model-like fashion by means of the active population. A In cells treated with EGF, TGFα, EREG, or NRGβ1, the population of immobile HER2 increases at the cost of freely diffusing HER2. This increase scales with the formation of phosphorylated HER2. B Temporal profile, strength, and decay of HER2 activation in cells treated with the EGFR-targeting ligands EGF and TGFα. C Temporal profile, strength, and decay of HER2 activation in cells treated with the ligand EREG and NRGβ1

Article Snippet: For stimulated cells, 20 nM epidermal growth factor (EGF) (#AF-100-15), transforming growth factor alpha (TGFα) (#100-16A), neuregulin beta 1 (NRGβ1) (#100-03), or epiregulin (EREG) (#100-04) (all from PeproTech, Waltham, MA, USA) were added 5 min after measurement start.

Techniques: Activation Assay, Derivative Assay, Single-particle Tracking