Journal: bioRxiv

Article Title: Transcriptional control of cardiac neural crest cells condensation and outflow tract septation by the Smad1/5/8 inhibitor Dullard

doi: 10.1101/548511

Figure Lengend Snippet: (A) Ai Schematic representation of the migration routes the cardiac NCC (green) have taken to reach the heart region (red) in a E10.5 mouse embryo. Aii Schematics of the embryonic heart at E11.5 showing the distal-proximal axis of the OFT. Aiii Schematic representation of transverse sections through the OFT showing discrete stages of NCC condensation and endocardium septation along the OFT distal-proximal axis. (B) Pecam and GFP immuno-labelling and DAPI staining on transverse sections throughout the medial OFT of E11.5 Wnt1 Cre or Pax3 Cre ; Dullard flox/+ ; Rosa26mTmG embryos. (C) Normalized expression levels of Dullard assayed by q-RT-PCR on single cells isolated from E11.5 Wnt1 Cre ; Dullard flox/+ and Wnt1 Cre ; Dullard flox/flox ; Rosa26mTmG hearts (dots: value for a single cell; boxplot: mean± s.e.m.). The probe monitoring Dullard expression specifically binds to exons 2-3, which are the exons recombined by the Cre recombinase. (D) Dullard mRNA distribution detected using RNAscope probes, in transverse sections of E11.5 control and mutant OFTs, assessed by RNAscope. Dullard mRNA levels were significantly reduced in mutant cardiac cushions compared to controls; however, mRNA signals were still detected given the binding of Z pair probes to non-recombined exons 5 to 8 and UTR region. (E) Ei. Schematics of E11.5 heart showing the position of the transverse sections used to quantify the levels of the phosphorylated forms of Smad1/5/8 (PSmads) in iii. Eii. Immuno-labelling for PSmads and GFP, and DAPI staining on transverse sections across the OFT at 3 distinct distal-proximal levels in E11.5 embryos with the indicated genotype. Pale green dotted lines delineate the area colonized by cardiac NCC. Eiii. Quantification of PSmads levels in cardiac NCC along the OFT distal-proximal axis of E11.5 embryos with the indicated genotype (dots: values obtained on a given section; n>4 embryos per genotype recovered from at least 3 liters; the black line is the linear regression, the coloured areas delineate the 95% confidence intervals, ***: p-value < 0,001 for a two way-Anova statistical test). Ao: aortic artery, Pa: pulmonary artery.

Article Snippet: The primary antibodies used were raised against: GFP (chicken, Aves Labs, GFP-1020, 1/500), Pecam (rat monoclonal, Santa-Cruz Biotechnology, sc-18916, 1/200), Phospho-Smad1/5/8 (rabbit monoclonal, Cell Signalling Technology, 13820S, 1/500), myosin heavy chain, MyHC (mouse monoclonal, DSHB, MF20,1/300), Phospho-Histone H3 (rabbit, Cell Signalling Technology, 9701, 1/500), Cleaved Caspase-3 (rabbit monoclonal, Cell Signalling Technology, Asp175, 1/500).

Techniques: Migration, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Transcriptional control of cardiac neural crest cells condensation and outflow tract septation by the Smad1/5/8 inhibitor Dullard

doi: 10.1101/548511

Figure Lengend Snippet: (A) Top: Western blot detecting the phosphorylated forms of Smad1/5/8, GFP or Gapdh in C2C12 muscle cells exposed with or without BMP2 for 1h. These cells were non transfected (ct) or transfected with either a GFP expressing plasmid (GFP), a GFP tagged version of the wild-type Dullard (Dull) or of Dullard carrying D67E mutation in its phosphatase domain (Dull D67E). Dullard inhibits BMP2 mediated phosphorylation of Smad1/5/8; this inhibition is dependent on the functionality of its phosphatase domain. Bottom-left: Immunofluorescence for PSmads and GFP and DAPI staining in C2C12 transfected with GFP or GFP-Dullard and exposed for 1 h to BMP2 showing that only cells transfected with Dullard do not have nuclear phosphorylated Smad1/5/8. Right: Quantification of the number of PSmads positive C2C12 cells exposed to BMP2 for 1h and transfected with GFP (ct), Dullard or Dullard carrying the D67E mutation (n=3 independent experiments; Student t-test **: p-value < 0.01. (B) Bi. Schematics of E11.5 heart showing the position of the transverse sections used to quantify the levels of the phosphorylated forms of Smad1/5/8 (PSmads) in iii. Bii. Immuno-labelling for PSmads and GFP and DAPI staining on transverse sections across the OFT at 3 distinct distal-proximal levels in E11.5 of control Pax3 Cre ; Dullard flox/+ ; Rosa26mTmG and mutant Pax3 Cre ; Dullard flox/flox ; Rosa26mTmG hearts. Pale green dotted lines delineate the area colonized by cardiac NCC. Biii. Quantification of PSmads levels in cardiac NCC along the distal-proximal axis of the OFT of E11.5 embryos with the indicated genotype (dots: values obtained on a given section; n>4 embryos per genotype recovered from at least 3 liters; the black line is the linear regression, the coloured areas delineate the 95% confidence intervals, ***: p-value < 0,001 for a two way-Anova statistical test). Ao: aortic artery, Pa: pulmonary artery.

Article Snippet: The primary antibodies used were raised against: GFP (chicken, Aves Labs, GFP-1020, 1/500), Pecam (rat monoclonal, Santa-Cruz Biotechnology, sc-18916, 1/200), Phospho-Smad1/5/8 (rabbit monoclonal, Cell Signalling Technology, 13820S, 1/500), myosin heavy chain, MyHC (mouse monoclonal, DSHB, MF20,1/300), Phospho-Histone H3 (rabbit, Cell Signalling Technology, 9701, 1/500), Cleaved Caspase-3 (rabbit monoclonal, Cell Signalling Technology, Asp175, 1/500).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Inhibition, Immunofluorescence, Staining

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 1. Upregulation of polyribonucleotide nucleotidyltransferase 1 (PNPT1) in host cells infected by various viruses and depletion of PNPT1 in host cells markedly reduced viral replication. Immunostaining (a) and Western blot analysis (b) of PNPT1 in BEAS-2B cells with or without adenovirus (Adv-5) infection (24 h). Immunostaining (c) and Western blot analysis (d) of PNPT1 in NIH-3T3 cells with or without murine cytomegalovirus (MCMV) infection (48 h). Immunostaining (e) and Western blot analysis (f) of PNPT1 in Neuro-2a cells with or without murine hepatovirus (MHV-A59) infection (12 h). (g) Quantitative reverse transcription polymerase chain reaction analysis of viral DNA level in BEAS-2B cells (left), NIH-3T3 cells (middle) and Neuro-2a cells (right) with or without Adv-5, MCMV or MHV-A59 infection for 12 h, 24 h or 48 h at indicated multiplicity of infection, respectively. Cells were treated with control shRNA (sh-CTL) or PNPT1 shRNA (sh-PNPT1) prior to viral infection. Scale bar 20 μm. Data from three independent experiments are presented as mean ± standard deviation. ∗∗∗∗P < 0.0 0 01.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Infection, Immunostaining, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Control, shRNA, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 2. A reduction in polyribonucleotide nucleotidyltransferase 1 (PNPT1) promotes relocation of mitochondria double-stranded RNAs (mt-dsRNAs) from mitochondria to cytoplasm. (a–c) Immunostaining of mt-dsRNAs (J2) in BEAS-2B cells infected or not infected with adenovirus (Adv-5) at 24 h (a), NIH-3T3 cells infected or not infected with murine cytomegalovirus (MCMV) at 48 h (b), and Neuro-2a cells infected or not infected with murine hepatovirus (MHV-A59) at 12 h (c). Scale bar 20 μm. (d) Quantitative reverse transcription polymerase chain reaction analysis of cytosolic mt-dsRNAs in BEAS-2B cells infected or not infected with Adv-5 at 24 h. (e) In-situ staining of cytosolic mt- ND5 heavy (left) and light strands (right) in BEAS-2B cells infected or not infected with Adv-5 at 24 h. ATP5A1 served as a mitochondria marker. Scale bar 50 μm. Data from three independent experiments are presented as mean ± standard deviation.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Immunostaining, Infection, Reverse Transcription, Polymerase Chain Reaction, In Situ, Staining, Marker, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 3. Cytosolic mitochondria double-stranded RNAs activate RNA-activated protein kinase (PKR) and, subsequently, eukaryotic initiation factor 2 α (eIF2 α) to terminate general protein production. (a) Left: Fluorescence microscopy images of BEAS-2B cells infected with adenovirus (Adv-5) for 1 h, then washed with phosphate buffered saline; nascent protein synthesis at 24 h post-infection was detected by OPP-647 (red) and DAPI (blue) staining. Right: Quantification of OPP-647 staining. Scale bar, 75 μm. (b) Western blot analysis of PNPT1, phosphorylated PKR (p-PKR), PKR, phosphorylated eIF2 α and eIF2 α levels in BEAS-2B cells infected or not infected with Adv-5 after 24 h infection. (c) Western blot analysis of PNPT1, p-PKR, PKR, p-eIF2 α and eIF2 α levels in NIH/3T3 cells infected or not infected with murine cytomegalovirus (MCMV) after 48 h infection. (d) Western blot analysis of PNPT1, p-PKR, PKR, p-eIF2 α and eIF2 α levels in Neuro-2a cells infected or not infected with murine hepatovirus (MHV-A59) after 12 h infection. In (a–d), cells were treated with control shRNA (sh-CTL) or PNPT1 shRNA (sh-PNPT1) prior to viral infection.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Fluorescence, Microscopy, Infection, Saline, Staining, Western Blot, Control, shRNA

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 4. Identification of lanatoside C (LanC) as a potent inhibitor of polyribonucleotide nucleotidyltransferase 1 (PNPT1) using bioactive compounds approved by the US Food and Drug Administration. (a) Prediction of PNPT1 inhibition based on functional PNPT1 structural analysis. (b) BEAS-2B cells were infected with adenovirus (Adv-5) at multiplicity of infection (MOI) of 0.5 and treated with various compounds at indicated concentrations for 24 h. The effects of each compound on induction of cellular mitochondria double-stranded RNAs (mt-dsRNAs) labelled with J2 antibody were analysed by immunofluorescence staining. (c) Left: Representative images of cellular mt- dsRNA staining by J2 antibody as a readout for PNPT1 inhibition (red, mt-dsRNA; blue, nuclei). Scale bar 50 μm. Right: Quantification of cellular mt-dsRNAs labelled with J2 antibody was analysed by immunofluorescence staining. (d) BEAS-2B cells were infected with Adv-5 at MOI of 0.5 and treated with different com pounds at indicated concentrations for 24 h. The inhibition of viral replication was analysed using quantitative reverse transcription polymerase chain reaction. (e) Dose-dependent inhibition of LanC and atazanavir sulfate on Adv-5 replication (MOI of 0.5) in BEAS-2B cells. (f) Micro-scale thermophoresis analysis of binding affinity of LanC with functional PNPT1 trimer. Data from three independent experiments are presented as mean ± standard deviation. ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Inhibition, Functional Assay, Infection, Staining, Reverse Transcription, Polymerase Chain Reaction, Binding Assay, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 6. Lanatoside C (LanC), an inhibitor of polyribonucleotide nucleotidyltransferase 1 (PNPT1), suppresses virus replication by activating RNA-activated protein kinase (PKR) and, subsequently, eukaryotic initiation factor 2 α (eIF2 α). (a) Immunostaining of double-stranded RNA (red) in BEAS-2B cells treated with LanC at indicated concentration for 24 h. Scale bar 20 μm. (b) Quantitative reverse transcription polymerase chain reaction analysis of cytosolic mitochondria double-stranded RNAs (mt-dsRNAs) in BEAS- 2B cells infected and not infected with adenovirus (Adv-5). (c) In-situ staining of cytosolic mt- ND5 heavy (left) and light strands (right) in BEAS-2B cells infected and not infected with Adv-5. ATP5A1 served as a mitochondria marker. Scale bar 50 μm. (d) Western blot analysis of PNPT1, p-PKR, PKR, p-eIF2 and eIF2 expression in BEAS-2B cells treated with LanC at indicated concentrations for 24 h. (e) Left: Fluorescence microscopy images of BEAS-2B cells infected with Adv-5 virus for 1 h, washed with phosphate buffered saline, then treated with LanC at indicated concentrations for another 24 h; nascent protein synthesis was detected by staining with OPP-647 (red), and genomic DNA was stained using DAPI (blue). Right: Quantification of OPP-647 staining. Scale bar 50 μm. Data from three independent experiments are presented as mean ± standard deviation. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0 0 01.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Virus, Immunostaining, Concentration Assay, Reverse Transcription, Polymerase Chain Reaction, Infection, In Situ, Staining, Marker, Western Blot, Expressing, Fluorescence, Microscopy, Saline, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 7. Lanatoside C (LanC) suppresses production of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike (S) protein in BEAS-2B cells. BEAS-2B cells were infected with SARS-CoV-2 pseudo-virions at multiplicity of infection of 0.01, and treated with LanC at indicated concentrations for 24 h. (a) The effects of suppression of viral replication were analysed quantitatively using quantitative reverse transcription polymerase chain reaction. (b) The effects of suppression of viral replication were detected with green fluorescent protein (GFP). (c) Western blot analysis of S-protein, polyribonucleotide nucleotidyltransferase 1 (PNPT1), phosphorylated RNA-activated protein kinase (p-PKR), PKR, phosphorylated eukaryotic initiation factor 2 α (p-eIF2 α) and eIF2 α expression after LanC treatment. Data from three independent experiments are presented as mean ± standard deviation. ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0 0 01.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Infection, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Expressing, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 8. Schematic working model of lanatoside C (LanC), an inhibitor of polyribonucleotide nucleotidyltransferase 1 (PNPT1), blocking pan-viral replication in host cells. PKR, RNA-activated protein kinase; eIF2 α, eukaryotic initiation factor 2 α.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Blocking Assay

Journal: Clinical science (London, England : 1979)

Article Title: Inhibition of HDAC3 prevents diabetic cardiomyopathy in OVE26 mice via epigenetic regulation of DUSP5-ERK1/2 pathway

doi: 10.1042/CS20170064

Figure Lengend Snippet: Representative Western blot of whole cell p-ERK1/2 and ERK1/2, nuclear p-ERK1/2 and ERK1/2 (A, C), and densitometric analyses of Western blot of p-ERK1/2 protein levels (B, D). The expression of DUSP5 detected by real-time PCR (E) and Western blot (F, G). The acetylated level of histone H3 on the DUSP5 gene promoter region revealed by ChIP (H). Normal mouse IgG was used as a negative control and data were normalized to input DNA samples. Data are presented as the mean ± S.D. n=5–7. *P < 0.05 vs. WT-Veh; #P < 0.05 vs. OVE-Veh at the same time point. Schematic illustration of the working hypothesis for the regulation of nuclear ERK1/2 signaling and diabetes-induced cardiac injury by HDAC3 and DUSP5 (I). Diabetes increased ERK1/2 phosphorylation and nuclear accumulation of phosphorylated ERK1/2 where it turns on the cardiac apoptosis, hypertrophy and fibrosis signaling pathways, leading to the final cardiac remodeling and dysfunction. HDAC3 (or HDACs) inhibitor stimulates DUSP5 expression by increasing the acetylated histone H3, leading to the dephosphorylation of nuclear ERK1/2 and thus reducing cardiac remodeling and dysfunction. In addition, phosphorylated ERK1/2 also inhibits DUSP5 expression via stimulating HDACs, making a self-reinforcing cycle.

Article Snippet: Primers [ANP: Mm01256744_m1; collagen I: Mm01302043_g1; fibronectin-1 (FN-1): Mm01255747_g1; DUSP5: Mm01266106; GAPDH: Mm99999915_g1] for PCR were obtained from Thermo Fisher (Grand Island, NY, USA).

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Negative Control, Phospho-proteomics, Protein-Protein interactions, De-Phosphorylation Assay