Journal: PLOS Pathogens

Article Title: Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release

doi: 10.1371/journal.ppat.1011132

Figure Lengend Snippet: HeLa cells were mock-infected and infected with EV-A71 (MOI 1) for 0, 12, and 24 h. PK-15 cells were mock-infected and infected with SVV (MOI 1) or FMDV (MOI 0.5) for 0, 6, and 12 h. The ΔΨm (A), mitochondrial Ca 2+ concentration (B), mitochondrial ROS (C), and mPTP opening (D) were detected using JC-1, Rhod-2 AM, mitoSOX, and calcein-AM, respectively. Data for CCCP (A) and lono (D) positive controls are shown. Error bars show standard deviation. **, P <0.01.

Article Snippet: The commercial antibodies used in this study included an anti-FLAG monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-FLAG polyclonal antibody (Sigma-Aldrich), anti-HA monoclonal antibody (Thermo Scientific, Waltham, MA, USA), anti-cGAS monoclonal antibody (Santa Cruz Biotechnology), anti-STING monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), anti-p-STING (S366) monoclonal antibody (Cell Signaling Technology), anti-IFI16 monoclonal antibody (Cell Signaling Technology), anti-TBK1 monoclonal antibody (Cell Signaling Technology), anti-IRF3 monoclonal antibody (Cell Signaling Technology), anti-p-IRF3 monoclonal antibody (Cell Signaling Technology), anti-dsDNA monoclonal antibody (Abcam, Cambridge, MA, USA), anti-ATG7 polyclonal antibody (ABclonal Technology Co., Ltd, Wuhan, China), anti-Tom20 monoclonal antibody (ABclonal), and anti-β-actin monoclonal antibody (Thermo Scientific), anti-Bax monoclonal antibody (ABclonal), anti-PPID polyclonal antibody (ABclonal), anti-EV-A71 3C polyclonal antibody (ABclonal), anti-SVV VP1 polyclonal antibody was prepared in our laboratory [ ], anti-FMDV VP1 polyclonal antibody was prepared in our laboratory [ ], and anti-2C polyclonal antibody was produced in rabbits by immunization with SVV 2C protein.

Techniques: Infection, Concentration Assay, Standard Deviation

Journal: PLOS Pathogens

Article Title: Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release

doi: 10.1371/journal.ppat.1011132

Figure Lengend Snippet: HeLa cells were mock-infected or infected with EV-A71 (MOI 1) for 0, 12, and 24 h, while PK-15 cells were mock-infected or infected with SVV (MOI 1) or FMDV (MOI 0.5) for 0, 6, and 12 h. The lysates were immunoprecipitated with anti-cGAS antibody. The mtDNA and gDNA in cGAS pulldown samples were detected and analyzed by qPCR. The EGFP DNA was used as an internal control (A). HeLa cells were mock-infected or infected with EV-A71 (MOI 1) for 24 h. PK-15 cells were mock-infected or infected with SVV (MOI 1) or FMDV (MOI 0.5) for 12 h. The mtDNA release was evaluated by IFA. Cells were double-immunostained for detection of Tom20 (red) and dsDNA (green); cellular nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (blue) (B, C, and D). Scale bar, 10 μm. Oropharyngeal tonsils collected from pigs infected with SVV (E) or FMDV (F) were analyzed for mtDNA release. Error bars show standard deviation. **, P <0.01.

