sars cov 2 nucleocapsid  (Sino Biological)

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: SARS-CoV-2-infected cardiomyocytes exhibit upregulated necroptosis, but no evidence of mitochondrial permeability transition

doi: 10.1016/j.jmccpl.2025.100833

Figure Lengend Snippet: Infection of iPSC-CMs by SARS-CoV-2 in vitro results in increased cellular cytotoxicity. (A) Immunofluorescence staining shows infection of iPSC-derived cardiomyocytes. Nuclei (blue), α-Actinin (grey) and Nucleocapsid (red). Scale bar: 50 μm.(B) Quantification of infected cells at 24 h post-infection. (C) Infection of iPSC-derived cardiomyocytes is confirmed by expression of SARS-CoV-2 Spike RNA. (D) Western blot analysis confirms Spike Protein expression, quantified in (E). Full western blots in A. (F) Cytotoxicity upon SARS-CoV-2 infection determined by activity of LDH released into the medium. Protein expression is normalized to β-actin, gene expression is normalized to GAPDH. n = 3 iPSC-CM differentiation rounds for all but (B), where n = 1 iPSC-CM differentiation round, statistical significance determined by Wilcoxon test (B), Kruskal-Wallis test (C, D), students t -test (F). p < 0.0001 = ****. Data are displayed as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Infection of iPSC-CMs by SARS-CoV-2 in vitro results in increased cellular cytotoxicity. (A) Immunofluorescence staining shows infection of iPSC-derived cardiomyocytes. Nuclei (blue), α-Actinin (grey) and Nucleocapsid (red). Scale bar: 50 μm.(B) Quantification of infected cells at 24 h post-infection. (C) Infection of iPSC-derived cardiomyocytes is confirmed by expression of SARS-CoV-2 Spike RNA. (D) Western blot analysis confirms Spike Protein expression, quantified in (E). Full western blots in Suppl. Fig. 3A. (F) Cytotoxicity upon SARS-CoV-2 infection determined by activity of LDH released into the medium. Protein expression is normalized to β-actin, gene expression is normalized to GAPDH. n = 3 iPSC-CM differentiation rounds for all but (B), where n = 1 iPSC-CM differentiation round, statistical significance determined by Wilcoxon test (B), Kruskal-Wallis test (C, D), students t -test (F). p < 0.0001 = ****. Data are displayed as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following antibodies were used in a 1:500 dilution: α-Actinin (Sigma, #A7811) and SARS-CoV-2 Nucleocapsid (SinoBiological, #40143-T62).

Techniques: Infection, In Vitro, Immunofluorescence, Staining, Derivative Assay, Expressing, Western Blot, Activity Assay, Gene Expression

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: SARS-CoV-2-infected cardiomyocytes exhibit upregulated necroptosis, but no evidence of mitochondrial permeability transition

