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: The Journal of general virology

Article Title: Epstein-Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes.

doi: 10.1099/vir.0.82741-0

Figure Lengend Snippet: Fig. 1. STAT1 is tyrosine-phosphorylated in LCLs after IFN-a stimulation, but can bind DNA in the absence of stimulation. (a) Total cell lysates were generated from three cell lines: Kit 225, KEM LCL and EB LCL. These cell lines were either unstimulated or incubated with IFN-a (1000 IU) for 30 min. These lysates were analysed by SDS-PAGE and Western blotting using antibodies specific to phospho-STAT1 (Y701), pan-STAT1 and LMP1. The Kit 225 T-cell line was used as a positive control for the presence of tyrosine- phosphorylated STAT1 following IFN-a stimulation, and LMP1 detection was used as a positive marker for EBV. (b) STAT1 DNA binding was measured in the BL41, BL41+B95.8 and IARC-171 cell lines by using a DNA-affinity precipitation assay. These cell lines were either unstimulated or incubated with IFN-a (1000 IU) for 30 min. DNA-bound proteins were analysed by SDS-PAGE and Western blotting using antibodies specific to phospho-STAT1 (Y701) and pan-STAT1. Typically, 1107 cell equivalents were applied to each lane of the gel. These results are representative of four separate experiments. (c) STAT1 DNA binding was measured in the BL41, BL41+B95.8 and IARC-171 cell lines by using an EMSA. These cell lines were either unstimulated or incubated with IFN-a (1000 IU) for 30 min. Nuclear extract (10 mg) was then incubated with 2 ng 32P-radiolabelled GRR oligonucleotide or SIE oligonucleotide probe. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradio- graphy. Only protein–DNA complexes are shown, as free probe has been removed from the figure. The arrow indicates a specific protein–DNA complex. The results shown are representative of three separate experiments.

Article Snippet: Antibodies to phospho-STAT1 (Y701) (sc-7988-R), phospho-STAT1 (S727) (sc-16570-R), pan-STAT1 (sc-346) and phospho-ERK1/2 (Y204) (sc-7383) ERK1/2 (sc-93) were from Santa Cruz Biotechnology and were used at a concentration of 0.2 mg ml21.

Techniques: Generated, Incubation, SDS Page, Western Blot, Positive Control, Marker, Binding Assay, Affinity Precipitation

Journal: The Journal of general virology

Article Title: Epstein-Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes.

doi: 10.1099/vir.0.82741-0

Figure Lengend Snippet: Fig. 2. EBV induces a constitutive STAT1 DNA-binding complex that is capable of stimulating transcriptional activation without requiring tyrosine phosphorylation. (a) Supershift analysis of protein–DNA complexes was measured in unstimulated and IFN-a- treated IARC-171 LCL cells. Nuclear protein (10 mg) was pre-incubated for 30 min with 2 mg STAT1 supershift antibody (sc- 592 X) prior to incubation with 2 ng 32P-radiolabelled GRR oligonucleotide or SIE oligonucleotide probe. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradiography. The results shown are representative of two separate experiments. (b) The specificity of STAT1–DNA complexes was measured in unstimulated and IFN-a-treated IARC-171 LCL cells. Nuclear protein (10 mg) was pre-incubated for 30 min with 2 mg STAT1 supershift antibody (sc-592 X) or 100 ng cold GRR oligonucleotide prior to incubation with 2 ng 32P-radiolabelled GRR oligonucleotide or mGRR oligonucleotide probe. Protein–DNA complexes were then separated by using a native 4 % polyacrylamide gel and were visualized by autoradiography. (c) Supershift of protein–DNA complexes was measured in unstimulated and IFN-a-treated IARC-171 LCL cells. Nuclear protein (10 mg) was pre-incubated for 30 min with 2 mg STAT1 supershift antibody (BD #610119) prior to incubation with 2 ng 32P-radiolabelled GRR oligonucleotide. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradiography. Arrows indicate specific protein–DNA and supershifted protein–DNA complexes. The results shown are representative of two separate experiments. (d) STAT transcriptional activation was measured in IARC-171 LCL cells by using a STAT reporter assay. Cells (1107) were transfected with 20 mg empty vector-luc reporter, 5 mg GRR (5)-luc reporter, 10 mg GRR (5)-luc reporter or 20 mg GRR (5)-luc reporter. One microgram of phRL-SV40 reporter was also co-transfected and luciferase activity was assayed 20 h post-transfection. Relative luciferase activity was calculated as a ratio of firefly over Renilla luciferase. The results are mean values representative of at least three experiments. Error bars indicate 1 SEM.