Article Snippet: The commercial antibodies used in this study included an anti-FLAG monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-FLAG polyclonal antibody (Sigma-Aldrich), anti-HA monoclonal antibody (Thermo Scientific, Waltham, MA, USA), anti-cGAS monoclonal antibody (Santa Cruz Biotechnology), anti-STING monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), anti-p-STING (S366) monoclonal antibody (Cell Signaling Technology), anti-IFI16 monoclonal antibody (Cell Signaling Technology), anti-TBK1 monoclonal antibody (Cell Signaling Technology), anti-IRF3 monoclonal antibody (Cell Signaling Technology), anti-p-IRF3 monoclonal antibody (Cell Signaling Technology), anti-dsDNA monoclonal antibody (Abcam, Cambridge, MA, USA), anti-ATG7 polyclonal antibody (ABclonal Technology Co., Ltd, Wuhan, China), anti-Tom20 monoclonal antibody (ABclonal), and anti-β-actin monoclonal antibody (Thermo Scientific), anti-Bax monoclonal antibody (ABclonal), anti-PPID polyclonal antibody (ABclonal), anti-EV-A71 3C polyclonal antibody (ABclonal), anti-SVV VP1 polyclonal antibody was prepared in our laboratory [ ], anti-FMDV VP1 polyclonal antibody was prepared in our laboratory [ ], and anti-2C polyclonal antibody was produced in rabbits by immunization with SVV 2C protein.

Techniques: Infection, Immunoprecipitation, Control, Standard Deviation

Journal: PLOS Pathogens

Article Title: Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release

doi: 10.1371/journal.ppat.1011132

Figure Lengend Snippet: HT-29 WT and PPID -/- cells were mock-infected or infected with EV-A71 (MOI 1) for 24 h, while PK-15 WT or PPID -/- cells were mock-infected or infected with SVV (MOI 1) or FMDV (MOI 0.5) for 12 h. The mtDNA release was evaluated by qPCR (A). The expression of the IFN-β protein was detected by ELISA kit, and the expression of PPID was confirmed by Western blotting (B). (C) HT-29 cells were mock-infected or infected with EV-A71 (MOI 1), while PK-15 cells were mock-infected or infected with SVV (MOI 1) or FMDV (MOI 0.5). After 1 h of incubation of the virus or DMEM, cells were treated with DMSO or VBIT4 inhibitor (10 uM) for 24 or 12 h. The mtDNA release was then detected by qPCR. The expression of Bax protein in the WT and Bax -/- cells was confirmed by western blotting (D) and these cells were used to measure mtDNA release after infection with EV-A71 (HT-29 cells at MOI 1) or SVV (PK-15 cells at MOI 1) or FMDV (PK-15 cells at MOI 0.5) for 12 h (E). Error bars show standard deviation. **, P <0.01.

Article Snippet: The commercial antibodies used in this study included an anti-FLAG monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-FLAG polyclonal antibody (Sigma-Aldrich), anti-HA monoclonal antibody (Thermo Scientific, Waltham, MA, USA), anti-cGAS monoclonal antibody (Santa Cruz Biotechnology), anti-STING monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), anti-p-STING (S366) monoclonal antibody (Cell Signaling Technology), anti-IFI16 monoclonal antibody (Cell Signaling Technology), anti-TBK1 monoclonal antibody (Cell Signaling Technology), anti-IRF3 monoclonal antibody (Cell Signaling Technology), anti-p-IRF3 monoclonal antibody (Cell Signaling Technology), anti-dsDNA monoclonal antibody (Abcam, Cambridge, MA, USA), anti-ATG7 polyclonal antibody (ABclonal Technology Co., Ltd, Wuhan, China), anti-Tom20 monoclonal antibody (ABclonal), and anti-β-actin monoclonal antibody (Thermo Scientific), anti-Bax monoclonal antibody (ABclonal), anti-PPID polyclonal antibody (ABclonal), anti-EV-A71 3C polyclonal antibody (ABclonal), anti-SVV VP1 polyclonal antibody was prepared in our laboratory [ ], anti-FMDV VP1 polyclonal antibody was prepared in our laboratory [ ], and anti-2C polyclonal antibody was produced in rabbits by immunization with SVV 2C protein.