doi: 10.1016/j.jmccpl.2025.100833

Figure Lengend Snippet: PDH phosphorylation is unaffected following SARS-CoV2 infection. (A) Cytotoxicity after Cyclosporine A (CsA) treatment [0.5 μM, 2 h] as determined by activity of LDH released into the medium. (B) Quantification of pyruvate dehydrogenase A1 (PDHA1) protein expression and its phosphorylation on serine 293. Values normalized to α-tubulin. (C) Representative Western Blot. (D) Quantification of CI = Complex I, CII = Complex II, CIII = Complex III, CIV = Complex IV and CV = Complex V normalized to α-tubulin. Full western blots in B. (E) Protein expression of the respiratory chain following SARS-CoV-2 infection. Full western blots in B. (F) Apoptosis rate determined by TUNEL staining 24 h post-infection with an MOI of 0.1. (G) Quantification of procaspase 3 and cleaved caspase 3 protein expression. Values normalized to α-tubulin. Corresponding western blots in C. n = 3 iPSC-CM differentiation rounds for all but (A, B), where n = 1 iPSC-CM differentiation round. Statistical significance determined by two-way ANOVA (A, B), Mann-Whitney test (F) or t-test (D, G). p ≤ 0.05 = *, p < 0.0001 = ****. Data are displayed as mean ± SD. PDH phosphorylation is unaffected following SARS-CoV2 infection. (A) Cytotoxicity after Cyclosporine A (CsA) treatment [0.5 μM, 2 h] as determined by activity of LDH released into the medium. (B) Quantification of pyruvate dehydrogenase A1 (PDHA1) protein expression and its phosphorylation on serine 293. Values normalized to α-tubulin. (C) Representative Western Blot. (D) Quantification of CI = Complex I, CII = Complex II, CIII = Complex III, CIV = Complex IV and CV = Complex V normalized to α-tubulin. Full western blots in Suppl. Fig. 3B. (E) Protein expression of the respiratory chain following SARS-CoV-2 infection. Full western blots in Suppl. Fig. 3B. (F) Apoptosis rate determined by TUNEL staining 24 h post-infection with an MOI of 0.1. (G) Quantification of procaspase 3 and cleaved caspase 3 protein expression. Values normalized to α-tubulin. Corresponding western blots in Suppl. Fig. 3C. n = 3 iPSC-CM differentiation rounds for all but (A, B), where n = 1 iPSC-CM differentiation round. Statistical significance determined by two-way ANOVA (A, B), Mann-Whitney test (F) or t-test (D, G). p ≤ 0.05 = *, p < 0.0001 = ****. Data are displayed as mean ± SD.

Article Snippet: The following antibodies were used in a 1:500 dilution: α-Actinin (Sigma, #A7811) and SARS-CoV-2 Nucleocapsid (SinoBiological, #40143-T62).

Techniques: Phospho-proteomics, Infection, Activity Assay, Expressing, Western Blot, TUNEL Assay, Staining, MANN-WHITNEY

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: SARS-CoV-2-infected cardiomyocytes exhibit upregulated necroptosis, but no evidence of mitochondrial permeability transition

doi: 10.1016/j.jmccpl.2025.100833

Figure Lengend Snippet: Increased necroptosis following SARS-CoV-2 infection. (A) Cytotoxicity after Necrostatin-1 (Nec-1) [2.5 μM] or MCC950 [0.05 μM] treatment as determined by activity of LDH released into the medium. (B) Spike- (C) RIP3 and (D) NLR family pyrin domain containing 3 (NLRP3) inflammasome gene expression following SARS-CoV-2 infection. Gene expression normalized to GAPDH. (E) Quantification of RIP3 and RIP3-S227 protein expression after Nec-1 treatment. (F) Representative WB for RIP3 and RIP3-S227 following SARS-CoV-2 infection. Full western blots in D. (G) Quantification of MLKL and MLKL-S358 protein expression with a representative WB (H). Full western blots in E. n = 3 iPSC-CM differentiation rounds, statistical significance determined by two-way ANOVA (A) or Kruskal-Wallis test (B–E) or Mann-Whitney (G). p < 0.01 = **, p < 0.0001 = ****. Data are displayed as mean ± SD. Increased necroptosis following SARS-CoV-2 infection. (A) Cytotoxicity after Necrostatin-1 (Nec-1) [2.5 μM] or MCC950 [0.05 μM] treatment as determined by activity of LDH released into the medium. (B) Spike- (C) RIP3 and (D) NLR family pyrin domain containing 3 (NLRP3) inflammasome gene expression following SARS-CoV-2 infection. Gene expression normalized to GAPDH. (E) Quantification of RIP3 and RIP3-S227 protein expression after Nec-1 treatment. (F) Representative WB for RIP3 and RIP3-S227 following SARS-CoV-2 infection. Full western blots in Suppl. Fig. 3D. (G) Quantification of MLKL and MLKL-S358 protein expression with a representative WB (H). Full western blots in Suppl. Fig. 3E. n = 3 iPSC-CM differentiation rounds, statistical significance determined by two-way ANOVA (A) or Kruskal-Wallis test (B–E) or Mann-Whitney (G). p < 0.01 = **, p < 0.0001 = ****. Data are displayed as mean ± SD.

