Journal: bioRxiv

Article Title: A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

doi: 10.1101/2025.02.17.638751

Figure Lengend Snippet: (A) Schematic illustration of the multi-stage process of the phage-display antibody screen used to identify antibodies that preferentially bind to DNA-damaged cells. (B) Flow cytometry histogram of CD46 levels in MCF10A cells exposed to various doses of irradiation (0, 2, 5, 10 Gy) and stained with PE-conjugated anti-CD46 antibody or IgG-PE as isotype control. (C) Flow cytometry histogram of CD46 levels in SUDHL4 cells treated with various chemotherapy for 48H. Conditions include untreated control, IgG-PE isotype control, and treatments with 1uM etoposide (ETO), 10nM gemcitabine (GEM), 0.2mM Hydroxyurea (HU), and 2nM vincristine (VCR). (D) Representative flow cytometry histograms of CD46, CD55, and CD59 in SUDHL4 cells treated with or without etoposide (ETO). Cells were stained with PE-conjugated specific antibodies or IgG-PE as isotype control. (E) Quantification of relative integrated mean fluorescence intensities (iMFI) for CD46, CD55, and CD59 in SUDHL4 cells treated with ETO, normalized to untreated control. Statistical analysis was performed using ratio paired t-tests of IgG-corrected iMFI values. Data are presented as mean ± SD from three independent experiments. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For mCRP neutralizing experiments, CD46 blocking antibody derived from the MC120.6 CD46 hybridoma (SFR Biosciences), CD55 antibody (BRI216) (Bio-Rad), or CD59 (YTH53.1) (Bio-rad) were added at 10µg/mL final concentration for 1 hour prior to the assay.

Techniques: Flow Cytometry, Irradiation, Staining, Control, Fluorescence

Journal: bioRxiv

Article Title: A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

doi: 10.1101/2025.02.17.638751

Figure Lengend Snippet: (A) Mean cell viability of 10 DLBCL cell lines treated with 10 ug/mL rituximab (RTX) over 2 hours, measured by CTG assay. Black dots represent CDC-mediated cell death, while clear dots indicate RTX-induced direct cell death. Error bars represent SD (n≥2 biological replicates). (B) Heatmap showing log-transformed normalized transcript per million (nTPM) RNA expression values for CD20, CD46, CD55, and CD59 in DLBCL cell lines, obtained from The Human Protein Atlas, ranked by %CDC activity derived from . (C) Dose-response curve of RTX-induced cell death in SUDHL4 cells pre-treated with etoposide (ETO) for 48H, measured by CTG assay. HS, human serum; hiHS, heat-inactivated human serum. Data fitted using a four-parameter variable slope model (n=3 biological replicates). (D) Cell death induced by RTX (37 ng/mL) in SUDHL4 cells pre-treated with various chemotherapeutic agents: doxorubicin (DOX), etoposide (ETO), gemcitabine (GEM), hydroxyurea (HU), and vincristine (VCR) for 48H. Both CDC and direct cell death are shown (n=3 biological replicates). Statistical significance was calculated by 2-way ANOVA relative to respective control followed by uncorrected Fisher’s LSD. Significance of % CDC and not % apoptosis is shown. (E) Representative flow cytometry histogram of CD20 expression in SUDHL4 cells treated with or without ETO. Cells were stained with APC-conjugated anti-CD20 antibody or IgG-APC as isotype control. (F) Relative mean fluorescence intensity (MFI) of CD20 in SUDHL4 cells treated with ETO, normalized to untreated control (n=4 biological replicates). Statistical significance was calculated by ratio paired t-test of IgG-corrected MFI values. (G) Percentage of CDC in SUDHL4 cells treated with 37 ng/mL RTX including ETO-treated with blocking antibodies against CD46, CD55, or CD59 (n=3 biological replicates). Statistical significance among groups was determined using a mixed effect model, followed by post hoc Dunnett’s multiple comparisons test. Data are presented as mean ± SD. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For mCRP neutralizing experiments, CD46 blocking antibody derived from the MC120.6 CD46 hybridoma (SFR Biosciences), CD55 antibody (BRI216) (Bio-Rad), or CD59 (YTH53.1) (Bio-rad) were added at 10µg/mL final concentration for 1 hour prior to the assay.