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Virus, Standard Deviation

Journal: PLOS Pathogens

Article Title: Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release

doi: 10.1371/journal.ppat.1011132

Figure Lengend Snippet: PK-15 cells were transfected with 2 μg of empty vector, the indicated SVV protein expressing plasmids (A) or FMDV protein expressing plasmids (B), and HeLa cells were transfected with 2 μg of empty vector or EV-A71 protein expressing plasmids (C). At 24 hpt, the mtDNA release was analyzed by qPCR. The expression of viral proteins was determined by Western blotting. (D) HeLa cells were transfected with 2 μg of empty vector, EMCV 2B or CA16 2B expressing plasmids for 24 h. The mtDNA release was detected by qPCR. (E) HT-29 WT and PPID -/- cells were transfected with 2 μg of empty vector, EMCV 2B, CA16 2B, or EV-A71 2B expressing plasmids for 24 h, and PK-15 WT or PPID -/- cells were transfected with 2 μg of empty vector, SVV 2B or FMDV 2B expressing plasmids for 24 h. The mtDNA release was detected by qPCR. Error bars show standard deviation. **, P <0.01.

Article Snippet: The commercial antibodies used in this study included an anti-FLAG monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-FLAG polyclonal antibody (Sigma-Aldrich), anti-HA monoclonal antibody (Thermo Scientific, Waltham, MA, USA), anti-cGAS monoclonal antibody (Santa Cruz Biotechnology), anti-STING monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), anti-p-STING (S366) monoclonal antibody (Cell Signaling Technology), anti-IFI16 monoclonal antibody (Cell Signaling Technology), anti-TBK1 monoclonal antibody (Cell Signaling Technology), anti-IRF3 monoclonal antibody (Cell Signaling Technology), anti-p-IRF3 monoclonal antibody (Cell Signaling Technology), anti-dsDNA monoclonal antibody (Abcam, Cambridge, MA, USA), anti-ATG7 polyclonal antibody (ABclonal Technology Co., Ltd, Wuhan, China), anti-Tom20 monoclonal antibody (ABclonal), and anti-β-actin monoclonal antibody (Thermo Scientific), anti-Bax monoclonal antibody (ABclonal), anti-PPID polyclonal antibody (ABclonal), anti-EV-A71 3C polyclonal antibody (ABclonal), anti-SVV VP1 polyclonal antibody was prepared in our laboratory [ ], anti-FMDV VP1 polyclonal antibody was prepared in our laboratory [ ], and anti-2C polyclonal antibody was produced in rabbits by immunization with SVV 2C protein.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Standard Deviation

Journal: PLOS Pathogens

Article Title: Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release

doi: 10.1371/journal.ppat.1011132

Figure Lengend Snippet: IFN-β and ISG54 mRNA expression in cGAS -/- HeLa cells infected with SVV (A), and cGAS -/- HT-29 cells infected with EV-A71 (B); IFN-β protein expression is also shown (C). Western blotting was used to detect the expression of SVV VP1 protein (D) and EV-A71 3C protein (E) and viral titers were determined by TCID 50 assay. Bone marrow-derived macrophages from the WT or cGAS -/- mice were infected with FMDV and IFN-β protein in the supernatant was determined by ELISA (F). The expression of FMDV VP1 protein was detected by Western blotting, and the viral titers were determined by TCID 50 assay (G). WT and ρ 0 PK-15 cells were infected with SVV or FMDV and WT and ρ 0 HT-29 cells were infected with EV-A71. IFN-β protein expression level was determined by ELISA (H), and the viral titers were determined by TCID 50 assay (I). Error bars show standard deviation. **, P <0.01.

Article Snippet: The commercial antibodies used in this study included an anti-FLAG monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-FLAG polyclonal antibody (Sigma-Aldrich), anti-HA monoclonal antibody (Thermo Scientific, Waltham, MA, USA), anti-cGAS monoclonal antibody (Santa Cruz Biotechnology), anti-STING monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), anti-p-STING (S366) monoclonal antibody (Cell Signaling Technology), anti-IFI16 monoclonal antibody (Cell Signaling Technology), anti-TBK1 monoclonal antibody (Cell Signaling Technology), anti-IRF3 monoclonal antibody (Cell Signaling Technology), anti-p-IRF3 monoclonal antibody (Cell Signaling Technology), anti-dsDNA monoclonal antibody (Abcam, Cambridge, MA, USA), anti-ATG7 polyclonal antibody (ABclonal Technology Co., Ltd, Wuhan, China), anti-Tom20 monoclonal antibody (ABclonal), and anti-β-actin monoclonal antibody (Thermo Scientific), anti-Bax monoclonal antibody (ABclonal), anti-PPID polyclonal antibody (ABclonal), anti-EV-A71 3C polyclonal antibody (ABclonal), anti-SVV VP1 polyclonal antibody was prepared in our laboratory [ ], anti-FMDV VP1 polyclonal antibody was prepared in our laboratory [ ], and anti-2C polyclonal antibody was produced in rabbits by immunization with SVV 2C protein.

