Journal: Antiviral research

Article Title: Antiviral activity of the HSP90 inhibitor VER-50589 against enterovirus 71.

doi: 10.1016/j.antiviral.2023.105553

Figure Lengend Snippet: Fig. 1. VER-50589 exhibits antiviral effects against EV71 in vitro. (A) Chemical structure of VER-50589. (B–E) RD and HeLa cells were cultured with five- fold dilutions of VER-50589 or ribavirin. After 48 h, cytotoxicity was assessed using an MTT assay. (F–H) RD cells were infected with EV71 (MOI = 0.1) for 2 h and incubated with five-fold dilutions of VER-50589 or ribavirin for 20 h. VP1 protein levels were measured using western blotting (F). EV71 mRNA levels were evaluated using qPCR (G, H). (l) Immu nofluorescence assay detected EV71 mRNA in EV71- infected RD cells treated with VER-50589. (J) EV71- infected RD cells were treated with different con centrations of VER-50589 for 48 h, and the cell freeze-thaw liquid was harvested for the plaque assay. (K)The number of infectious clones of the samples was calculated. (L–M) EV71-infected RD cells were treated with VER-50589 (4 μM) or ribavirin (4 μM) under the same experimental conditions. VP1 protein and viral mRNA levels were measured using western blotting and qPCR, respectively. ***P < 0.001; **P < 0.01; *P < 0.05.

Article Snippet: The following antibodies were used according to manufacturer’s recommendations: anti-phospho-AKT (CST, Cat: 4060S), anti-total AKT (CST, Cat: 4691S), anti-HSP90 (Abcom, Cat: ab208035), anti-phospho-ERK1/2 (Santa Cruz, Cat: sc-81492), anti-total ERK1/2 (Santa Cruz, Cat: sc-514302), anti-phospho-RAF (CST, Cat: 2969S), antitotal RAF (CST, Cat: 148145S), anti-total VP1 antibody (CST, cat: 9167), goat anti-rabbit IgG (Cat: SA00001-2, Proteintech, China), goat antimouse IgG (Cat: SA00001-1, Proteintech, China), β-actin (Cat: 60008- 1, Proteintech, China), and GAPDH (Cat: 10494-1, Proteintech, China).

Techniques: In Vitro, Cell Culture, MTT Assay, Infection, Incubation, Western Blot, Plaque Assay, Clone Assay

Journal: Antiviral research

Article Title: Antiviral activity of the HSP90 inhibitor VER-50589 against enterovirus 71.

doi: 10.1016/j.antiviral.2023.105553

Figure Lengend Snippet: Fig. 4. VER-50589 inhibits HSP90 and mediates AKT and RAF phosphorylation. (A–B) RD cells were transfected with HSP90-specific siRNA and cultured for 24 h. The p-AKT, AKT, RAF, p-RAF, and HSP90 levels were detected using western blotting (A). HSP90 mRNA levels were detected using qPCR (B). (C–D) RD cells were transfected with HSP90-specific siRNA for 24 h and infected with EV71 (MOI = 8) for 24 h. The VP1, p-AKT, AKT, p-RAF, RAF, and HSP90 levels were detected using western blotting (C). The mRNA levels of EV71 were detected using qPCR (D). (E) Cherry-HSP90 plasmid-carrying RD cells were infected with EV71 (MOI = 8) for 24 h. The expression levels of HSP90, p-AKT, AKT, p-RAF, RAF, and VP1 were detected using western blotting. (F) RD cells were infected with the EV71 virus with different MOI (0.01, 0.1, 1, and 10) and subjected to western blotting. (G) RD cells were infected with EV71 (MOI = 0.1) and harvested at 8 h, 12 h, 18 h, and 22 h. Protein levels were detected using western blotting. (H) RD cells were treated with five-fold dilution of VER-50589 and cultured for 20 h. Different protein levels were detected using western blotting. (I) RD cells were incubated with EV71 (MOI = 0.1) for 2 h, and the medium was replaced with one containing a five-fold series dilution of VER-50589 for another 20 h incubation. Proteins were extracted for western blotting analysis.