Techniques: CTG Assay, Transformation Assay, RNA Expression, Activity Assay, Derivative Assay, Control, Flow Cytometry, Expressing, Staining, Fluorescence, Blocking Assay

Journal: bioRxiv

Article Title: A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

doi: 10.1101/2025.02.17.638751

Figure Lengend Snippet: (A) Immunoblot of Chk1 and phosphorylated Chk1 Ser345 (pChk1) in SUDHL4 cells treated with or without etoposide (ETO) for 48 hours. (B) Bar chart showing relative integrated mean fluorescence intensity (iMFI) of CD46, CD55, and CD59 in SUDHL4 cells treated with ETO alone or ETO + Chk1 inhibitor (Chk1i, Rabusertib). Statistical analysis was performed using repeated measures one-way ANOVA with Dunnett’s multiple comparisons test relative to the ETO condition. Error bars represent SD of 4 biological replicates. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Representative flow cytometry histograms in of CD46, CD55, and CD59 expression in SUDHL4 cells under control conditions, ETO treatment, or ETO + Chk1i treatment. Cells were stained with PE-conjugated specific antibodies or IgG-PE as isotype control. (D) Flow cytometry histograms of CD59 expression in multiple DLBCL cell lines (SUDHL6, SUDHL2, Karpas-231) under control conditions, ETO treatment, or ETO + Chk1i treatment. Cells were stained with PE-conjugated anti-CD59 antibody or IgG-PE as isotype control.

Article Snippet: For mCRP neutralizing experiments, CD46 blocking antibody derived from the MC120.6 CD46 hybridoma (SFR Biosciences), CD55 antibody (BRI216) (Bio-Rad), or CD59 (YTH53.1) (Bio-rad) were added at 10µg/mL final concentration for 1 hour prior to the assay.

Techniques: Western Blot, Fluorescence, Flow Cytometry, Expressing, Control, Staining

Journal: bioRxiv

Article Title: A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

doi: 10.1101/2025.02.17.638751

Figure Lengend Snippet: (A-C) Relative mRNA expression of CD46 (A), CD55 (B), and CD59 (C) in SUDHL4 cells treated with etoposide (ETO) or ETO + Chk1 inhibitor (Chk1i, Rabusertib). (D) Schematic diagram of CD59 gene transcriptional regulators (adapted from Du et al.). (E) Sp1 and NF-κB primarily regulate transcripts T1-T4. TP53 regulates the T5 transcript but has minimal impact on overall CD59 expression. CREB primarily regulates T6-T8 transcripts and plays additional roles in CD59 transcription beyond T6-T8 regulation. An enhancer region (-500 to -1000 bp) interacts with multiple factors (CREB, NF-κB, TP53) through CBP/p300 scaffolding, facilitating coordinated regulation of CD59 expression. (E) Relative mRNA expression of CD59 exons T1-4, T5, and T6-8 in SUDHL4 cells after ETO or ETO + Chk1i treatment. (F) Percentage of complement-dependent cytotoxicity (%CDC) in SUDHL4 cells pretreated with ETO or ETO + 10 nM Mithramycin A (MitA). (G,H) Bar chart showing CD59 protein levels (MFI relative to control) (G) and relative CD59 mRNA expression (H) in SUDHL4 cells treated with control, ETO, or ETO + 10 nM MitA. (I) Immunoblot analysis of Sp1 protein and cleaved caspase-3 in SUDHL4 cells after treatment with indicated compounds. For all panels, statistical significance was calculated by ANOVA followed by post hoc Dunnett’s multiple comparisons test relative to the ETO condition. Error bars represent SD of biological replicates. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For mCRP neutralizing experiments, CD46 blocking antibody derived from the MC120.6 CD46 hybridoma (SFR Biosciences), CD55 antibody (BRI216) (Bio-Rad), or CD59 (YTH53.1) (Bio-rad) were added at 10µg/mL final concentration for 1 hour prior to the assay.

Techniques: Expressing, Scaffolding, Control, Western Blot

Journal: bioRxiv

Article Title: A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

doi: 10.1101/2025.02.17.638751

Figure Lengend Snippet: Schematic representation of the mechanism by which genotoxic agents antagonize rituximab-mediated complement-dependent cytotoxicity (CDC) through CD59 upregulation. Left panel: Under normal conditions, rituximab can induce CDC, resulting in membrane attack complex (MAC) formation and cell lysis. CD59 expression is driven by Sp1 and regulated by repressor complexes. Right panel: In the presence of DNA damage, genotoxic agents activate Chk1. Activated Chk1 prevents repressor complexes from associating with Sp1-bound promoter sites. Unrestricted Sp1 drives increased mCRP gene expression, particularly CD59. Elevated CD59 levels block MAC formation, inhibiting rituximab-driven CDC.