Techniques: Expressing, Infection, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: PLOS Pathogens

Article Title: Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release

doi: 10.1371/journal.ppat.1011132

Figure Lengend Snippet: (A) The 3-weeks-old WT and cGAS -/- mice were showed, and the expression of cGAS in the WT and cGAS -/- mice was detected by Western blotting. (B and C) The three-day-old WT mice were subcutaneously inoculated with FMDV (10 8 TCID 50 ) or EV-A71 (10 8 TCID 50 ) for 0, 1, or 3 d (n = 3/group), the mice carcasses without the head, tail, limbs, and viscera were collected for detection of mtDNA release (B) and IFN-β mRNA expression (C). (D and E) The three-day-old WT (n = 8) and cGAS -/- (n = 8) mice were subcutaneously inoculated with FMDV (10 8 TCID 50 ) or EV-A71 (10 8 TCID 50 ). The IFN-β mRNA level in the carcasses without the head, tail, limbs, and viscera from FMDV-infected mice was measured and compared at 3 dpi by qPCR (D). The IFN-β mRNA level in the carcasses without the head, tail, limbs, and viscera from EV-A71-infected mice was measured and compared at 1 dpi by qPCR (E). FMDV (F) and EV-A71 (G) titers in the mice carcasses without the head, tail, limbs, and viscera were determined at 2 dpi by TCID 50 assay. The mortality of WT (n = 10) and cGAS -/- (n = 10) mice infected by FMDV (F) or EV-A71 (G) was determined, respectively. Error bars show standard deviation. **, P <0.01.

Article Snippet: The commercial antibodies used in this study included an anti-FLAG monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-FLAG polyclonal antibody (Sigma-Aldrich), anti-HA monoclonal antibody (Thermo Scientific, Waltham, MA, USA), anti-cGAS monoclonal antibody (Santa Cruz Biotechnology), anti-STING monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), anti-p-STING (S366) monoclonal antibody (Cell Signaling Technology), anti-IFI16 monoclonal antibody (Cell Signaling Technology), anti-TBK1 monoclonal antibody (Cell Signaling Technology), anti-IRF3 monoclonal antibody (Cell Signaling Technology), anti-p-IRF3 monoclonal antibody (Cell Signaling Technology), anti-dsDNA monoclonal antibody (Abcam, Cambridge, MA, USA), anti-ATG7 polyclonal antibody (ABclonal Technology Co., Ltd, Wuhan, China), anti-Tom20 monoclonal antibody (ABclonal), and anti-β-actin monoclonal antibody (Thermo Scientific), anti-Bax monoclonal antibody (ABclonal), anti-PPID polyclonal antibody (ABclonal), anti-EV-A71 3C polyclonal antibody (ABclonal), anti-SVV VP1 polyclonal antibody was prepared in our laboratory [ ], anti-FMDV VP1 polyclonal antibody was prepared in our laboratory [ ], and anti-2C polyclonal antibody was produced in rabbits by immunization with SVV 2C protein.