Article Snippet: The following antibodies were used in a 1:500 dilution: α-Actinin (Sigma, #A7811) and SARS-CoV-2 Nucleocapsid (SinoBiological, #40143-T62).

Techniques: Infection, Activity Assay, Gene Expression, Expressing, Western Blot, MANN-WHITNEY

Journal: Signal Transduction and Targeted Therapy

Article Title: Inducible CD147 up-regulation boosts extended SARS-CoV-2 infection triggering severe COVID-19 independent of ACE2

doi: 10.1038/s41392-025-02551-x

Figure Lengend Snippet: SARS-CoV-2-induced CD147 up-regulation causes extended virus infection and pathological injury. a A flow chart for establishing hCD147 mice model of COVID-19. b The virus loads in the lung tissues of hCD147 mice were detected by Taqman-based RT-PCR at 3 and 7 dpi, *** p < 0.001. c , d ,The expression of CD147 was evaluated by western blot in the lung tissues of virus-infected hCD147 mice at 3 and 7 dpi, and the lung tissues of healthy hCD147 mice were used as the control ( c ). The relative expression level of CD147 was analyzed by Image Lab software, *** p < 0.001 ( d ). e The scRNA-seq identified the expression of CD147 in virus-infected ( n = 4,203) and virus-uninfected ( n = 13,354) cells of lung tissues from hCD147 mice. Wilcoxon test, p < 0.0001. f The scRNA-seq identified the expression of CD147 in virus-infected and virus-uninfected AT2 cells of lung tissues from hCD147 mice. g –i Infected, border, and uninfected regions in the lung tissues of virus-infected hCD147 mice were defined using spatial transcriptomics ( g ). The total expression of CD147 was quantified in three defined regions ( h ), and the expression of CD147 in AT2, M2 macrophages, and fibroblasts was determined ( i ). The number of spots used for data analysis in uninfected, border, and infected group were 312, 3,554, and 1,833, respectively. Wilcoxon test, p < 0.05, p < 0.001. j The BEAS-2B cells with SARS-CoV-2 pseudovirus infection in the upper chamber were co-cultured with the BEAS-2B cells in the lower chamber for 48 h, and the expression of CD147 in cells collected from the lower chamber were detected by western blot. k , l The expression of CD147 was determined by western blot in BEAS-2B cells with SARS-CoV-2 pseudovirus infection ( k ) or RBD incubation ( l ). m The relative luciferase signals in pseudovirus-infected BEAS-2B cells were determined by dual-luciferase reporter assays with RBD incubation, *** p < 0.001. n The relative luciferase signals in pseudovirus-infected BEAS-2B cells were determined by dual-luciferase reporter assays, ns, not significant, *** p < 0.001. o The virus loads in authentic virus-infected BEAS-2B cells were determined by Taqman-based RT-PCR, ns, not significant, ** p < 0.01, *** p < 0.001. p A flow chart for establishing a virus-infected hCD147 mouse model by primarily inoculating with RBD protein followed by authentic virus infection. q The expression of CD147 was detected in the lung tissues of hCD147 mice with His-RBD ( n = 4) or His-tag ( n = 4) inoculation and hCD147 mice with His-RBD ( n = 4) or His-tag ( n = 4) inoculation followed by authentic virus infection. r The virus loads in the lung tissues of virus-infected hCD147 mice were determined, * p < 0.05. s , t The infiltration of F4/80+iNOS+ M1 macrophages and Ly6G+ neutrophils in lung tissues of virus-infected hCD147 mice was evaluated by multicolor immunofluorescence staining, Ly6G, yellow; F4/80, green; iNOS, red, scale bar, 100 μm ( s ), and their infiltrated percentages were quantified, ** p < 0.01, *** p < 0.001 ( t ). u The level of cytokines and chemokines in lung tissues of virus-infected hCD147 mice was performed by RT-PCR, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: His-RBD protein (500 ng/mL, 40592-V08H, Sino Biological) and 6×His-tag (500 ng/mL, QYAOBIO) were added in the medium for 24 h. Then, the cell medium was discarded and the authentic SARS-CoV-2 virus (Beta, 100 TCID 50 ) was added into each well for 2 h. Subsequently, the cell supernatant was replaced with 2% FBS medium and the cells were cultured at 37 °C for 48 h. Finally, the samples were collected and the virus loads were detected by Taqman-based real-time PCR.