Article Snippet: For mCRP neutralizing experiments, CD46 blocking antibody derived from the MC120.6 CD46 hybridoma (SFR Biosciences), CD55 antibody (BRI216) (Bio-Rad), or CD59 (YTH53.1) (Bio-rad) were added at 10µg/mL final concentration for 1 hour prior to the assay.

Techniques: Membrane, Lysis, Expressing, Blocking Assay

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Comparisons of urinary complement proteins between MN patients and HC or IgAN patients, respectively.

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques:

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Comparisons of urinary complement protein levels and clinical parameters between the group with no remission and the group with clinical remission in the discovery cohorts after immunosuppressive therapy.

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques:

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Correlation analysis of 11 urinary complement proteins with clinical and pathological parameters in patients with MN.

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques:

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Comparisons of urinary complement protein levels and clinical parameters between the group with no remission and the group with clinical remission in the validation cohorts after immunosuppressive therapy.

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques:

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Comparison of CD59 and C5b-9 immunohistochemistry in renal tissues from patients with clinical remission versus those without remission of membranous nephropathy (MN) (A) . The clinical remission group had higher expression of CD59 and lower expression of C5b-9; the no remission group had lower expression of CD59 and higher expression of C5b-9. Comparison of CD59 immunohistochemistry staining intensity between the group with clinical remission and the group with no remission (B) . CD59 was significantly higher in the clinical remission group than in the no remission group. Comparison of C5b-9 immunohistochemistry staining intensity between the group with clinical remission and the group with no remission (C) . C5b-9 was significantly lower in the clinical remission group than in the no remission group. The staining intensity was measured by average OD (AOD).

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques: Comparison, Immunohistochemistry, Expressing, Staining

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Comparison of CD59 and C5b-9 immunofluorescence immunohistochemistry in renal tissues from patients with clinical remission versus those without remission of membranous nephropathy (MN).

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques: Comparison, Immunofluorescence, Immunohistochemistry

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Comparisons of urinary complement proteins between MN patients and HC or IgAN patients, respectively.

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques:

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Comparisons of urinary complement protein levels and clinical parameters between the group with no remission and the group with clinical remission in the discovery cohorts after immunosuppressive therapy.

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques:

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Correlation analysis of 11 urinary complement proteins with clinical and pathological parameters in patients with MN.

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques:

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Comparisons of urinary complement protein levels and clinical parameters between the group with no remission and the group with clinical remission in the validation cohorts after immunosuppressive therapy.

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques:

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Comparison of CD59 and C5b-9 immunohistochemistry in renal tissues from patients with clinical remission versus those without remission of membranous nephropathy (MN) (A) . The clinical remission group had higher expression of CD59 and lower expression of C5b-9; the no remission group had lower expression of CD59 and higher expression of C5b-9. Comparison of CD59 immunohistochemistry staining intensity between the group with clinical remission and the group with no remission (B) . CD59 was significantly higher in the clinical remission group than in the no remission group. Comparison of C5b-9 immunohistochemistry staining intensity between the group with clinical remission and the group with no remission (C) . C5b-9 was significantly lower in the clinical remission group than in the no remission group. The staining intensity was measured by average OD (AOD).

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques: Comparison, Immunohistochemistry, Expressing, Staining

Journal: Frontiers in Medicine

Article Title: Urine complement analysis implies complement activation is involved in membranous nephropathy

doi: 10.3389/fmed.2025.1515928

Figure Lengend Snippet: Comparison of CD59 and C5b-9 immunofluorescence immunohistochemistry in renal tissues from patients with clinical remission versus those without remission of membranous nephropathy (MN).

Article Snippet: Finally, the nuclei were stained with hematoxylin for 10 min. For immunofluorescence, primary anti-CD59 or anti-C5b-9 antibody diluted 1:100 was added first and incubated overnight at room temperature, followed by incubation with the corresponding fluorescein isothiophosphate (FITC-)-coupled secondary antibodies for 1 h at 37°C, and finally staining of nuclei with DAPI for 5 min. Primary anti-CD59 antibody, goat anti-mouse IgG H&L (FITC), and goat anti-rabbit IgG H&L (TRITC) were obtained from Zenbio under item numbers 220,768, 511,101 and 511,202, respectively.