Techniques: Expressing, Western Blot, Infection, Standard Deviation

Journal: PLOS Pathogens

Article Title: Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release

doi: 10.1371/journal.ppat.1011132

Figure Lengend Snippet: (A) HEK-293T cells were transfected with 1 μg of empty vector or the indicated FLAG-2C-expressing plasmids, along with 1 μg of empty vector or HA-cGAS plus HA-STING expressing plasmids. At 24 hpt, the IFN-β protein amount in the supernatant was determined by ELISA, and the expression of 2C was confirmed by Western blotting. (B) STING-HEK-293T cells were transfected with increasing amount (0, 3, or 6 μg) of the indicated FLAG-2C-expressing plasmids. At 24 hpt, cells were transfected with poly(dA:dT) (2 μg/ml) for 12 h. The cells lysates were immunoprecipitated with anti-STING antibody. The antibody-antigen complexes were detected using anti-STING, TBK1, and FLAG antibodies, respectively. (C) Schematic representation of the structure and conserved functional sites in EV-A71 2C protein. The redder the color was, the more conservative the sites were. (D, E) HEK-293T cells were transfected with 1 μg of empty vector or EV-A71 (D), CA16 (E), and EMCV (E) FLAG-2C- or FLAG-2C mutants-expressing plasmids, along with 1 μg of empty vector or HA-cGAS plus HA-STING expressing plasmids. At 24 hpt, the IFN-β protein amount in the supernatant was determined by ELISA, and the expression of 2C was confirmed by Western blotting. (F) STING-HEK-293T cells were transfected with 6 μg of empty vector, FLAG-2C- or FLAG-2C mutants-expressing plasmids. At 24 hpt, cells were transfected with poly(dA:dT) (2 μg/ml) for 12 h. The cells lysates were immunoprecipitated with anti-STING antibody. The antibody-antigen complexes were detected using anti-STING, TBK1, and FLAG antibodies, respectively. (G) HEK-293T cells were transfected with 1 μg of empty vector, FLAG-2C- or FLAG-2C mutants-expressing plasmids, and 1 μg of HA-cGAS plus HA-STING expressing plasmids. At 24 hpt, expression of IRF3, p-IRF3, and FLAG-2C protein was determined by Western blotting. The IRF3 dimerization was detected using native PAGE. Error bars show standard deviation. **, P <0.01.

Article Snippet: The commercial antibodies used in this study included an anti-FLAG monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-FLAG polyclonal antibody (Sigma-Aldrich), anti-HA monoclonal antibody (Thermo Scientific, Waltham, MA, USA), anti-cGAS monoclonal antibody (Santa Cruz Biotechnology), anti-STING monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), anti-p-STING (S366) monoclonal antibody (Cell Signaling Technology), anti-IFI16 monoclonal antibody (Cell Signaling Technology), anti-TBK1 monoclonal antibody (Cell Signaling Technology), anti-IRF3 monoclonal antibody (Cell Signaling Technology), anti-p-IRF3 monoclonal antibody (Cell Signaling Technology), anti-dsDNA monoclonal antibody (Abcam, Cambridge, MA, USA), anti-ATG7 polyclonal antibody (ABclonal Technology Co., Ltd, Wuhan, China), anti-Tom20 monoclonal antibody (ABclonal), and anti-β-actin monoclonal antibody (Thermo Scientific), anti-Bax monoclonal antibody (ABclonal), anti-PPID polyclonal antibody (ABclonal), anti-EV-A71 3C polyclonal antibody (ABclonal), anti-SVV VP1 polyclonal antibody was prepared in our laboratory [ ], anti-FMDV VP1 polyclonal antibody was prepared in our laboratory [ ], and anti-2C polyclonal antibody was produced in rabbits by immunization with SVV 2C protein.

Techniques: Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunoprecipitation, Functional Assay, Clear Native PAGE, Standard Deviation

Journal: PLOS Pathogens

Article Title: Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release

doi: 10.1371/journal.ppat.1011132

Figure Lengend Snippet: In this model, cytosolic mtDNA released after EV-A71, SVV, and FMDV infection binds to cGAS to activate cGAS-mediated signal transduction, resulting in the antiviral responses. Virus immune evasion strategies include SVV 2C induced the reduction of cGAS by activation of autophagy and FMDV 2B and 3C pro proteins which inhibit STING expression to block the antiviral response. Furthermore, EV-A71, CA16, and EMCV 2C can antagonize activation of the cGAS-STING signaling pathway by impairing the interaction of STING with TBK1, while FMDV L pro and EV-A71 2A proteins inhibit cGAS-STING-induced IFN-β protein expression.

Article Snippet: The commercial antibodies used in this study included an anti-FLAG monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-FLAG polyclonal antibody (Sigma-Aldrich), anti-HA monoclonal antibody (Thermo Scientific, Waltham, MA, USA), anti-cGAS monoclonal antibody (Santa Cruz Biotechnology), anti-STING monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), anti-p-STING (S366) monoclonal antibody (Cell Signaling Technology), anti-IFI16 monoclonal antibody (Cell Signaling Technology), anti-TBK1 monoclonal antibody (Cell Signaling Technology), anti-IRF3 monoclonal antibody (Cell Signaling Technology), anti-p-IRF3 monoclonal antibody (Cell Signaling Technology), anti-dsDNA monoclonal antibody (Abcam, Cambridge, MA, USA), anti-ATG7 polyclonal antibody (ABclonal Technology Co., Ltd, Wuhan, China), anti-Tom20 monoclonal antibody (ABclonal), and anti-β-actin monoclonal antibody (Thermo Scientific), anti-Bax monoclonal antibody (ABclonal), anti-PPID polyclonal antibody (ABclonal), anti-EV-A71 3C polyclonal antibody (ABclonal), anti-SVV VP1 polyclonal antibody was prepared in our laboratory [ ], anti-FMDV VP1 polyclonal antibody was prepared in our laboratory [ ], and anti-2C polyclonal antibody was produced in rabbits by immunization with SVV 2C protein.

Techniques: Infection, Transduction, Virus, Activation Assay, Expressing, Blocking Assay

Journal: eBioMedicine

Article Title: Tanomastat exerts multi-targeted inhibitory effects on viral capsid dissociation and RNA replication in human enteroviruses

doi: 10.1016/j.ebiom.2024.105277

Figure Lengend Snippet: Tanomastat targets EV-A71 RNA replication. ( a ) RD cells were infected with EV-A71 at M.O.I of 1 at 4 °C. After pre-adsorption, the infected cells were incubated for 2 h at 37 °C and post-treated with 0.1% DMSO vehicle control or Tanomastat at the relevant concentrations (from 1 μM to 50 μM). ( b ) In entry-bypass assay, RD cells were transfected with EV-A71 RNA and treated with 0.1% DMSO vehicle control or Tanomastat at the relevant concentrations (from 10 μM to 50 μM). In a and b , the infectious virus titres were determined by viral plaque assay. ( c–e ) RD cells were infected with EV-A71 at M.O.I of 1 and post-treated with 0.1% DMSO vehicle control or Tanomastat at the relevant concentrations (from 1 μM to 40 μM). Protein bands were separated by SDS-PAGE, followed by Western blot analysis using anti-EV-A71 VP2 monoclonal antibody, anti-EV-A71 VP1 polyclonal antibody, and anti- β -Actin monoclonal antibody. Band intensities below the detectable limit are denoted as not detectable (n.d.). The relative VP2 and VP1 band intensities were normalized against β -Actin. Results are representative of two independent experiments. ( f and g ) RD cells were transfected with EV-A71 3D polymerase replication competent or defective RNA replicons and treated with Tanomastat at the relevant concentrations (from 1 μM to 10 μM). 0.1% DMSO, CHX and GuHCl served as vehicle, general translation inhibitor and RNA replication-specific inhibitor controls, respectively. ( h ) RD cells were transfected with the EV-A71 bicistronic luciferase construct and treated with Tanomastat at the relevant concentrations (from 10 μM to 50 μM). 0.1% DMSO and apigenin served as vehicle and EV-A71 IRES translation inhibitor controls, respectively, Luminescence readings were used to derive the normalized F Luc/R Luc ratio, which is reflective of IRES activity. Each data point denotes the mean of triplicates, and the error bar denotes the standard deviation. One-way ANOVA followed by Dunnett’s test was used to determine the statistical significance of the treatments when compared against 0.1% DMSO vehicle control. P-values, mean difference, and 95% CI are reported in Supplementary Table S5 . Bar graphs represent mean ± standard deviation with ∗ P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Article Snippet: After blocking with 2% bovine serum albumin (BSA) (Sigma–Aldrich) dissolved in Tris-buffered saline-Tween 20, the membrane was incubated in the following primary antibodies: mouse anti-EV-A71 VP2 (Merck Millipore Cat#MAB979, RRID:AB_95300), rabbit anti-EV-A71 VP1 (Genetex Cat#GTX132339, RRID:AB_2886617), mouse anti-MMP-9 (Thermo Fisher Scientific Cat#MA5-15886, RRID:AB_11157246), and mouse anti- β -Actin (Sigma–Aldrich Cat#A2228, RRID:AB_476697).

Techniques: Infection, Adsorption, Incubation, Control, Transfection, Virus, Viral Plaque Assay, SDS Page, Western Blot, Luciferase, Construct, Activity Assay, Standard Deviation

Journal: eBioMedicine

Article Title: Tanomastat exerts multi-targeted inhibitory effects on viral capsid dissociation and RNA replication in human enteroviruses

doi: 10.1016/j.ebiom.2024.105277

Figure Lengend Snippet: Tanomastat displays in vivo prophylactic efficacy and viral load inhibition at tested drug doses in sucking BALB/c mice challenged with lethal dose of EV-A71. ( a ) 5-day-old BALB/c neonatal mice were infected with EV-A71 at a dose of 2 × 10 7 per mice via i.p. At 0 d.p.i, a single dose of 10 mg/kg or 30 mg/kg Tanomastat was administered via oral gavage 2 h pre-infection. A second dose was administered 24 h post-infection, and subsequent doses were administered daily up to 120 h.p.i. DMSO was used as treatment vehicle control. The ( b ) percent survival, ( c ) body weight, and ( d ) clinical scorings were recorded for up to 14 d.p.i. EV-A71-infected mice were sacrificed on 5 d.p.i for quantification of viral titres in ( e ) brain and ( f ) hind limb muscle tissues, as well as for histopathology evaluation in spinal cord and hind limb muscle tissues. H&E staining showed severe necrosis in ( g ) DMSO control-treated mice, whereas mild necrosis was observed in 10 mg/kg and 30 mg/kg Tanomastat-treated mice. IHC staining muscle tissue using anti-EV-A71 VP2 monoclonal antibody showed extensive antigen positive in ( h ) DMSO control-treated mice, whilst similarly low antigen distribution was present in 10 mg/kg and 30 mg/kg Tanomastat-treated mice. Mantel–Cox test or Kruskal–Wallis test was used to determine the statistical significance of the treatments when compared against DMSO vehicle control. P-values, HR, and 95% CI are reported in Supplementary Table S7 . Horizontal lines in scatter dot plots represent geometric mean ± geometric standard deviation with ∗ P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Data displayed are representative images of each group (n ≥ 7). Magnification for H&E and IHC staining are conducted at 10X.

Article Snippet: After blocking with 2% bovine serum albumin (BSA) (Sigma–Aldrich) dissolved in Tris-buffered saline-Tween 20, the membrane was incubated in the following primary antibodies: mouse anti-EV-A71 VP2 (Merck Millipore Cat#MAB979, RRID:AB_95300), rabbit anti-EV-A71 VP1 (Genetex Cat#GTX132339, RRID:AB_2886617), mouse anti-MMP-9 (Thermo Fisher Scientific Cat#MA5-15886, RRID:AB_11157246), and mouse anti- β -Actin (Sigma–Aldrich Cat#A2228, RRID:AB_476697).

Techniques: In Vivo, Inhibition, Infection, Control, Histopathology, Staining, Immunohistochemistry, Standard Deviation