Journal: PLoS Pathogens

Article Title: EV71 infection induces neurodegeneration via activating TLR7 signaling and IL-6 production

doi: 10.1371/journal.ppat.1008142

Figure Lengend Snippet: ( A ) The mice cerebral cortex sections from WT and TLR7 -/- mice mock-infected or EV71-infected for 3 or 5 days were subjected to hematoxylin-eosin (H&E) staining. The typical inflammatory cells infiltration or neurodegeneration were indicated by yellow arrows. The presentative images were acquired using light microscopy. Bar = 100 μm. ( B ) The WT and TLR7 -/- mice were sacrificed on 3, 5, and 7 days post-infection (each group, n = 3). The RNA was extracted from the brain tissues and the levels of IL-1β , IL-6 , TNF-α , and CXCL-1 mRNAs were measured by qPCR. Data are shown as mean ± SD. **, P < 0.01; ***, P < 0.001; ns, non-significant. ( C and D ) The WT and TLR7 -/- mice brain sections on 3 and 5 days post-infection were subjected to IHC staining with anti-IL-6 antibody (Brown) (C) and anti-cleaved-Caspase-3 (cl-Caspase-3) antibody (Brown) (D). The presentative images were acquired using light microscopy. Bar = 100 μm. ( E ) The proteins were extracted from individual mice brain tissues and then detected by Western blotting with targeted antibodies. Relative Caspase-3 or cl-Caspase-3 protein expression to internal control is quantified using Image J software. ( F ) The brain sections from mice on day 3 post EV71 incubation were subjected to immunostained with TUNNEL (Green) and DAPI (Blue). The presentative images were acquired using fluorescence microscopy. Bar = 20 μm. ( G ) The RNA was extracted from the brain tissues on day 3 and 5 post EV71 incubation. The levels of apoptosis-associated genes mRNAs were measured by qPCR. Data are shown as fold changes of RNA expression compared to mock samples (PBS WT group).

Article Snippet: Cell apoptosis inducer, etoposide (Catalog number: HY-13629), and TLR8 agonist, VTX-2337 (Catalog number: HY-13773) were purchased from MedchemExpress (NJ, USA).

Techniques: Infection, Staining, Light Microscopy, Immunohistochemistry, Western Blot, Expressing, Control, Software, Incubation, Fluorescence, Microscopy, RNA Expression

Journal: PLoS Pathogens

Article Title: EV71 infection induces neurodegeneration via activating TLR7 signaling and IL-6 production

doi: 10.1371/journal.ppat.1008142

Figure Lengend Snippet: ( A ) U251 cells were seeded on 20-mm cover slips and probed with mouse anti-GFAP or isotype IgG antibodies (Green) and DAPI (Blue). The cells were visualized by confocal laser scanning microscopy. Bar = 20 μm. ( B – G ) U251 cells (2×10 6 ) were transfected with 2 μg plasmid transcribing siRNA specific to TLR7 (shTLR7) or its control (shGFP) and then mock-infected or infected with EV71 (MOI = 0.5) for 12 or 24 h, respectively. The total mRNA of treated cells was extracted. TLR7 mRNA and EV71 VP1 RNA (B) as well as cytokines mRNA levels (C) were determined by qPCR. The supernatants of treated cells were collected and IL-1β and IL-6 protein levels were measured by ELISA (D). Cell viability and LDH release of treated cells for 12, 24 or 48 h were examined using CCK8 and LDH assay, respectively (E). Data are shown as mean ± SD and correspond to a representative experiment out of three performed. ns, non-significant; *, P < 0.05; **, P < 0.01. The RNA was extracted from the transfected cells at 12 or 24 h post EV71 infection. The levels of apoptosis-associated genes mRNAs were measured by qPCR (F). Data are shown as fold changes of RNA expression compared to mock samples. The protein was extracted from the transfected cells at 24 h post EV71 infection and then detected by Western blotting with targeted antibodies (G). Relative PARP protein expression to internal control is quantified using Image J software.

Article Snippet: Cell apoptosis inducer, etoposide (Catalog number: HY-13629), and TLR8 agonist, VTX-2337 (Catalog number: HY-13773) were purchased from MedchemExpress (NJ, USA).