Techniques: Virus, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Control, Software, Cell Culture, Incubation, Luciferase, Multicolor Immunofluorescence Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Inducible CD147 up-regulation boosts extended SARS-CoV-2 infection triggering severe COVID-19 independent of ACE2

doi: 10.1038/s41392-025-02551-x

Figure Lengend Snippet: SARS-CoV-2-induced CD147 up-regulation causes extended virus infection and pathological injury. a A flow chart for establishing hCD147 mice model of COVID-19. b The virus loads in the lung tissues of hCD147 mice were detected by Taqman-based RT-PCR at 3 and 7 dpi, *** p < 0.001. c , d ,The expression of CD147 was evaluated by western blot in the lung tissues of virus-infected hCD147 mice at 3 and 7 dpi, and the lung tissues of healthy hCD147 mice were used as the control ( c ). The relative expression level of CD147 was analyzed by Image Lab software, *** p < 0.001 ( d ). e The scRNA-seq identified the expression of CD147 in virus-infected ( n = 4,203) and virus-uninfected ( n = 13,354) cells of lung tissues from hCD147 mice. Wilcoxon test, p < 0.0001. f The scRNA-seq identified the expression of CD147 in virus-infected and virus-uninfected AT2 cells of lung tissues from hCD147 mice. g –i Infected, border, and uninfected regions in the lung tissues of virus-infected hCD147 mice were defined using spatial transcriptomics ( g ). The total expression of CD147 was quantified in three defined regions ( h ), and the expression of CD147 in AT2, M2 macrophages, and fibroblasts was determined ( i ). The number of spots used for data analysis in uninfected, border, and infected group were 312, 3,554, and 1,833, respectively. Wilcoxon test, p < 0.05, p < 0.001. j The BEAS-2B cells with SARS-CoV-2 pseudovirus infection in the upper chamber were co-cultured with the BEAS-2B cells in the lower chamber for 48 h, and the expression of CD147 in cells collected from the lower chamber were detected by western blot. k , l The expression of CD147 was determined by western blot in BEAS-2B cells with SARS-CoV-2 pseudovirus infection ( k ) or RBD incubation ( l ). m The relative luciferase signals in pseudovirus-infected BEAS-2B cells were determined by dual-luciferase reporter assays with RBD incubation, *** p < 0.001. n The relative luciferase signals in pseudovirus-infected BEAS-2B cells were determined by dual-luciferase reporter assays, ns, not significant, *** p < 0.001. o The virus loads in authentic virus-infected BEAS-2B cells were determined by Taqman-based RT-PCR, ns, not significant, ** p < 0.01, *** p < 0.001. p A flow chart for establishing a virus-infected hCD147 mouse model by primarily inoculating with RBD protein followed by authentic virus infection. q The expression of CD147 was detected in the lung tissues of hCD147 mice with His-RBD ( n = 4) or His-tag ( n = 4) inoculation and hCD147 mice with His-RBD ( n = 4) or His-tag ( n = 4) inoculation followed by authentic virus infection. r The virus loads in the lung tissues of virus-infected hCD147 mice were determined, * p < 0.05. s , t The infiltration of F4/80+iNOS+ M1 macrophages and Ly6G+ neutrophils in lung tissues of virus-infected hCD147 mice was evaluated by multicolor immunofluorescence staining, Ly6G, yellow; F4/80, green; iNOS, red, scale bar, 100 μm ( s ), and their infiltrated percentages were quantified, ** p < 0.01, *** p < 0.001 ( t ). u The level of cytokines and chemokines in lung tissues of virus-infected hCD147 mice was performed by RT-PCR, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The mobile phases contained the mutants of CD147 (His-CD147-R54A, His-CD147-E84A, His-CD147-E92A, His-CD147-Q100A, His-CD147-S112A, our laboratory), the mutants of RBD (His-RBD-G413A, His-RBD-K417A, His-RBD-K424A, His-RBD-G447A, His-RBD-Y489A, Sino Biological), His-RBD (WT) (40592-V08H, Sino Biological), His-RBD (Beta) (40592-V08H85, Sino Biological), His-RBD (Gamma) (40592-V08H86, Sino Biological), His-RBD (JN.1) (40592-V08H155, Sino Biological), rhesus macaque CD147 (Sino Biological), human ACE2 (Sino Biological), and rhesus macaque ACE2 (Sino Biological).

