Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 1 TRIM21 attenuates the JEV mediated upregulation of the p-IRF3 level and IFN-β level in human microglial cells. (A) PCR amplification of TRIM21 and TRIM21 (ΔRING) primers was carried out and the product run on 1% agarose gel (upper panel). Expression of wild-type TRIM21 as well as the TRIM21 (ΔRING) domain was confirmed by Western blotting (lower panel). (B) CHME3 cells were transfected with 4 μg of TRIM21 plasmid or TRIM21 (ΔRING) for 48 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21, anti-IRF-3 and anti-β- tubulin antibodies by Western blotting. Representative image is shown. (C) Cells transfected with TRIM21 or TRIM21 (ΔRING) were infected with JEV, and total RNA was isolated post 48 h of transfection. Real-time PCR for IFN-β1 was performed, and an average of three independent sets of experiments is plotted and shown. (D) Luciferase assay for IFN-β for cells transfected with TRIM21 or TRIM21 (ΔRING) and infected with JEV along with respective controls was performed. Luciferase activity normalized against β-gal activity was averaged and plotted (*p <0.05, **p < 0.01, ***p < 0.001 from control).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Amplification, Agarose Gel Electrophoresis, Expressing, Western Blot, Transfection, Plasmid Preparation, SDS Page, Infection, Isolation, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Control

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 4 JEV induces TRIM21 protein levels in human microglial cells in a time-dependent manner. (A) CHME3 cells were either un-infected or infected with JEV at MOI 5 for 6, 12, 24 and 36 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21 antibody and anti-β- tubulin antibody (loading control) by Western blotting. A representation of three independent experiments is shown along with densitometry analysis with normalization of TRIM21 against β-tubulin, averaging and plotting. (B) CHME3 cells were infected with JEV for 24 h (MOI 5). Total RNA was isolated from harvested cells. cDNA prepared by reverse transcription of control and infected samples was used as a template for qPCR against primers for TRIM21 gene. Average fold change in the TRIM21 mRNA level from three independent experiments is plotted and shown (*p < 0.05, **p < 0.01, ***p < 0.001 from control, #p from 6 h, $p from 12 h).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Infection, SDS Page, Control, Western Blot, Isolation, Reverse Transcription

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 5 TRIM21 knockdown facilitates JEV-mediated IRF3 activation and upregulation of the IFN-β level. (A) Cells were either transfected with negative control RNA (NC) or transfected with 10nM siRNA against TRIM21 for 48 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21 antibody and anti-β-tubulin antibody by Western blotting. A representative image is shown. (B) Cells were either non-transfected (C), transfected with negative control RNA (NC) or with TRIM21 siRNA for 24 h followed by JEV infection for 24 h. Cell lysates were resolved on SDS-PAGE and probed with anti-p-IRF3 antibody, anti-IRF3 and anti-β-tubulin antibodies (loading control) by Western blotting. A representative of three independent experiments is shown. Densitometry analyses of Western blot experiments were performed with normalizing p-IRF-3 and p-IRF-3 against β-tubulin. (C) Real-time PCR for IFN-β1 for siRNA-transfected and JEV-infected cells along with the respective controls was performed and averaged for three independent sets of experiments. (D) Luciferase assay for IFN-β for cells transfected with siRNA against TRIM21 and infected with JEV along with respective controls was performed. Luciferase activity normalized against β-gal activity was averaged and plotted (**p < 0.01, ***p < 0.001 from control).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Knockdown, Activation Assay, Transfection, Negative Control, SDS Page, Western Blot, Infection, Control, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 6 Model showing a plausible role of TRIM21 as a negative regulator of IRF3 activation and IFN-β production following JEV infection in human microglial cells. JEV infection causes activation of the RIG-1 receptor, initiating a downstream signaling mechanism leading to the activation of IRF-3. Phosphorylated IRF-3 dimerizes and translocates into the nucleus, where it leads to the transcription and production of IFN-β. JEV infection also induces the TRIM21 protein, which negatively regulates IRF-3 phosphorylation, leading to reduced IFN-β production. The upregulation of TRIM21 is proposed to be a feedback mechanism to inhibit the innate immune response in JEV infection.

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Activation Assay, Infection, Phospho-proteomics

Journal: BMC Cancer

Article Title: Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B Leukemic Cells

doi: 10.1186/1471-2407-11-268

Figure Lengend Snippet: JSI-124-induced apoptosis was not mediated by c-Jun activation in B-cell malignancies . A . BJAB, I-83, and NALM-6 cells were pre-treated with 20 μM SP600125 followed by 1 μM JSI-124. Decreased levels of phospho-and total c-Jun protein levels were detected by western blotting in the cells treated with the combination of SP600125 and JSI-124 compared to SP600125 alone. Representative original data from three independent experiments are shown. The XIAP and STAT3proteins level, as determined by immunoblotting with antibodies against XIAP and serine phosphor-STAT3/STAT3, did not change with this treatment B . Apoptotic cell population was measured using flow cytometric analysis for AnnexinV/PI staining for apoptosis after 24 hour treatment with JSI-124, either alone or in combination with SP600125. DMSO-treated cells were taken as the control. Cells that were 7-AAD-negative and Annexin V-positive were undergoing apoptosis. The percentage of cells in each quadrant is indicated in the quadrant. Representative original data are shown. C . Cells were treated with same as above and were analyzed for cell cycle by FACS as described in Materials and Methods. Experiments were done at least three times and representative original data are shown (* represents significant difference of p value of < 0.05 between JSI-124 treated and untreated cells and ** represents a p value of < 0.01).

Article Snippet: Rabbit polyclonal antibodies against phosphorylated or total c-Jun, JNK, p38, phosphor and total Erk1/2, XIAP and STAT3 antibodies, as well as siRNA for c-Jun (#6203) and Stat3 siRNA I (#6580) and control siRNA (#6201 fluorescein conjugate) were purchased from Cell Signaling Technology, VEGF antibody was purchased from ThermoScientific and Santa Cruz.

Techniques: Activation Assay, Western Blot, Staining, Control

Journal: BMC Cancer

Article Title: Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B Leukemic Cells

doi: 10.1186/1471-2407-11-268

Figure Lengend Snippet: JSI-124 mediated c-Jun activation was not dependent on STAT3 expression in B-cell leukemia . A and B , Cells were transfected with siRNA-STAT or siRNA-c-Jun or siRNA non-target control. Then cells were treated with JSI-124 for additional 6 h. Cell extract was assayed for Western blotting for STAT3 and c-Jun. Knock-down of STAT3 or c-Jun was confirmed by western blotting with antibody to STAT3 or c-Jun. C , The same transfected cells were treated with JSI-124 for an additional 24 hours and then cells were analyzed for cell cycle analysis for G1 and G2 phase fractions in the cells transfected with siRNA-c-Jun or non-targeting control siRNA. Standard error was determined on the basis of three independent experiments and an asterisk represents significant difference (a p value of < 0.05) between JSI-124 treated and untreated controls. D , Cells were stimulated with JSI-124 and nuclear extracts incubated with the AP1 probe. Pulled down with streptavidin beads, the bound proteins were detected by SDS/polyacrylamide gel electrophoresis and western blotting. To control for specificity, a biotin-labeled scrambled probe was used along with no probe beads control. Blots were probed with antibody to c-Jun.

Article Snippet: Rabbit polyclonal antibodies against phosphorylated or total c-Jun, JNK, p38, phosphor and total Erk1/2, XIAP and STAT3 antibodies, as well as siRNA for c-Jun (#6203) and Stat3 siRNA I (#6580) and control siRNA (#6201 fluorescein conjugate) were purchased from Cell Signaling Technology, VEGF antibody was purchased from ThermoScientific and Santa Cruz.

Techniques: Activation Assay, Expressing, Transfection, Control, Western Blot, Knockdown, Cell Cycle Assay, Incubation, Polyacrylamide Gel Electrophoresis, Labeling