Article Snippet: The following antibodies were used according to manufacturer’s recommendations: anti-phospho-AKT (CST, Cat: 4060S), anti-total AKT (CST, Cat: 4691S), anti-HSP90 (Abcom, Cat: ab208035), anti-phospho-ERK1/2 (Santa Cruz, Cat: sc-81492), anti-total ERK1/2 (Santa Cruz, Cat: sc-514302), anti-phospho-RAF (CST, Cat: 2969S), antitotal RAF (CST, Cat: 148145S), anti-total VP1 antibody (CST, cat: 9167), goat anti-rabbit IgG (Cat: SA00001-2, Proteintech, China), goat antimouse IgG (Cat: SA00001-1, Proteintech, China), β-actin (Cat: 60008- 1, Proteintech, China), and GAPDH (Cat: 10494-1, Proteintech, China).

Techniques: Phospho-proteomics, Transfection, Cell Culture, Western Blot, Infection, Plasmid Preparation, Expressing, Virus, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Toxoplasma gondii dysregulates IFN-gamma-inducible gene expression in human fibroblasts: insights from a genome-wide transcriptional profiling.

doi: 10.4049/jimmunol.178.8.5154

Figure Lengend Snippet: FIGURE 4. IFN--inducible IRF1 expression in Toxoplasma- and Neospora-infected HFFs. A, Top pan- els are IFM images showing IRF1 expression (anti- IRF1 in red) in uninfected cells after IFN- treatment (100 U/ml) for the indicated amount of time. Same ex- posure time was used for all IRF1 images. Correspond- ing Hoechst nuclear staining is shown in middle panels. Bottom panels for 0- and 0.5-h time points are longer exposure images for the same cells shown at top and demonstrate the presence of IRF1 in the host cell nu- cleus. B, IFN--induced IRF1 expression in HFFs in- fected with GFP-expressing Toxoplasma strains RH, Pru, and CL14 representing types I, II, and III, respec- tively. HFFs were infected for 18 h and then treated with IFN- for 2 or 6 h. Top panels in each time point show parasite GFP (green) and anti-IRF1 (red); lower panels in each vertical pair are images after merging Hoechst nuclear staining (blue). C, IFN--induced IRF-1 expression (red) in HFFs infected with N. cani- num for 18 h. The NC-1 strain used lacks GFP expres- sion and infected cells can be identified in the phase images. D, Western blots showing basal expression lev- els of STAT1 (91 kDa), IRF1 (48 kDa), and SOCS1 (24 kDa), in HFFs uninfected or infected with indicated par- asite strains for 18 h at a MOI of 3. Approximately 70–80% of the cells were infected with one to three vacuoles per cell containing two to eight parasites per vacuole at the time of lysate preparation. GAPDH (36 kDa) was used as a loading control. E, Western blots showing SOCS1 expression levels in HFFs uninfected or infected with RH or Pru strains as in D following IFN- stimulation for 2 and 4 h. Data shown are rep- resentative of three experiments performed.

Article Snippet: Proteins were transferred to nitrocellulose membrane and probed with the following Abs: mouse monoclonal IgG against total STAT1; pTyr701STAT1 and IRF1 that were also used in IFM; rabbit polyclonal antipSer727-STAT1, anti-pTyr1022/1023-JAK1, and anti-pTyr1007/1008-JAK2 IgG (Cell Signaling Technology); rabbit polyclonal anti-suppressor of cytokine signaling (SOCS)1 IgG (Santa Cruz Biotechnology; clone H-93); and mouse monoclonal anti-GAPDH IgG (Calbiochem; clone 6C5).

Techniques: Expressing, Infection, Staining, Western Blot, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Toxoplasma gondii dysregulates IFN-gamma-inducible gene expression in human fibroblasts: insights from a genome-wide transcriptional profiling.

doi: 10.4049/jimmunol.178.8.5154

Figure Lengend Snippet: FIGURE 5. STAT1 nuclear trafficking and autoin- duction in Toxoplasma- and Neospora-infected HFFs following exposure to IFN-. A and B, IFM images of anti-total STAT1 staining using a mAb that recognizes STAT1 irrespective of its phosphorylation state. Unin- fected HFFs (A) and HFFs infected with a type II Tox- oplasma Pru for 18 h (MOI of 0.5) (B) are compared. Times indicate the duration of IFN- treatment. Phase images in B allow for the delineation of infected cells. White triangles indicate parasite vacuoles. C, Western blots with uninfected HFFs or HFFs infected with Pru strain for 18 h (MOI of 3) and then treated with IFN- for 24 h or left untreated. D, IFM for total STAT1 in HFFs infected with N. caninum for 18 h (MOI of 0.5). Triangles indicate parasite vacuoles.

Article Snippet: Proteins were transferred to nitrocellulose membrane and probed with the following Abs: mouse monoclonal IgG against total STAT1; pTyr701STAT1 and IRF1 that were also used in IFM; rabbit polyclonal antipSer727-STAT1, anti-pTyr1022/1023-JAK1, and anti-pTyr1007/1008-JAK2 IgG (Cell Signaling Technology); rabbit polyclonal anti-suppressor of cytokine signaling (SOCS)1 IgG (Santa Cruz Biotechnology; clone H-93); and mouse monoclonal anti-GAPDH IgG (Calbiochem; clone 6C5).

Techniques: Infection, Staining, Phospho-proteomics, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Toxoplasma gondii dysregulates IFN-gamma-inducible gene expression in human fibroblasts: insights from a genome-wide transcriptional profiling.

doi: 10.4049/jimmunol.178.8.5154

Figure Lengend Snippet: FIGURE 7. STAT1 phosphorylation at the Tyr701

Article Snippet: Proteins were transferred to nitrocellulose membrane and probed with the following Abs: mouse monoclonal IgG against total STAT1; pTyr701STAT1 and IRF1 that were also used in IFM; rabbit polyclonal antipSer727-STAT1, anti-pTyr1022/1023-JAK1, and anti-pTyr1007/1008-JAK2 IgG (Cell Signaling Technology); rabbit polyclonal anti-suppressor of cytokine signaling (SOCS)1 IgG (Santa Cruz Biotechnology; clone H-93); and mouse monoclonal anti-GAPDH IgG (Calbiochem; clone 6C5).

Techniques: Phospho-proteomics

Journal: Acta Neuropathologica Communications

Article Title: Kinesin light chain-1 serine-460 phosphorylation is altered in Alzheimer’s disease and regulates axonal transport and processing of the amyloid precursor protein

doi: 10.1186/s40478-019-0857-5

Figure Lengend Snippet: Characterisation of KLC1 serine-460 phospho-specific antibody. HEK293 cells were transfected with either control vector, FLAG-KLC1S460A or FLAG-KLC1wt and treated with either vehicle or okadaic acid (OA) as indicated. The different samples were then probed on immunoblots with antibodies to serine-460 phosphorylated KLC1 (KLC1-ser460p), FLAG to detect total KLC1 (FLAG-KLC1), active and total ERK1/2, and tubulin. The KLC1 serine-460 phospho-specific antibody detects KLC1wt but not KLC1S460A and signals with the antibody are increased in KLC1wt cells treated with okadaic acid that activates the KLC1 serine-460 kinase ERK1/2

Article Snippet: The following primary antibodies were used in this study: Mouse anti-total extracellular-signal-regulated kinase 1/2 (ERK1/2) (L34F12 Cell Signaling 1/2000), rabbit anti-active ERK1/2 (phosphorylated on threonine-202 and tyrosine-204) (Cell Signaling 1/2000), mouse anti-FLAG (M2 Sigma 1/2000), rabbit anti-total KLC1 (sc-25735/H-75 Santa Cruz 1/2000), mouse anti-neuron specific enolase (NSE Dako 1/50,000), mouse anti-tubulin (DM1A Sigma 1/20,000).

Techniques: Transfection, Control, Plasmid Preparation, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Kinesin light chain-1 serine-460 phosphorylation is altered in Alzheimer’s disease and regulates axonal transport and processing of the amyloid precursor protein

doi: 10.1186/s40478-019-0857-5

Figure Lengend Snippet: KLC1 levels are reduced and the relative levels of KLC1 serine-460 phosphorylation are increased in Alzheimer’s disease frontal cortex. Representative immunoblots showing total KLC1, KLC1 serine-460 phosphorylation (KLC1S460p) and NSE levels in post-mortem human control (Ctrl) and Alzheimer’s disease frontal cortex. Braak stages are indicated. Graphs show quantification of total KLC1 and KLC1 phosphorylated on serine-460 in the different samples. KLC1 and KLC1S460p signals were normalised to NSE levels. These normalised KLC1 levels were then used to quantify changes to total KLC1 and KLC1S460p (expressed as the ratio of KLC1S460p/total KLC1). Data were analysed by Welch’s ANOVA and Games-Howell post hoc test. N = 13–15, error bars are s.e.m., * p < 0.05 ** p < 0.01, *** p < 0.001, ns not significant

Article Snippet: The following primary antibodies were used in this study: Mouse anti-total extracellular-signal-regulated kinase 1/2 (ERK1/2) (L34F12 Cell Signaling 1/2000), rabbit anti-active ERK1/2 (phosphorylated on threonine-202 and tyrosine-204) (Cell Signaling 1/2000), mouse anti-FLAG (M2 Sigma 1/2000), rabbit anti-total KLC1 (sc-25735/H-75 Santa Cruz 1/2000), mouse anti-neuron specific enolase (NSE Dako 1/50,000), mouse anti-tubulin (DM1A Sigma 1/20,000).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Acta Neuropathologica Communications

Article Title: Kinesin light chain-1 serine-460 phosphorylation is altered in Alzheimer’s disease and regulates axonal transport and processing of the amyloid precursor protein

doi: 10.1186/s40478-019-0857-5

Figure Lengend Snippet: Mutation of KLC1 serine-460 to mimic permanent phosphorylation, inhibits axonal transport of APP in cultured rat cortical neurons. ( a ) Representative kymographs showing axonal transport of APP-EGFP in APP-EGFP+KLC1wt and APP-EGFP+KLC1S460D co-transfected neurons; scale bar and times are indicated. Bar charts show % for total, anterograde and retrograde APP-EGFP movement. N = 17 EGFP+KLC1wt and 20 APP-EGFP+KLC1S460D co-transfected neurons. Statistical significance was determined by Student’s t-test. Error bars are s.e.m.; ** p < 0.01; ns not significant. ( b ) Violin plots show velocities of APP-EGFP movement in anterograde and retrograde directions in the different transfected cells. Median and interquartile ranges are indicated by hashed lines. N = 163 anterogradely and 62 retrogradely moving APP-EGFP in KLC1wt and 179 anterogradely and 54 retrogradely moving APP-EGFP in KLC1S460D co-transfected cells. Statistical significance was determined by Mann-Whitney U test; ns not significant

Article Snippet: The following primary antibodies were used in this study: Mouse anti-total extracellular-signal-regulated kinase 1/2 (ERK1/2) (L34F12 Cell Signaling 1/2000), rabbit anti-active ERK1/2 (phosphorylated on threonine-202 and tyrosine-204) (Cell Signaling 1/2000), mouse anti-FLAG (M2 Sigma 1/2000), rabbit anti-total KLC1 (sc-25735/H-75 Santa Cruz 1/2000), mouse anti-neuron specific enolase (NSE Dako 1/50,000), mouse anti-tubulin (DM1A Sigma 1/20,000).

Techniques: Mutagenesis, Phospho-proteomics, Cell Culture, Transfection, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: Kinesin light chain-1 serine-460 phosphorylation is altered in Alzheimer’s disease and regulates axonal transport and processing of the amyloid precursor protein

doi: 10.1186/s40478-019-0857-5

Figure Lengend Snippet: Mutation of endogenous Drosophila KLC serine-433 to mimic permanent phosphorylation inhibits axonal transport of APP in wing sensory neurons. ( a ) Alignment showing high conservation of the amino acid sequences encompassing rat KLC1 serine-460 and the homologous Drosophila KLC serine-433 (indicated in red). ( b ) CRISPR genome editing approach to mutate KLC serine-433 to aspartic acid. Drosophila Klc sequence (upper line) along with the ssODN (lower line) are shown. Serine-433 codon (KLC) and aspartic acid codon (ssODN) are shown in red, the protospacer sequence in grey shade and the PAM site in blue shade. A G-to-A transition is introduced to mutate the PAM. ( c ) Representative kymographs showing axonal transport of APP-YFP in Klcwt and KlcS433D homozygous backgrounds in 2-day old Drosophila ; scale bar and times are indicated. Bar chart shows the relative proportions of stationary, anterograde and retrograde moving APP-YFP in the Klcwt background. ( d ) Bar charts show total, anterograde and retrograde APP-YFP movement runs in Klcwt and KlcS433D backgrounds. ( e ) Violin plots show velocities of APP-YFP runs in anterograde and retrograde directions in the different backgrounds. Median and interquartile ranges are indicated by hashed lines. N = 8 wings for each genotype. For velocity studies, N = 577 anterogradely and 347 retrogradely moving APP-YFP in Klcwt , and 343 anterogradely and 247 retrogradely moving APP-YFP in the KlcS433D background. Statistical significance was determined by one-way ANOVA with Holm-Sidak’s multiple comparison test in ( c ), Mann-Whitney U test in ( d ) and two-tailed Student’s t test in ( e ). Error bars are s.e.m.; **p < 0.01; **** p < 0.0001; ns not significant

Article Snippet: The following primary antibodies were used in this study: Mouse anti-total extracellular-signal-regulated kinase 1/2 (ERK1/2) (L34F12 Cell Signaling 1/2000), rabbit anti-active ERK1/2 (phosphorylated on threonine-202 and tyrosine-204) (Cell Signaling 1/2000), mouse anti-FLAG (M2 Sigma 1/2000), rabbit anti-total KLC1 (sc-25735/H-75 Santa Cruz 1/2000), mouse anti-neuron specific enolase (NSE Dako 1/50,000), mouse anti-tubulin (DM1A Sigma 1/20,000).

Techniques: Mutagenesis, Phospho-proteomics, CRISPR, Sequencing, Comparison, MANN-WHITNEY, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Kinesin light chain-1 serine-460 phosphorylation is altered in Alzheimer’s disease and regulates axonal transport and processing of the amyloid precursor protein

doi: 10.1186/s40478-019-0857-5

Figure Lengend Snippet: Expression of KLC1 serine-460 to mimic permanent phosphorylation promotes amyloidogenic processing of APP. ( a ) APP processing was monitored in HEK293 cells essentially as described using an APP-GAL4 reporter assay that drives GAL4-UAS-dependent expression of firefly luciferase . Luciferase signals were normalised to co-transfected Renilla signals to correct for transfection efficiency. Bar chart shows relative luciferase signals in cells transfected with the GAL4-UAS-luciferase+Renilla reporters plus either APP-GAL4 + KLC1wt or APP-GAL4 + KLC1S460D as indicated. N = 15 from 3 independent experiments. Data were analysed by Student’s t test, error bars are s.e.m., * p < 0.05. ( b ) Expression of KLC1S460D increases Aβ production. Levels of Aβ (1–38), Aβ (1–40) and Aβ (1–42) we quantified in conditioned media from cells co-transfected with APP+KLC1wt or APP+ KLC1S460D. N = 7; data were analysed by Student’s t test, error bars are s.e.m., * p < 0.05

Article Snippet: The following primary antibodies were used in this study: Mouse anti-total extracellular-signal-regulated kinase 1/2 (ERK1/2) (L34F12 Cell Signaling 1/2000), rabbit anti-active ERK1/2 (phosphorylated on threonine-202 and tyrosine-204) (Cell Signaling 1/2000), mouse anti-FLAG (M2 Sigma 1/2000), rabbit anti-total KLC1 (sc-25735/H-75 Santa Cruz 1/2000), mouse anti-neuron specific enolase (NSE Dako 1/50,000), mouse anti-tubulin (DM1A Sigma 1/20,000).

Techniques: Expressing, Phospho-proteomics, Reporter Assay, Luciferase, Transfection