Article Snippet: Antibodies to phospho-STAT1 (Y701) (sc-7988-R), phospho-STAT1 (S727) (sc-16570-R), pan-STAT1 (sc-346) and phospho-ERK1/2 (Y204) (sc-7383) ERK1/2 (sc-93) were from Santa Cruz Biotechnology and were used at a concentration of 0.2 mg ml21.

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Incubation, Autoradiography, Reporter Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: The Journal of general virology

Article Title: Epstein-Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes.

doi: 10.1099/vir.0.82741-0

Figure Lengend Snippet: Fig. 3. LMP1 induces STAT1 protein expression, nuclear translo- cation and DNA binding without triggering tyrosine phosphoryla- tion. (a) LMP1 expression was measured in stable DG75 transfectants (with inducible LMP1 expression) following removal of 1 mg tetracycline ml”1. Total lysates were generated from cells that were washed five times in RPMI 1640 medium and recultured in the presence of tetracycline (+) or absence of tetracycline for either 72 h (”3) or 96 h (”4). IARC-171 LCLs were used as a positive control for LMP1. These lysates were analysed by SDS- PAGE and Western blotting using antibodies specific to LMP1 and actin. Typically, 2105 cells were applied to each lane of the gel. These results are representative of three experiments. (b) STAT1 tyrosine phosphorylation and nuclear expression were measured in stable DG75 transfectants with inducible LMP1 expression. These cells were recultured in the presence of tetracycline (+) or absence of tetracycline for either 72 h (”3) or 96 h (”4). Cells were also incubated with IFN-a (1000 IU) for 30 min or left unstimulated. STAT1 tyrosine phosphorylation and nuclear expression were also measured in unstimulated IARC-171 LCL cells. Nuclear extracts were generated and were then analysed by SDS-PAGE and Western blotting using antibodies specific to phospho-STAT1 (Y701), pan-STAT1 and actin. Nuclear extract (10 mg) was applied to each lane of the gel. These results are representative of three experiments. (c) STAT1 DNA binding was measured in stable DG75 transfectants with inducible LMP1 expression by using an EMSA. These cells were recultured in the presence of tetracycline (+) or absence of tetracycline for either 72 h (”3) or 96 h (”4). STAT1 DNA binding was also measured in IARC-171 LCL cells. Cells were incubated with IFN-a for 30 min or left unstimulated. Nuclear extract (10 mg) was incubated with 2 ng 32P-radiolabelled GRR oligonucleotide probe. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradiography. Only the protein–DNA complexes are shown, as free probe has been removed from the figure. The arrow indicates a specific protein– DNA complex. The results shown are representative of three separate experiments.

Article Snippet: Antibodies to phospho-STAT1 (Y701) (sc-7988-R), phospho-STAT1 (S727) (sc-16570-R), pan-STAT1 (sc-346) and phospho-ERK1/2 (Y204) (sc-7383) ERK1/2 (sc-93) were from Santa Cruz Biotechnology and were used at a concentration of 0.2 mg ml21.

Techniques: Expressing, Binding Assay, Generated, Positive Control, SDS Page, Western Blot, Phospho-proteomics, Incubation, Autoradiography

Journal: The Journal of general virology

Article Title: Epstein-Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes.

doi: 10.1099/vir.0.82741-0

Figure Lengend Snippet: Fig. 4. STAT1 is constitutively serine-phosphorylated in LCLs, but lacks detectable lysine acetylation. (a) STAT1 immunoprecipitates were generated from two B-cell lines, DG75 and IARC-171 LCL. These cell lines were either unstimulated or incubated with IFN-a (1000 IU) for 30 min. Beads only and irrelevant antibody (Irr. Ab; ATF-3) controls were also incubated with nuclear extracts of untreated IARC-171 LCL cells. STAT1 immunoprecipitates were then analysed by SDS-PAGE and Western blotting using antibodies specific to phospho-STAT1 (S727) and pan-STAT1. Typically, 5106 cell equivalents were loaded in each lane of the gel. These results are representative of three separate experiments. (b) The presence of serine- phosphorylated STAT1 in DNA-bound protein complexes was measured in unstimulated and IFN-a-treated IARC-171 LCL cells. Nuclear protein (10 mg) was pre-incubated for 30 min with 2 mg phospho-STAT1 (S727) antibody prior to incubation with 2 ng 32P-radiolabelled GRR oligonucleotide. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradiography. Arrows indicate specific protein–DNA and supershifted protein–DNA complexes. The results shown are representative of two separate experiments. (c) STAT1 immunoprecipitates were generated from IARC-171 LCL cells that were unstimulated, incubated with IFN-a (1000 IU) for 30 min and/or treated with trichostatin A (TSA) (2 mM) for 24 h. STAT1 immunoprecipitates were then analysed by SDS-PAGE and Western blotting using antibodies specific to acetyl- lysine and pan-STAT1. Typically, 5106 cell equivalents were loaded in lanes 1–6 of the gel, and 2.5105 cell equivalents of nuclear extracts from unstimulated and TSA-treated IARC-171 LCL cells were loaded in lanes 7 and 8 as controls. These results are representative of four separate experiments.

Article Snippet: Antibodies to phospho-STAT1 (Y701) (sc-7988-R), phospho-STAT1 (S727) (sc-16570-R), pan-STAT1 (sc-346) and phospho-ERK1/2 (Y204) (sc-7383) ERK1/2 (sc-93) were from Santa Cruz Biotechnology and were used at a concentration of 0.2 mg ml21.

Techniques: Generated, Incubation, SDS Page, Western Blot, Autoradiography

Journal: The Journal of general virology

Article Title: Epstein-Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes.

doi: 10.1099/vir.0.82741-0

Figure Lengend Snippet: Fig. 5. STAT1 is serine-phosphorylated downstream of PI3K and MEK and seems to restrict IFN-stimulated STAT1 DNA binding. (a) Total cell lysates were generated from IARC-171 LCL cells incubated for 24 h with different combinations of PD98059 and LY294002. These combinations were: PD98059 (50 mM) alone; LY294002 (20 mM) alone; and PD98059 (50 mM)+LY294002 (20 mM). Total cell lysates were incubated with DMSO for 24 h as a control. These lysates were then analysed by SDS-PAGE and Western blotting using antibodies specific to phospho-STAT1 (S727), pan-STAT1, phospho- ERK1/2 (Y204), pan-ERK1/2, phospho-S6, pan-S6 and actin. Typically, 5105 cells were applied to each lane of the gel. These results are representative of four experiments. (b) The effect of serine phosphorylation on STAT1 DNA binding was measured in IARC-171 LCL cells by using an EMSA. These cells were unstimulated, treated with a combination of PD98059 (50 mM) and LY294002 (20 mM) for 24 h and/or incubated with IFN-a (1000 IU) for 30 min. Nuclear extract (10 mg) was then incubated with 2 ng 32P-radiolabelled GRR oligonucleotide probe. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradiography. Nuclear extract (10 mg) was also analysed by SDS-PAGE and Western blotting using antibodies specific to STAT1 and actin. This demonstrates that the nuclear levels of STAT1 were equal in each sample analysed. The results shown are representative of five separate experiments.

Article Snippet: Antibodies to phospho-STAT1 (Y701) (sc-7988-R), phospho-STAT1 (S727) (sc-16570-R), pan-STAT1 (sc-346) and phospho-ERK1/2 (Y204) (sc-7383) ERK1/2 (sc-93) were from Santa Cruz Biotechnology and were used at a concentration of 0.2 mg ml21.