Techniques: Virus, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Control, Software, Cell Culture, Incubation, Luciferase, Multicolor Immunofluorescence Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Inducible CD147 up-regulation boosts extended SARS-CoV-2 infection triggering severe COVID-19 independent of ACE2

doi: 10.1038/s41392-025-02551-x

Figure Lengend Snippet: Cryo-EM structure demonstrates CD147-spike interaction. a Cryo-EM map of the CD147-spike complex. The RBD up protomer, green; the two RBD down protomers, sandy brown and blue; CD147, magenta. b The overall structure of the CD147-spike complex shown as cartoons in side (left) and top (right) views. c The overlaid structures of CD147-spike and spike-closed are shown in surface representation. CD147 is depicted in magenta, with the spike protomer in the up and down conformations shown in green and cyan, respectively. The contact patch recognized by CD147 is highlighted in yellow on the down RBD. d The structure and key residues within the binding interface of the CD147-spike complex. The key residues forming hydrogen bonds are presented by stick models, with the hydrogen-bonding interactions labeled in yellow lines. e The binding ability of spike RBD with wildtype CD147 or its mutants (CD147-R54A, E84A, E92A, Q100A, and S112A) and CD147 with wildtype spike RBD or its mutants (G413A, K417A, K424A, G447A, and Y489A) was determined by SPR assay. f The binding ability of CD147 with wildtype spike RBD or its mutants (spike RBD-Beta, Gamma, and JN.1) was determined by SPR assay. g The virus loads and relative luciferase signals in CD147/ACE2 double knockout Vero E6 cells transfected with either wild type CD147 or mutated CD147 were determined by Taqman-based RT-PCR and dual-luciferase reporter assays, respectively, *** p < 0.001. h The overlaid structures of the CD147-spike complex, colored as in (a), and the CD147-Meplazumab Fab complex (PDB ID: 5X0T), colored in gray. The up RBD and Meplazumab Fab are depicted as surfaces, with the binding epitopes on CD147 (residues 61-75, colored in red) recognized by Meplazumab. The steric hindrance between Meplazumab and the spike bound to CD147 is indicated with orange arrows. i The competitive inhibitory role of MPZ for the binding of CD147 and SARS-CoV-2 spike (RBD) protein was determined by ELISA assay

Article Snippet: The mobile phases contained the mutants of CD147 (His-CD147-R54A, His-CD147-E84A, His-CD147-E92A, His-CD147-Q100A, His-CD147-S112A, our laboratory), the mutants of RBD (His-RBD-G413A, His-RBD-K417A, His-RBD-K424A, His-RBD-G447A, His-RBD-Y489A, Sino Biological), His-RBD (WT) (40592-V08H, Sino Biological), His-RBD (Beta) (40592-V08H85, Sino Biological), His-RBD (Gamma) (40592-V08H86, Sino Biological), His-RBD (JN.1) (40592-V08H155, Sino Biological), rhesus macaque CD147 (Sino Biological), human ACE2 (Sino Biological), and rhesus macaque ACE2 (Sino Biological).

Techniques: Cryo-EM Sample Prep, Binding Assay, Labeling, SPR Assay, Virus, Luciferase, Double Knockout, Transfection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay