Journal: Journal of Cell Science

Article Title: SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

doi: 10.1242/jcs.193698

Figure Lengend Snippet: EV71 enhances SIRT1 expression and translocation. (A) A diagram of the EV71 genome structure. EV71 genome contains a single ORF flanked by a 5′UTR and a 3′UTR. The ORF encodes a 250-kDa polyprotein that is processed into P1, P2 and P3 regions, which are further cleaved into mature proteins (VP1 to VP4, 2A to 2C, and 3A to 3D pol ). (B) RD cells were infected with EV71 at a multiplicity of infection (MOI) of 5 for different times. Photographs of infected cells were taken using a digital camera (at 100× magnification). (C–E) RD cells were infected with EV71 at an MOI of 5 for different times (C). RD cells were infected with EV71 for 12 h at different MOI (D). SK-N-SH A372 cells were infected with EV71 at an MOI of 5 for different times (E). The relative amount of SIRT1 and VP1 mRNAs were determined by qRT-PCR (upper panels). SIRT1 and VP1 proteins were detected by western blot analyses using corresponding antibodies (lower panels). (F,G) RD cells (F) and SK-N-SH A372 cells (G) were infected with EV71 at an MOI of 5 for 6 h. Cytoplasm extracts (CE) and nuclear extracts (NE) were prepared. SIRT1, β-actin and lamin A were detected by western blot analyses using corresponding antibodies. Each experiment was performed in triplicate wells and repeated at least three times. The intensity of western blot bands signals were quantified with Image J. RI, relative intensity.

Article Snippet: Human rhabdomyosarcoma (RD) cells and human neuroblastoma (SK-N-SH or A375) cells were purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China) and cultured in modified Eagle's medium (MEM).

Techniques: Expressing, Translocation Assay, Infection, Quantitative RT-PCR, Western Blot

Journal: Journal of Cell Science

Article Title: SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

doi: 10.1242/jcs.193698

Figure Lengend Snippet: SIRT1 is sumoylated with Sumo1 and EV71 facilitates SIRT1 sumoylation. (A) RD cells were harvested and incubated in RIPA buffer for 20 min. Cell lysates were centrifuged at 20,000 g for 15 min to remove cellular debris. IgG, anti-Sumo1 antibody, anti-Sumo2+3 antibody (i.e. an antibody that could recognize both Sumo-2 and Sumo-3) or protein G (IP) was added to supernatants for immunoprecipitation (IP). Sumoylated SIRT1 (Sumo-SIRT1) and SIRT1 were detected with an anti-SIRT1 antibody (IB). (B) RD cells were co-transfected with pcDNA3.1-SIRT1 and plasmid expressing HA–Sumo1, HA–Sumo2 or HA–Sumo3. Cell lysates were prepared and immunoprecipitated with anti-HA antibody. Precipitated sumoylated SIRT1 and SIRT1 were detected with an anti-SIRT1 antibody. (C) RD cells were infected with EV71 for different times. Infected cell lysates were prepared for western blotting using anti-SIRT1 or anti-VP1 antibodies. (D) RD cells were infected with EV71 for different times. Cell lysates were prepared and immunoprecipitated with anti-SIRT1 antibody, and Sumo–SIRT1 was detected with anti-Sumo1 antibody. (E) RD cells were infected with EV71 for different times. Whole-cell lysates (WCL) were prepared for detecting EV71 replication (top). Nuclear extracts (NE) (middle) and cytoplasm extracts (CE) (bottom) were prepared. The levels of Sumo–SIRT1, SIRT1, lamin A and GAPDH were determined by western blot analyses with the corresponding antibodies. Each treatment was repeated three or more times. The intensity of the western blot signals was quantified with Image J. RI, relative intensity.

Article Snippet: Human rhabdomyosarcoma (RD) cells and human neuroblastoma (SK-N-SH or A375) cells were purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China) and cultured in modified Eagle's medium (MEM).

Techniques: Incubation, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Infection, Western Blot

Journal: Journal of Cell Science

Article Title: SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

doi: 10.1242/jcs.193698

Figure Lengend Snippet: SIRT1 inhibits EV71 replication in cytoplasm of infected cells. (A) RD cells were transfected with pcDNA3.1(+)-SIRT1 at 0, 0.5, 1, 1.5 and 2 µg for 24 h and infected with EV71 at an MOI of 5 for 12 h. Cell lysates were prepared. SIRT1, EV71 VP1 and β-actin were detected by western blot analyses with corresponding antibodies. (B) RD cells were transfected with siR-Ctrl, siR-SIRT1#1 or siR-SIRT1#2, for 24 h, and infected with EV71 at an MOI of 5 for 12 h. SIRT1, VP1 and β-actin in cell lysates were detected by western blot analysis. (C) 293T and RD cells were transfected with plasmids expressing wild-type SIRT1 (WT-SIRT1) and SIRT1 with a mutant NLS (mtNLS-SIRT1). The cells were fixed, permeabilized and immunostained with antibody against SIRT1 (a,d,g,j), with Cy3-conjugated goat anti-rabbit-IgG used as a secondary antibody. The nucleus was stained with DAPI (b,e,h,k). The immunofluorescence results were analyzed by confocal laser-scanning microscopy. (D) RD cells were transfected with plasmids expressing mtNLS-SIRT1 at 0, 0.5, 1 and 1.5 µg for 24 h, and infected with EV71 infected at an MOI of 5 for 12 h. VP1 and β-actin in cell lysates were detected by western blot analysis. (E) RD cells were transfected with plasmids expressing mtNLS-SIRT1 at different concentrations for 24 h, and infected with EV71 at an MOI of 5 for 12 h or (F) RD cells were transfected with siR-Ctrl, siR-SIRT1#1 or siR-SIRT1#2 at 5 µM for 24 h, and infected with EV71 at an MOI of 5 for 12 h. Cells were harvested and total mRNA was isolated by using Trizol. The levels of GAPDH mRNA, EV71 VP1 double-strand RNA, positive-strand RNA and negative-strand RNA were determined by qRT-PCR. Ratios of positive-strand RNA to GAPDH mRNA, positive-strand RNA to GAPDH mRNA and negative-strand RNA to GAPDH mRNA were calculated. Results are mean±s.e.m. ( n =5). The intensity of western blot signals was quantified with Image J. RI, relative intensity.

Article Snippet: Human rhabdomyosarcoma (RD) cells and human neuroblastoma (SK-N-SH or A375) cells were purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China) and cultured in modified Eagle's medium (MEM).

Techniques: Infection, Transfection, Western Blot, Expressing, Mutagenesis, Staining, Immunofluorescence, Confocal Laser Scanning Microscopy, Isolation, Quantitative RT-PCR

Journal: Journal of Cell Science

Article Title: SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

doi: 10.1242/jcs.193698

Figure Lengend Snippet: SIRT1 inhibits EV71 genome RNA replication by interacting with 3D pol . (A) RD cells were infected with EV71 or not (Mock) at an MOI of 5 for 8 h or 12 h, fixed, permeabilized, and immunostained with antibody against 3D pol (a,e,i) or SIRT1 (b,f,j). Nuclei were stained by DAPI (c,g,k). FITC-conjugated donkey anti-mouse-IgG or Cy3-conjugated goat anti-rabbit-IgG was used as a secondary antibody. Immunofluorescence was detected by confocal laser-scanning microscopy. (B) Schematic diagram of the truncated EV71 3D pol constructs used in this study, with amino acid numbers indicated. DNA fragments containing mutant EV71 3D pol genes (3D-NT1, 3D-NT2, 3D-NT3, 3D-NT4 and 3D-NT5) were sub-cloned into plasmid peGFP-C1 to generate plasmids peGFP-C1-3D-NT1 to -NT5, respectively. N-terminal and C-terminal domains are indicated. (C) 293T cells were co-transfected with pcDNA3.1(+)-SIRT1 and peGFP-C1, peGFP-C1-3D pol and peGFP-C1-3D-NT1–NT5 for 24 h. Cell extracts were prepared for co-immunoprecipitation (IP) assays using anti-GFP antibody and precipitated with protein G. Interactions between SIRT1 and EV71 proteins were determined by western blotting using anti-SIRT1 antibody or anti-GFP antibody. (D) Purified GST–3D pol was incubated with p300 protein in acetylation assay buffer. Acetylated GST–3D pol (0.02 µg) was incubated with His–SIRT1 (0.02 µg) in deacetylation assay buffer at 37°C for 30 min. The levels of acetylated (ac-K) GST–3D pol and nonacetylated GST–3D pol were determined by western blotting with anti-acetylated-lysine antibody or anti-GST antibody. (E–G) Purified GST or GST–3D pol were incubated in acetylation assay buffer with p300 protein; then, acetylated GST and GST–3D pol were incubated with His–SIRT1 in deacetylation assay buffer at 37°C for 30 min (E,F), or acetylated GST-3D pol was incubated with His–H363Y-SIRT1 in deacetylation assay buffer for 30 min at 37°C (G). Mixtures were applied onto glutathione–Sepharose columns, gently rotated at 4°C for 3 h, incubated in RNA elongation assay buffer with DIG-UTP and then spotted onto Hybond-N membrane. Synthetic RNAs were detected using a Luminescent Image Analyzer. (H) RD cells were incubated with DMSO, resveratrol (an activator of SIRT1), EX-527 (an inhibitor of SIRT1) or nicotinamide (an inhibitor of SIRT1) for 6 h, and infected with EV71 at an MOI of 5 for 10 h. The levels of VP1 and β-actin were determined by western blot analysis. The intensity of the western blot signals was quantified with Image J. RI, relative intensity.

Article Snippet: Human rhabdomyosarcoma (RD) cells and human neuroblastoma (SK-N-SH or A375) cells were purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China) and cultured in modified Eagle's medium (MEM).

Techniques: Infection, Staining, Immunofluorescence, Confocal Laser Scanning Microscopy, Construct, Mutagenesis, Clone Assay, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Purification, Incubation, Acetylation Assay, Membrane

Journal: Journal of Cell Science

Article Title: SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

doi: 10.1242/jcs.193698

Figure Lengend Snippet: SIRT1 binds directly to EV71 5′UTR, but not 3′UTR. (A–C) Cell extracts of RD, 293T or SK-N-SH cells were prepared and used as inputs, or incubated with no RNA, biotin-16-UTP, non-biotinylated EV71 5′UTR or biotinylated EV71 5′UTR (A). RD cell lysates were prepared and used as input, or were incubated with nonbiotinylated EV71 3′UTR RNA or biotinylated EV71 3′UTR RNA (B). RD cell lysates were prepared and used as input, or were incubated with biotinylated EV71 3′UTR RNA along with different concentrations of non-biotinylated EV71 3′UTR RNA or nonbiotinylated yeast tRNA (C). Protein–RNA pulldown assays were carried out with anti-SIRT1 antibody and precipitated with protein G. Interactions between SIRT1 and EV71 5′UTR were determined by western blotting with anti-SIRT1 antibody. (D) RD cells were infected with EV71 at an MOI of 10 for 12 h. Cell extracts were prepared and used for mRNA RNA extraction (Total RNA), or were used for protein–RNA pulldown assays with anti-SIRT1 antibody, anti-Flag antibody, without antibody or with water and followed by mRNA extraction. Then standard RT-PCR analysis using primers specific to EV71 5′UTR RNA or ribosomal protein S16 (RPS16) was performed.

Article Snippet: Human rhabdomyosarcoma (RD) cells and human neuroblastoma (SK-N-SH or A375) cells were purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China) and cultured in modified Eagle's medium (MEM).

Techniques: Incubation, Western Blot, Infection, RNA Extraction, Extraction, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Cell Science

Article Title: SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

doi: 10.1242/jcs.193698

Figure Lengend Snippet: SIRT1 is colocalized with EV71 RNA in cytoplasm. (A) RD cells were infected or not (Mock) with EV71 at an MOI of 5 for 8 or 12 h. Cells were fixed, permeabilized and immunostained with antibody against EV71 dsRNA (a,e,i) or SIRT1 (b,f,j). FITC-conjugated donkey anti-mouse-IgG or Cy3-conjugated goat anti-rabbit-IgG was used as a secondary antibody. Nuclei were stained with DAPI (c,g,k). m and n show enlargements of h and l. Immunofluorescence was detected using a confocal laser-scanning microscopy. (B) SK-N-SH cells were infected with EV71 at an MOI of 20 for 12 h. Cells were fixed, permeabilized and immunostained with antibody against EV71 dsRNA (a,e) or SIRT1 (b,f). FITC-conjugated donkey anti-mouse-IgG or Cy3-conjugated goat anti-rabbit-IgG was used as a secondary antibody. Nuclei were stained with DAPI (c,g). i shows an enlargement of h. Immunofluorescence was detected with a confocal laser-scanning microscopy. (C) A proposed mechanism underlying the regulation of EV71 replication.

Article Snippet: Human rhabdomyosarcoma (RD) cells and human neuroblastoma (SK-N-SH or A375) cells were purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China) and cultured in modified Eagle's medium (MEM).