Techniques: Binding Assay, Generated, Incubation, Control, SDS Page, Western Blot, Phospho-proteomics, Autoradiography

Journal: AIDS

Article Title: HIV-1 trans-activator protein dysregulates IFN-γ signaling and contributes to the suppression of autophagy induction

doi: 10.1097/qad.0b013e328340fd61

Figure Lengend Snippet: Fig. 2. Inhibition of STAT1 phosphorylation by fludarabine in primary human blood macrophages. (a) Primary human blood macrophages were treated with the indicated doses of fludarabine for 24 h, and cell viability was measured by MTT assay. The percentage cell proliferation was compared with the viability of the untreated cells. The data presented are expressed as the mean SD on blood cells from three inde- pendent donors. (b) Primary human blood macrophages were treated with fludarabine at the indicated doses for 24 h, followed by IFN-g (100 U/ml) challenge for another 30 min. Cell lysates were examined by western blot using anti- phosphor-STAT1 (p-STAT1) or anti-total STAT1 (STAT1) anti- bodies, as indicated. Total STAT1 was probed as the loading control. The results shown are representative of three inde- pendent experiments using blood macrophages from different individuals. (c) The western blot was quantified by laser densitometry. The intensities of the phosphor-STAT1 were normalized to the corresponding total STAT1. The ratio of p- STAT1/STAT1 in each sample was compared with that in cells that did not undergo fludarabine and IFN-g treatment. The data are expressed as the mean SD of three independent experiments using macrophages from different individuals. A P value less than 0.01 () is considered to represent statistical significance.

Article Snippet: Specific antibodies for LC3B I&II, pSTAT1 (Tyr701), and total STAT1 were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Inhibition, Phospho-proteomics, MTT Assay, Western Blot, Control

Journal: AIDS

Article Title: HIV-1 trans-activator protein dysregulates IFN-γ signaling and contributes to the suppression of autophagy induction

doi: 10.1097/qad.0b013e328340fd61

Figure Lengend Snippet: Fig. 4. HIV-1 Tat suppressed IFN-g induced STAT1 phosphorylation and autophagy. (a) PBMac were treated with HIV-1 Tat (100ng/ml) for 4 h, followed by IFN-g (100 U/ml) incubation for the indicated time intervals. Cell lysates were examined by western blot using anti-phosphor-STAT1 or anti-total STAT1 antibodies, as indicated. Total STAT1 was probed as the loading control. The levels of phosphorylation were normalized with total STAT1. The level of change in individual treatments with reference to the untreated cells is shown. ‘Fold change’ refers to the measurement of the indicated band by laser densitometry. The results shown are representative of three independent experiments using macrophages from different individuals. (b) Primary human blood macrophages were treated with HIV-1 Tat at the indicated concentrations for 4 h, followed by IFN-g (100 U/ml) challenge for another 16 h. Cell lysates were examined by western blot using anti-LC3B or anti-actin antibodies, as indicated. The level of actin was probed as the loading control. The results shown are representative of five independent experiments using macrophages from different individuals. (c) The western blot was quantified by laser densitometry. The intensities of the LC3B-II were normalized to the corresponding actin. The ratio of LC3B-II/actin in each sample was compared with that in cells that did not undergo fludarabine and IFN-g treatment. The data are expressed as the mean SD of five independent experiments using macrophages from different individuals. A P value below 0.01 () is considered to represent statistical significance. (d) Primary blood macrophages were untreated (UT) or treated with IFN-g (100 U/ml) for 16 h after pretreatment with or without HIV-1 Tat (50 or 100 ng/ml). Cells were fixed and stained with LC3B antibodies and DAPI. Images were taken with a Carl Zeiss fluorescence microscope system. Representative images from four fields per well are shown. (e) A cell with autophagosomes was defined as a cell containing five or more LC3B punctas. The bullets in the figure represent the data from each individual experiment. The numbers in the figure are expressed as the mean SD of five independent experiments using macrophages from different individuals. A P value below 0.01 () is considered to represent statistical significance. Untreated (UT), Tat 100 ng/ml (Tat), IFN-g 100 U/ml (IFN-g).

Article Snippet: Specific antibodies for LC3B I&II, pSTAT1 (Tyr701), and total STAT1 were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Phospho-proteomics, Incubation, Western Blot, Control, Staining, Microscopy

Journal: AIDS

Article Title: HIV-1 trans-activator protein dysregulates IFN-γ signaling and contributes to the suppression of autophagy induction

doi: 10.1097/qad.0b013e328340fd61

Figure Lengend Snippet: Fig. 6. HIV-1 Tat perturbed IFN-g-induced autophagosome formation. IFN-g activated STAT1 phosphorylation led to the formation of an autophagosome and the retention of myco- bacteria. HIV-1 Tat can suppress IFN-g-induced STAT1 phos- phorylation, and may further suppress autophagosome formation.

Article Snippet: Specific antibodies for LC3B I&II, pSTAT1 (Tyr701), and total STAT1 were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Phospho-proteomics, Bacteria

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Utilizes a Nontraditional Signal Transducer and Activator of Transcription 1 Activation Cascade via Signaling through Epidermal Growth Factor Receptor and Integrins To Efficiently Promote the Motility, Differentiation, and Polarization of Infected Monocytes

doi: 10.1128/jvi.00622-17

Figure Lengend Snippet: Figure 1. STAT1 is upregulated following HCMV infection of monocytes. (A) 1004

Article Snippet: Western blot analyses were performed using primary antibodies specific 261 for phosphorylated and non-phosphorylated forms of STAT1 (phospho-STAT1 (Y701) 262 antibody (Cell Signaling Technology, Beverly, MA), phospho-STAT1 (S727) antibody 263 (Cell Signaling Technology), pan-STAT1 (Cell Signaling Technology), Lamin A/C 264 (Sigma), -tubulin (Santa Cruz), and actin (Santa Cruz).

Techniques: Infection

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Utilizes a Nontraditional Signal Transducer and Activator of Transcription 1 Activation Cascade via Signaling through Epidermal Growth Factor Receptor and Integrins To Efficiently Promote the Motility, Differentiation, and Polarization of Infected Monocytes

doi: 10.1128/jvi.00622-17

Figure Lengend Snippet: Figure 2. STAT1 is activated by HCMV infection of monocytes. (A) Monocytes 1020

Article Snippet: Western blot analyses were performed using primary antibodies specific 261 for phosphorylated and non-phosphorylated forms of STAT1 (phospho-STAT1 (Y701) 262 antibody (Cell Signaling Technology, Beverly, MA), phospho-STAT1 (S727) antibody 263 (Cell Signaling Technology), pan-STAT1 (Cell Signaling Technology), Lamin A/C 264 (Sigma), -tubulin (Santa Cruz), and actin (Santa Cruz).

Techniques: Infection

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Utilizes a Nontraditional Signal Transducer and Activator of Transcription 1 Activation Cascade via Signaling through Epidermal Growth Factor Receptor and Integrins To Efficiently Promote the Motility, Differentiation, and Polarization of Infected Monocytes

doi: 10.1128/jvi.00622-17

Figure Lengend Snippet: Figure 3. STAT1 activation in infected monocytes occurs in an IFN-independent 1035

Article Snippet: Western blot analyses were performed using primary antibodies specific 261 for phosphorylated and non-phosphorylated forms of STAT1 (phospho-STAT1 (Y701) 262 antibody (Cell Signaling Technology, Beverly, MA), phospho-STAT1 (S727) antibody 263 (Cell Signaling Technology), pan-STAT1 (Cell Signaling Technology), Lamin A/C 264 (Sigma), -tubulin (Santa Cruz), and actin (Santa Cruz).

Techniques: Activation Assay, Infection

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Utilizes a Nontraditional Signal Transducer and Activator of Transcription 1 Activation Cascade via Signaling through Epidermal Growth Factor Receptor and Integrins To Efficiently Promote the Motility, Differentiation, and Polarization of Infected Monocytes

doi: 10.1128/jvi.00622-17

Figure Lengend Snippet: Figure 5. The expression pattern of ISGs associated with the STAT1 pathway is 1090

Article Snippet: Western blot analyses were performed using primary antibodies specific 261 for phosphorylated and non-phosphorylated forms of STAT1 (phospho-STAT1 (Y701) 262 antibody (Cell Signaling Technology, Beverly, MA), phospho-STAT1 (S727) antibody 263 (Cell Signaling Technology), pan-STAT1 (Cell Signaling Technology), Lamin A/C 264 (Sigma), -tubulin (Santa Cruz), and actin (Santa Cruz).

Techniques: Expressing

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Utilizes a Nontraditional Signal Transducer and Activator of Transcription 1 Activation Cascade via Signaling through Epidermal Growth Factor Receptor and Integrins To Efficiently Promote the Motility, Differentiation, and Polarization of Infected Monocytes

doi: 10.1128/jvi.00622-17

Figure Lengend Snippet: Figure 6: STAT1 is required for HCMV-induced monocyte motility. (A and B) 1105

Article Snippet: Western blot analyses were performed using primary antibodies specific 261 for phosphorylated and non-phosphorylated forms of STAT1 (phospho-STAT1 (Y701) 262 antibody (Cell Signaling Technology, Beverly, MA), phospho-STAT1 (S727) antibody 263 (Cell Signaling Technology), pan-STAT1 (Cell Signaling Technology), Lamin A/C 264 (Sigma), -tubulin (Santa Cruz), and actin (Santa Cruz).

Techniques:

Journal: Retrovirology

Article Title: Innate immune defects in HIV permissive cell lines

doi: 10.1186/s12977-016-0275-8

Figure Lengend Snippet: Defects in 3 selected innate immunity pathways in cell lines. a The figure represents a simplified view of the TLR7/TLR8, IFN-gamma and TNF-alpha signaling pathways. Boxes representing genes display the transcriptional levels detected in RNA-seq libraries of resting CD4+ T cells, the four human laboratory cell lines HEK293T, Jurkat, SupT1 and CEM -mock (MO), heat-inactivated (HI) and HIV-infected (HIV)- and 4 samples corresponding to Activated CD4+ T cells at 8 and 24 h after TCR activation. Inset describes the order of the libraries as well as the color-code scale of expression levels (log10 transformation of the number of library size-normalized reads per kilobase of exonic sequence) ranging from 0 ( green ) to ≥2.8 ( red ; lower limit of the 9th-decile of expression values). The expression levels indicated for IFNG and TNF convey the basal expression level before adding the stimuli. b Experimental validation of the functional integrity of the selected innate immunity pathways. The table reports the stimuli applied and the functional read-out measured 24 h after stimulation. A positive sign indicates positive detection of functional read-outs (transcript levels by RT-qPCR or phosphorylation of STAT1 by Western blot analysis). NT not tested, nd not detected

Article Snippet: Total phospho-Stat1 (Mouse anti-P-Y701 Stat1, 1:1000, #612132, BD Biosciences) and Stat1 (Rabbit anti-Stat1 antibody, 1:1000, #9172, Primary Cell Signaling Technology) were detected using standard procedures with Tris-Buffered Saline (TBS)-0.2 % Tween-5 % BSA and PBS-0.2 % Tween-5 % milk as blocking buffers for Phospho-Stat1 and Stat1 respectively.

Techniques: RNA Sequencing Assay, Infection, Activation Assay, Expressing, Transformation Assay, Sequencing, Functional Assay, Quantitative RT-PCR, Western Blot

Journal: Retrovirology

Article Title: Innate immune defects in HIV permissive cell lines

doi: 10.1186/s12977-016-0275-8

Figure Lengend Snippet: Heatmap of expression values of paradigmatic genes involved in antiretroviral defense and signaling relevant to HIV biology. The figure shows the expression values of antiretroviral genes ( APOBEC3G, TRIM5, BST2, MX2, GBP5, and SAMHD1 ) and signaling genes ( JAK, STAT1, IFI16 and STING/TMEM173 ) in RNA-seq libraries of resting CD4+ T cells, cell lines HEK293T, Jurkat, SupT1 and CEM -mock (Mock), heat-inactivated (hiLV) and HIV-infected (LV)- and activated CD4+ T cells at 8 and 24 h after TCR activation . The color-code scale in the inset represents the expression levels (log10 transformation of the number of library size-normalized reads per kilobase of exonic sequence), ranging from green (low) to red (high) expression. Complete hierarchical clustering of genes was based on Pearson correlation of the expression levels. Complete hierarchical clustering of samples was kept as assessed in Additional file : Figure S4. JAK1, JAK2, TMEM173 and GBP5 had a fold change higher than 2 between resting CD4+ T cells and permissive cell lines (Benjamini–Hochberg adjusted p value <0.01, )

Article Snippet: Total phospho-Stat1 (Mouse anti-P-Y701 Stat1, 1:1000, #612132, BD Biosciences) and Stat1 (Rabbit anti-Stat1 antibody, 1:1000, #9172, Primary Cell Signaling Technology) were detected using standard procedures with Tris-Buffered Saline (TBS)-0.2 % Tween-5 % BSA and PBS-0.2 % Tween-5 % milk as blocking buffers for Phospho-Stat1 and Stat1 respectively.

Techniques: Expressing, RNA Sequencing Assay, Infection, Activation Assay, Transformation Assay, Sequencing

Journal: Oncoimmunology

Article Title: Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling

doi: 10.1080/2162402X.2017.1321185

Figure Lengend Snippet: Simultaneous downregulation of PTP1B and TC-PTP enhances moDCs maturation. (A) Comparisons between double heterozygous (1B:TC fl/wt ) and TC fl/wt moDCs, 1B fl/wt moDCs and wt moDCs respectively. MFI values of the expression of MHC class I, CD86 and CD80 ( n = 5). (B) Production of Th1 polarizing cytokines ( n = 5): (B) IL-12 and (C) IFNγ. (D) Quantification of IFNγ produced by activated OT-I CD8 + T cells co-cultured with mature and OVA-pulsed 1B:TC fl/wt, 1B fl/wt , TC fl/wt or wt moDCs for 48 h ( n = 8–9). (E) Quantification of IFNγ produced by activated OT-II CD4 + T cells co-cultured with mature and OVA-pulsed 1B:TC fl/wt , 1B fl/wt , TC fl/wt or wt moDCs for 48 h ( n = 5). (F) Activation status of Src kinase and IkBa downstream of TLR4 ( n = 3). Significant differences are represented by p values and (*) indicate significant differences between wt DCs and the rest of the groups. (G) Activation status of STAT1, STAT4 and Src in STAT1 inhibitor (fludarabine) or STAT4 inhibitor (lisofylline) treated or non-treated 1B:TC fl/wt , 1B fl/wt , TC fl/wt or wt moDCs ( n = 3). Production of Th1-polarizing cytokines by 1B:TC fl/wt , 1B fl/wt , TC fl/wt or wt moDCs previously treated with STAT1- or STAT4-inhibitor during the maturation process ( n = 3): (H) IL-12 and (I) IFNγ. The results are representative of at least three independent experiments. Significant differences among the groups are represented by p values and (*) indicate significant differences within a group. The comparisons were determined using One-Way ANOVA (Holm–Sidak multiple comparison test) for parametric and Dunn's multiple test for non-parametric.

Article Snippet: The following proteins were detected using antibodies from Cell Signaling Technologies (CST) phospho-specific and total STAT1 (total-stat1 CST cat# 9172 and phospho-stat1 CST cat# 9134), STAT3 (total-stat3 CST cat# 9139 and phosphor-stat3 CST cat# 9145), STAT5 (total-stat5 CST cat# 9310 and phosphor-stat5 CST cat# 9356), STAT4 (total-stat4 CST cat# 2653 and phosphor-stat4 CST cat# 5267), Src (total-Src CST cat# 2109 and phospho-Src CST cat# 2101), IkBα (total-IkBα CST #4812 and phospho-IkBα CST #2859), ERK (total-ERK CST cat# 9102 and phospho-ERK CST cat# 9106, p38 (total-p38 CST #9212 and phospho-p38 CST cat# 9211) and c-Jun (total-cJun CST cat# 9165 and phospho-cJun CST cat# 9261) proteins were detected using polyclonal antibodies (Cell Signaling Technology). β-Actin and Calnexin (a gift from Dr. John J.M.

Techniques: Expressing, Produced, Cell Culture, Activation Assay

Journal: Oncoimmunology

Article Title: Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling

doi: 10.1080/2162402X.2017.1321185

Figure Lengend Snippet: Pharmacological inhibition of both PTP1B and TC-PTP with specific inhibitor. (A) Specificity of 1B/TC inh (50 μM) to inhibit PTP1B and TC-PTP dephosphorylation ( n = 3). (B) Titration of 1B/TC inh in mouse moDCs using IL-12 production as indicator of activation ( n = 3). (C) IFNγ production by 1B/TC inh-treated mature moDCs (4 μM dose) ( n = 4). (D) Expression levels (MFI) of CD40, CD80, CD86, MHC class I and II on 1B/TC inh-treated mature moDCs (4 μM dose) ( n = 3). (E) Activation status STAT1 and STAT4 ( n = 3). (F) Activation status of Src kinase and IkBα ( n = 4). Therapeutic properties of 1B/TC inh-treated moDCs in a mouse model of E.G7 lymphoma: (G) Tumor volume before moDC treatment (10 d after implantation of 5 × 10 5 E.G7-OVA cells) and (H) after intraperitoneal (IP) injections of 5 × 10 6 1B/TC inh-treated or non-treated moDCs compared with control group (tumor-bearing mice without moDC treatments). The IP injections with moDCs were given 18 d after tumor implantation. (I) Images represent tumor-bearing mice 8 d after of moDC treatment. The results are representative of at least three independent experiments. The differences in more than two groups were determined using One-Way ANOVA (Holm–Sidak multiple comparison test) for parametric and Dunn's multiple test for non-parametric. The differences between two groups were determined with unpaired t test (two tails of distribution). Significant differences are represented by p values <0.05.

Article Snippet: The following proteins were detected using antibodies from Cell Signaling Technologies (CST) phospho-specific and total STAT1 (total-stat1 CST cat# 9172 and phospho-stat1 CST cat# 9134), STAT3 (total-stat3 CST cat# 9139 and phosphor-stat3 CST cat# 9145), STAT5 (total-stat5 CST cat# 9310 and phosphor-stat5 CST cat# 9356), STAT4 (total-stat4 CST cat# 2653 and phosphor-stat4 CST cat# 5267), Src (total-Src CST cat# 2109 and phospho-Src CST cat# 2101), IkBα (total-IkBα CST #4812 and phospho-IkBα CST #2859), ERK (total-ERK CST cat# 9102 and phospho-ERK CST cat# 9106, p38 (total-p38 CST #9212 and phospho-p38 CST cat# 9211) and c-Jun (total-cJun CST cat# 9165 and phospho-cJun CST cat# 9261) proteins were detected using polyclonal antibodies (Cell Signaling Technology). β-Actin and Calnexin (a gift from Dr. John J.M.

Techniques: Inhibition, De-Phosphorylation Assay, Titration, Activation Assay, Expressing, Tumor Implantation

Journal: Oncoimmunology

Article Title: Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling

doi: 10.1080/2162402X.2017.1321185

Figure Lengend Snippet: Application of 1B/TC inh-treated human DCs in cancer immunotherapy. (A) Activation status of STAT1 and STAT4 in 1B/TC inh-treated human moDCs derived from a pancreatic cancer (PaC) patient ( n = 3). (B) IL-12 production by 1B/TC inh-treated human moDCs from four PaC patients. (C) Frequency of antigen-specific (CEA and CA19–9) activated T cells measured based on IFNγ production detected in ELISpot assay. The differences in more than two groups were determined using One-Way ANOVA (Holm–Sidak multiple comparison test) for parametric and Dunn's multiple test for non-parametric. The differences between two groups were determined with unpaired t -test (two tails of distribution). Significant differences are represented by p values <0.05.

Article Snippet: The following proteins were detected using antibodies from Cell Signaling Technologies (CST) phospho-specific and total STAT1 (total-stat1 CST cat# 9172 and phospho-stat1 CST cat# 9134), STAT3 (total-stat3 CST cat# 9139 and phosphor-stat3 CST cat# 9145), STAT5 (total-stat5 CST cat# 9310 and phosphor-stat5 CST cat# 9356), STAT4 (total-stat4 CST cat# 2653 and phosphor-stat4 CST cat# 5267), Src (total-Src CST cat# 2109 and phospho-Src CST cat# 2101), IkBα (total-IkBα CST #4812 and phospho-IkBα CST #2859), ERK (total-ERK CST cat# 9102 and phospho-ERK CST cat# 9106, p38 (total-p38 CST #9212 and phospho-p38 CST cat# 9211) and c-Jun (total-cJun CST cat# 9165 and phospho-cJun CST cat# 9261) proteins were detected using polyclonal antibodies (Cell Signaling Technology). β-Actin and Calnexin (a gift from Dr. John J.M.

Techniques: Activation Assay, Derivative Assay, Enzyme-linked Immunospot