Techniques: Confocal Laser Scanning Microscopy, Transfection, Plasmid Preparation, Control, Infection, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, RNA Expression, Western Blot, Expressing, Software

Journal: PLoS Pathogens

Article Title: EV71 infection induces neurodegeneration via activating TLR7 signaling and IL-6 production

doi: 10.1371/journal.ppat.1008142

Figure Lengend Snippet: ( A ) The schematic of IL-6 antibody neutralization upon EV71 infection. 2×10 6 U251 cells were blocked with neutralized mouse anti-human IL-6 antibody (0.5 μg/ml) or mouse isotype IgG (0.5 μg/ml) at 2 h post EV71 infection (MOI = 0.5). ( B ) The supernatants of treated cells were collected and IL-6 protein level was measured by ELISA. ( C and D ) Cell viability (C) and LDH release (D) of treated cells for 12, 24 or 48 h were examined using CCK8 and LDH assay, respectively. Data are shown as mean ± SD and correspond to a representative experiment out of three performed. ns, non-significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. ( E ) The RNA was extracted from the transfected cells at 12 or 24 h post EV71 infection. The levels of apoptosis-associated genes mRNAs were measured by qPCR. Data are shown as fold changes of RNA expression compared to mock samples. ( F ) The protein was extracted from the transfected cells at 24 h post EV71 infection and then detected by Western blotting with targeted antibodies. Relative PARP protein expression to internal control is quantified using Image J software.

Article Snippet: Cell apoptosis inducer, etoposide (Catalog number: HY-13629), and TLR8 agonist, VTX-2337 (Catalog number: HY-13773) were purchased from MedchemExpress (NJ, USA).

Techniques: Neutralization, Infection, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Transfection, RNA Expression, Western Blot, Expressing, Control, Software

Journal: PLoS Pathogens

Article Title: EV71 infection induces neurodegeneration via activating TLR7 signaling and IL-6 production

doi: 10.1371/journal.ppat.1008142

Figure Lengend Snippet: The neurotropic EV71 preferentially infects and replicates in astrocytes of mice and human brain cerebral cortex. EV71 ssRNA is then recognized by TLR7 to trigger the signaling events, which in turn dominantly promote IL-6 protein production in astrocytes. The release of IL-6 protein in EV71-infected cells affects astrocyte self and neighboring astrocytes, resulting in the induction of cell apoptosis. The dysfunctional astrocytes disrupt brain homeostasis, which systematically develops neural inflammation and injury in brain.

Article Snippet: Cell apoptosis inducer, etoposide (Catalog number: HY-13629), and TLR8 agonist, VTX-2337 (Catalog number: HY-13773) were purchased from MedchemExpress (NJ, USA).

Techniques: Infection

Journal: Molecular Cell

Article Title: DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination

doi: 10.1016/j.molcel.2016.12.016

Figure Lengend Snippet:

Article Snippet: Mouse-anti-cMyc (9E10) , Santa Cruz , sc-40.

Techniques: Recombinant, Transfection, Plasmid Preparation, Purification, Western Blot, Sequencing, Software, Imaging, Microscopy, Laser-Scanning Microscopy

Journal: Nature Communications

Article Title: SUMO conjugation to the pattern recognition receptor FLS2 triggers intracellular signalling in plant innate immunity

doi: 10.1038/s41467-018-07696-8

Figure Lengend Snippet: SUMOylation is required for BIK kinase release from activated FLS2-BAK1 immune complex to initiate immune signalling. a FLS2-GFP and FLS2 K/R –GFP interacts with BAK1-myc in a flagellin dependant manner. Nicotiana benthamiana leaves transiently expressing FLS2-GFP or FLS2 K/R -GFP with BAK1-myc were treated with MgCl 2 or 1 µM flg22 for 10 min. Subsequently, total protein was subjected to immunoprecipitation with anti-GFP immunoaffinity beads (IP: αGFP) followed by immunoblot analysis with anti-myc (IB: αmyc) antibodies to detect BAK1-myc and anti-GFP (IB: αGFP) antibodies to detect FLS2-GFP. BAK1 protein levels in all samples were determined by probing with anti-myc antibodies to detect BAK1-myc in total protein extracts (BAK1-myc input). GFP was used as a negative control. b Non-SUMOylatable FLS2 K/R -GFP remains bound to BIK1 after flagellin treatment. FLS2-GFP and FLS2 K/R -GFP were transiently expressed with BIK1-myc in N. benthamiana and coimmunoprecipitation and immunoblotting was done as described in ( a ) except that BIK1 was analysed by antibodies against c-myc. Total protein of all samples was probed with anti-myc antibody to determine BIK1 protein levels (BIK1-myc input). GFP was used as a negative control. c SUMOylation of FLS2 is required for the induction of immune related ROS burst. Leaf discs from three-week-old plants of Col-0, fls2 , and two transgenic lines each of FLS2-GFP, FLS2 K/R -GFP were treated with 1 µM of flg22 for 10 min and ROS burst detected. Results shown are average ± SE ( n = 3). d Non-SUMOylatable FLS2 K/R -GFP plants exhibit significantly reduced levels of MAPK activation. Different genotypes indicated were treated with water or 1 µM of flg22 for 10 min and total protein were extracted after treatment. Immunoblots were probed with anti-p44/42 MAPK antibodies to detect activated MPK3 and 6 (upper panel); the lower panel was immunoblotted with anti-MPK3 antibody to ensure equal protein loading

Article Snippet: Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibodies from Cell Signaling (Cat. No. #4370; dilution: 1:2000) were used according to the manufacturer’s protocol.

Techniques: Expressing, Immunoprecipitation, Western Blot, Negative Control, Transgenic Assay, Activation Assay

Journal: Nature Communications

Article Title: SUMO conjugation to the pattern recognition receptor FLS2 triggers intracellular signalling in plant innate immunity

doi: 10.1038/s41467-018-07696-8

Figure Lengend Snippet: Identification of SUMO components controlling FLS2-mediated immune signalling. a SCE1 interacts with FLS2. Nicotiana benthamiana leaves transiently expressing FLS2-GFP/FLS2 K/R -GFP with SCE1-HA were treated with MgCl 2 or 1 µM flg22 (10 min). Immunoprecipitation was done with anti-GFP beads (IP: αGFP) and immunoblotted with anti-HA (IB: αHA) for SCE1-HA and anti-GFP (IB: αGFP) for GFP/GFP-fusion proteins. Total protein extracts were probed with anti-HA antibody (SCE1-HA input) for equal SCE1. b Desi3a co-localizes with FLS2-GFP to plasma membrane. N. benthamiana leaves co-infiltrated with mCherry-Desi3a and FLS2-GFP or GFP were detected for fluorescence after 3 days. Before imaging, cells were plasmolysed with 1 M NaCl (1 h). Images were obtained using confocal laser scanning microscope Carl Zeiss 880. Scale bar = 10 µm. Arrows indicate overlap of mCherry-Desi3a and FLS2-GFP signals. c FLS2 interacts with Desi3a. Coimmunoprecipitation from N. benthamiana leaves expressing FLS2-GFP/FLS2 K/R -GFP or GFP with Desi3a-HA were performed. Total proteins were probed with anti-HA antibody for equal Desi3a (Desi3a-HA input). d Desi3a is a bona fide SUMO protease. High molecular conjugates of His-SUMO1 chains were incubated with GST-Desi3a (Desi3a WT ) or Desi3a C168S at 30 °C and subsequently immunoblotted with anti-SUMO1 (IB: αSUMO1) to detect SUMO chains (upper panel), and with anti-GST to detect GST-fusion proteins (lower panel). e FLS2 is hyper-SUMOylated in desi3a-1 . Immunoprecipitates (IP: αGFP) from transgenic lines expressing proFLS2::FLS-GFP in fls2 or desi3a-1 were probed with anti-GFP (IB: αGFP) or anti-SUMO1/2 (IB: αSUMO1) antibodies. f HyperSUMOylation of FLS2 enhances ROS burst. Leaf discs from three-week-old plants of Col-0, FLS2-GFP ( fls2 ), FLS2-GFP ( desi3a-1 ) transgenic lines were treated with 1 µM flg22 (10 min) and ROS burst detected. Results shown are average ± SE ( n = 3). g HyperSUMOylation of FLS2 increases MAPK activation. Col-0, FLS2-GFP ( fls2 ) and FLS2-GFP ( desi3a-1 ) transgenic lines were treated with water or 1 µM flg22 (10 min) and total proteins were probed with anti-p44/42 MAPK antibody to detect activated MPK3/6 (upper panel) and with anti-MPK3 antibody for equal protein loading. h flg22 degrades Desi3a. 10-day-old seedlings of 35 S::Desi3a-HA ( desi3a-1 ) transgenic lines were treated with 200 μM cycloheximide or a combination of 200 μM cycloheximide + 1 μM flg22 and total proteins extracted at indicated time-points were immunobloted with anti-HA (IB: αHA) antibody

Article Snippet: Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibodies from Cell Signaling (Cat. No. #4370; dilution: 1:2000) were used according to the manufacturer’s protocol.

Techniques: Expressing, Immunoprecipitation, Clinical Proteomics, Membrane, Fluorescence, Imaging, Laser-Scanning Microscopy, Incubation, Transgenic Assay, Activation Assay

Journal: Nature Communications

Article Title: SUMO conjugation to the pattern recognition receptor FLS2 triggers intracellular signalling in plant innate immunity

doi: 10.1038/s41467-018-07696-8

Figure Lengend Snippet: Desi3a functions downstream of FLS2 to regulate FLS2-mediated immune responses. a fls2 desi3a-1 seedlings revert desi3a-1 response to flagellin. A representative image of Col-0, fls2, desi3a-1 and fls2 desi3a-1 seedlings in 1/2MS or 1/2MS with 250 nM flg22. Scale Bar = 1 cm. b Quantification of root growth inhibition for Col-0, fls2 , desi3a-1 and fls2 desi3a-1 . Four-day-old seedlings of the four genotypes were transferred to 1/2MS or 1/2MS + 250 nM flg22 and grown for additional 6 days and root lengths were measured. Data presented are means ± SE from at least 25 individual seedlings per genotype (Bars with different letters were significantly different from others; P < 0.05; two-way ANOVA with post hoc Tukey test). c fls2 desi3a-1 shows reduced ROS production than desi3a-1 . Leaf discs from three-week-old plants of Col-0, fls2, desi3a-1 and fls2 desi3a-1 were treated with 1 µM flg22 (10 min) and ROS burst was detected. Results shown are average ± SE ( n = 3). d Enhanced MAPK activation in desi3a-1 reverts to fls2 levels in fls2 desi3a-1 . Different genotypes were treated with water or 1 µM flg22 (10 min) and total proteins extracted were immunoblotted with anti-p44/42 MAPK antibody (upper panel) to detect activated MPK3/6, and with anti-MPK3 antibody for equal protein loading (lower panel). e fls2 desi3a-1 shows enhanced sensitivity to bacterial pathogen Pst DC3000 than desi3a-1 . Four-week-old rosettes of Col-0, fls2 , desi3a-1 and fls2 desi3a-1 plants were dipped in liquid cultures of Pst . and leaves from rosettes infected with Pst . were photographed 5 days post-infection (dpi). f Quantification of colony-forming units (CFUs) from infected rosettes was counted at day 0 and 3 dpi. Values are means ± SE from three independent biological replicates ( N = 3; bars with different letters were significantly different from others; P < 0.05; two-way ANOVA with post hoc Tukey test). g A schematic of flg22-mediated SUMO conjugation of the FLS2 receptor during PAMP signalling. In the presence of flg22 (+PAMP) FLS2 SUMOylation facilitates the release of BIK1 from the FLS2-BAK1 immune complex. This is concomitant with the flg22-dependant degradation of Desi type SUMO protease, Desi3a

Article Snippet: Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibodies from Cell Signaling (Cat. No. #4370; dilution: 1:2000) were used according to the manufacturer’s protocol.

Techniques: Inhibition, Activation Assay, Infection, Conjugation Assay

Journal: Advanced Science

Article Title: An Insect Effector Mimics Its Host Immune Regulator to Undermine Plant Immunity

doi: 10.1002/advs.202409186

Figure Lengend Snippet: Interactions between OsGF14e and OsEDR1l. A) Yeast‐two hybrid (Y2H) assay of yeast strain co‐transformed with the indicated plasmids and spotted on quadruple dropout media with X‐a‐gal. Ala substitutions were made at R137 and Y138 positions of OsGF14e to generate OsGF14e (R137A/Y138A). B) Co‐immunoprecipitation (Co‐IP) analysis. OsEDR1l‐GFP and OsGF14e‐mCherry fusion protein were transiently co‐expressed in Nicotiana benthamiana . Protein extracts (input) were immunoprecipitated by mCherry‐trap (mRFP‐Trap) beads (IP) and resolved by SDS‐PAGE. Immunoblots were developed with mCherry antibody (AbmCherry) to detect OsGF14e‐mCherry protein (arrowheads) and with green fluorescent protein (GFP) antibody (AbGFP) to detect OsEDR1l‐GFP (asterisks). The Co‐IP assays were repeated three times. C) Bimolecular fluorescence complementation (BiFC) analysis to determine the interaction in the leaf cells of N. benthamiana . Fluorescence indicates reconstitution of an intact fluorescent protein from the complementary OsGF14e‐cYFP and OsEDR1l‐nYFP (yellow fluorescent protein). A previous studied OsEDR1 (ref. , LOC_Os03g06410) served as a negative control. D) Co‐localization of OsGF14e and OsEDR1l to the cytoplasm and nucleus. OsEDR1l‐GFP and OsGF14e‐mCherry fusion protein were co‐expressed in rice protoplasts by PEG‐mediated transformation. Confocal laser‐scanning microscopy was used to investigate their distribution 24 h after transformation. OsEDR1l (green) and OsGF14e (red) were mainly co‐localized in the cytoplasm and nucleus (yellow). E‐G) OsEDR1l expression in gf14e mutants, oeGF14e and WT plants (E), OsEDR1l phosphorylation level in vitro (F), OsMPK3/6 phosphorylation levels in gf14e mutant, oeGF14e and WT plants (G). In vitro analysis of OsEDR1l phosphorylation level was performed using recombinant OsEDR1l and oeGF14 proteins, and recombinant OsGF14e (R137A/Y138A) protein was used as the negative control (F). Phospho‐p44/42 MAPK (Thr202/Tyr204) antibodies were used for phosphorylation test, and the Western blot band density of phosphorylated OsMPK3/6 protein in gf14e mutant and oeGF14e plants (relative to WT) was quantified from three biological replicates using ImageJ software (G). The rice plants were infested by BPH for 12 h. CBB staining was shown as the protein loading control, OsEDR1l phosphorylation (F), Each assay was repeated three times. Different letters indicate a significant difference among treatments ( p < 0.05; one‐way ANOVA followed by Duncan's multiple range test). Error bars represent standard errors.

Article Snippet: The mixture was separated by 12% SDS‐PAGE, and the phosphorylated levels of OsMAPK3/6.blot were detected in western blot using Phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibodies (Cell Signaling Technology, Lane Danvers, MA, USA).

Techniques: Y2H Assay, Transformation Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Fluorescence, Negative Control, Confocal Laser Scanning Microscopy, Expressing, Phospho-proteomics, In Vitro, Mutagenesis, Recombinant, Software, Staining, Control

Journal: Advanced Science

Article Title: An Insect Effector Mimics Its Host Immune Regulator to Undermine Plant Immunity

doi: 10.1002/advs.202409186

Figure Lengend Snippet: Interactions between Nl14 and OsEDR1l. A) Y2H assay of yeast strain co‐transformed with the indicated plasmids and spotted on quadruple dropout medium with X‐a‐gal. Ala substitutions were made at R130 and Y131 positions of Nl14 to generate Nl14 (R130A/Y131A). B) Co‐IP analysis. OsEDR1l‐GFP and Nl14‐mCherry fusion protein were transiently co‐expressed in Nicotiana benthamiana . Protein extracts (input) were immunoprecipitated by mCherry‐trap (mRFP‐Trap) beads (IP) and resolved by SDS‐PAGE. Immunoblots were developed with mCherry antibody (AbmCherry) to detect Nl14‐mCherry protein (arrowheads) and with green fluorescent protein (GFP) antibody (AbGFP) to detect OsEDR1l‐GFP (asterisks). C) Bimolecular fluorescence complementation (BiFC) analysis to determine the interaction in vivo. Fluorescence indicates reconstitution of an intact fluorescent protein from the complementary Nl14 ‐cYFP and OsEDR1l‐nYFP (yellow fluorescent protein). A previous studied OsEDR1 (ref. , LOC_Os03g06410) served as a negative control. D‐F) OsEDR1l expression in oeNl14 and WT plants (D), OsEDR1l phosphorylation level in vitro (E), and OsMPK3/6 phosphorylation levels in oeNl14 and WT plants (F). In vitro analysis of OsEDR1l phosphorylation level was performed using recombinant OsEDR1l and Nl14 proteins, and the recombinant Nl14 (R130A/Y131A) protein was used as the negative control (E). Phospho‐p44/42 MAPK (Thr202/Tyr204) antibodies were used for OsMPK3/6 phosphorylation test, and the Western blot band density of phosphorylated OsMPK3/6 protein in oeNl14 plants (relative to WT) was quantified from three biological replicates using ImageJ software (F). The rice plants were infested by BPH for 12h. CBB staining was shown as the protein loading control. Asterisks indicate significant differences between WT and oeNl14 plants (*, p < 0.05; Student's t ‐test) with error bars representing standard errors.

Article Snippet: The mixture was separated by 12% SDS‐PAGE, and the phosphorylated levels of OsMAPK3/6.blot were detected in western blot using Phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibodies (Cell Signaling Technology, Lane Danvers, MA, USA).

Techniques: Y2H Assay, Transformation Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page, Western Blot, Fluorescence, In Vivo, Negative Control, Expressing, Phospho-proteomics, In Vitro, Recombinant, Software, Staining, Control