Techniques: Infection, Staining, Immunofluorescence, Confocal Laser Scanning Microscopy

Journal: Cancers

Article Title: Oncogenic and Stemness Signatures of the High-Risk HCMV Strains in Breast Cancer Progression

doi: 10.3390/cancers14174271

Figure Lengend Snippet: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. ( A ) Time-course of the viral titer in the supernatant of MRC5 infected with the strains HCMV-B544 and HCMV-B693, as measured by IE1-qPCR. ( B ) Confocal microscopic images of HCMV-IE1 and pp65 staining in HMECs infected with HCMV-B544 and HCMV-B693 (day 1 post-infection). Uninfected HMECs were used as controls. Nuclei were counterstained with DAPI; magnification ×63, scale bar 10 μm. ( C ) HMECs time-course infection with HCMV-B544 and HCMV-B693 strains (MOI = 1). Magnification ×100, scale bar 100 μm. Uninfected HMECs were used as a control. ( D ) Presence of giant cells with blastomere-like morphology (1 and 6), mesenchymal cells (4 and 7), lipid droplet-packed cells (3, 8, and 9), cells displaying multiple nuclei (2) as well as cell budding (4, 5, and 6), and cells with filopodia protrusions (9) in CTH-B544 and CTH-B693 cells. The inverted light microscope scale bar represents 100 µm; magnification ×200.

Article Snippet: HMECs (A10565, Life Technologies, Carlsbad, CA, USA), CTH cells, MDA-MB231 as well as MCF7 (Institut Hiscia, Arlesheim, Switzerland), and MRC5 (RD-Biotech, Besançon, France) were cultured as previously described [ ].

Techniques: Infection, Staining, Light Microscopy

Journal: Virus Research

Article Title: Propagation and immunological characterization of coxsackievirus A10 in a serum-free HEK293A cell culture system

doi: 10.1016/j.virusres.2023.199101

Figure Lengend Snippet: Propagation profiles of CVA10 in serum-free roller bottle culture. HEK293A cells were infected with CVA10 at an MOI of 10 −4 . (A) Morphology of serum-free HEK293A cells in roller bottle. (B) Morphology of HEK293A cells after CVA10 infection. (C) TCID 50 values of three roller bottles after counting CPE in infected RD cells. The TCID 50 values were calculated using the Reed-Muench method. The bar represents 100 µm.

Article Snippet: Rhabdomyosarcoma (RD) cells and Madin-Darby Canine Kidney (MDCK) cells were obtained from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan.

Techniques: Infection, Endpoint Dilution Assay

Journal: Virus Research

Article Title: Propagation and immunological characterization of coxsackievirus A10 in a serum-free HEK293A cell culture system

doi: 10.1016/j.virusres.2023.199101

Figure Lengend Snippet: Propagation profiles of CVA10 in serum-free suspension culture. HEK293A cells were infected with CVA10 at an MOI of 10 −4 . (A) Suspension HEK293A cells in a spinner. (B) Suspension HEK293A cells after CVA10 infection. (C) TCID 50 values of four spinners after counting CPE in the infected RD cells. (D) Extracellular and intracellular TCID 50 values of serum-free bioreactor culture after counting CPE in infected RD cells. The TCID 50 values were calculated using the Reed-Muench method. The bar represents 100 µm.

Article Snippet: Rhabdomyosarcoma (RD) cells and Madin-Darby Canine Kidney (MDCK) cells were obtained from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan.

Techniques: Suspension, Infection, Endpoint Dilution Assay

Journal: Virus Research

Article Title: Propagation and immunological characterization of coxsackievirus A10 in a serum-free HEK293A cell culture system

doi: 10.1016/j.virusres.2023.199101

Figure Lengend Snippet: Propagation profiles of other HFMD-related viruses in serum-free HEK293A cell culture. HEK293A cells were infected with each virus (CVA6 and EV-A71) at MOI = 10 −4 . The culture and harvest conditions were identical to that described in the Method for CVA10 propagation. (A) Morphology of HEK293A cells after virus infection in roller bottle at 6 DPI. (B) Morphology of suspension HEK293A cells after virus infection in spinner at 4 DPI. (C) TCID 50 values of serum-free roller bottle cultures after counting CPE in infected RD cells. (D) TCID 50 values of serum-free spinner cultures after counting CPE in infected RD cells. The TCID 50 values were calculated using the Reed-Muench method. The bar represents 100 µm.

Article Snippet: Rhabdomyosarcoma (RD) cells and Madin-Darby Canine Kidney (MDCK) cells were obtained from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan.

Techniques: Cell Culture, Infection, Virus, Suspension, Endpoint Dilution Assay