Techniques: Comparison, Immunofluorescence, Immunohistochemistry

Journal: Journal of Virology

Article Title: Mitochondrial injury and complement dysregulation are drivers of pathological inflammation in viral myocarditis

doi: 10.1128/jvi.01804-24

Figure Lengend Snippet: Complement protective factors are downregulated following CVB3 infection. ( A ) Schematic illustration of cardiac protein expression analysis in control and 7 day CVB3-infected A/J male mice ( n = 3 per group). ( B ) Protein expression of complement protective factors CD59 and CD55 was assessed by Western blot analysis from cardiac tissue of sham or CVB3-infected A/J mice ( A ). Ponceau red stain of membrane was used to assess total protein. ( C ) HL1 cardiomyocytes were sham or CVB3-infected (MOI = 100) for 12 or 24 h. Proteins were harvested from cells and subjected to Western blot analysis for complement protective factors CD55 and CD59. VP1 and ACTB were used as viral replication marker and loading control, respectively. ( D ) Control and CVB3-infected human heart tissues were subjected to immunohistochemistry to detect the complement protective factors CD59 and CD55. Scale bar = 60 µm. CD59 and CD55 optical density was quantified in the right panel and presented as mean ± SD, n = 3. ( E ) In vitro cleavage assay was performed on HeLa cell lysates using purified CVB3 viral proteinase 2A (300 ng), 3C (100 ng), or 2A + 3C (left). Previously reported substrates EIF4G and SNAP29 were used as positive controls for 2A and 3C, respectively (right). Red arrows depict cleavage fragments. ( F ) Time-course in vitro cleavage assay was performed using HeLa cell lysates treated with purified viral proteinase 3C (100 ng). Lysates were subjected to Western blot analysis for CD55. Red arrowheads depict cleavage fragments.

Article Snippet: Primary antibodies used for staining include CD59 (Abcam, ab9183), CD55 (Abcam, ab231061), TOM20 (Abclonal, A19403), dsRNA J2 ms mAb (Scicon, 10010500), and cGAS (Abclonal, A8335).

Techniques: Infection, Expressing, Control, Western Blot, Staining, Membrane, Marker, Immunohistochemistry, In Vitro, Cleavage Assay, Purification

Journal: Journal of Virology

Article Title: Mitochondrial injury and complement dysregulation are drivers of pathological inflammation in viral myocarditis

doi: 10.1128/jvi.01804-24

Figure Lengend Snippet: Complement protective factors are downregulated following CVB3 infection. ( A ) Schematic illustration of cardiac protein expression analysis in control and 7 day CVB3-infected A/J male mice ( n = 3 per group). ( B ) Protein expression of complement protective factors CD59 and CD55 was assessed by Western blot analysis from cardiac tissue of sham or CVB3-infected A/J mice ( A ). Ponceau red stain of membrane was used to assess total protein. ( C ) HL1 cardiomyocytes were sham or CVB3-infected (MOI = 100) for 12 or 24 h. Proteins were harvested from cells and subjected to Western blot analysis for complement protective factors CD55 and CD59. VP1 and ACTB were used as viral replication marker and loading control, respectively. ( D ) Control and CVB3-infected human heart tissues were subjected to immunohistochemistry to detect the complement protective factors CD59 and CD55. Scale bar = 60 µm. CD59 and CD55 optical density was quantified in the right panel and presented as mean ± SD, n = 3. ( E ) In vitro cleavage assay was performed on HeLa cell lysates using purified CVB3 viral proteinase 2A (300 ng), 3C (100 ng), or 2A + 3C (left). Previously reported substrates EIF4G and SNAP29 were used as positive controls for 2A and 3C, respectively (right). Red arrows depict cleavage fragments. ( F ) Time-course in vitro cleavage assay was performed using HeLa cell lysates treated with purified viral proteinase 3C (100 ng). Lysates were subjected to Western blot analysis for CD55. Red arrowheads depict cleavage fragments.

Article Snippet: Western blotting was conducted using the following primary antibodies: Cox2 (D5H5) (Cell Signaling Technology, #13314), p-TBK1 (Cell Signaling Technology, #5483), TBK1 (Cell Signaling Technology, #3504), STING (Cell Signaling Technology, #13647), p-STING (Cell Signaling Technology, #19781), VP1 (Mediagnost, Cox mAb 31A2), NLRP3 (Adipogen, #AG-20B-0014-C100), VDAC (Abclonal, #A19707), TOM20 (Abclonal, #19403), ACTB (Sigma-Aldrich, A5316), FLAG (Sigma, F1804), CD59 (Abclonal, #A4090), CD55 (Abcam, #ab231061), C4B (Abcam, #ab181241), LC3B (Novus Biologicals, NB100-2220), CAR (Cell Signaling Technology, #16984), and MAVS (Cell Signaling Technology, #24930).

Techniques: Infection, Expressing, Control, Western Blot, Staining, Membrane, Marker, Immunohistochemistry, In Vitro, Cleavage Assay, Purification