Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: ( a ) Cells transfected with Flag-JWA (4 μg) or without transfection were incubated with EGCG (20–40 μM) for 24 h. JWA protein expression was assessed by Western blot analysis. β-actin expression served as a loading control. ( b ) NCI-H460 cells were treated with EGCG (20–40 μM) for 24 h and total cellular RNA was extracted. mRNA level of JWA was detected by real-time PCR. GAPDH was used as an internal control. ( c ) Western blot analysis of the protein level of JWA in EGCG-treated A549 cells for 24 h. β-actin expression served as a loading control. ( d ) After A549 cells were incubated with EGCG (20–40 μM) for 24 h, total RNAs were prepared and real-time PCR was applied to measure the JWA mRNA level. GAPDH was used as an internal control. ( e ) NCI-H460 cells were transfected with or without Flag-topoisomerase IIα plasmid (4 μg) and then treated with EGCG (20–40 μM) for 24 h. Protein from cell was subjected to western blot analysis. β-actin expression was served as a loading control. ( f ) Total RNAs from NCI-H460 cells incubated with EGCG (20–40 μM) for 24 h were extracted and subjected to real-time PCR using primer for topoisomerase IIα. GAPDH was used as an internal control. ( g ) A549 cells were treated with EGCG (20–40 μM) for 24 h and then protein from cell lysate was subjected to western blot analysis. β-actin expression was served as a loading control. ( h ) A549 cells were incubated in the absence or presence of EGCG (20–40 μM) for 24 h. Then cells were lysed for the detection expression of topoisomerase IIα mRNA by real-time PCR. GAPDH was used as an internal control. The A549 xenograft nude mice model was established and treated with EGCG or normal saline. ( i ) Tumor size was checked twice per week. ( j ) Protein obtained from tumor tissues was subjected to western blot. β-tubulin expression was served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Transfection, Incubation, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Plasmid Preparation, Saline

Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: ( a ) and ( b ) Expression of topoisomerase IIα was elevated and that of topoisomerase IIα was reduced in lung cancer tissues compared with those in matched normal tissues. β-Tubulin expression was served as a loading control. JWA and topoisomerase IIα expressions in cancer tissues (T) and paired non-cancerous normal tissues (N) of lung cancer patients were analyzed by Western blotting. The level of each protein was normalized against β-tubulin. NCI-H460 cells were transiently transfected with Flag-JWA or Flag- topoisomerase IIα plasmid (1–4 μg). Whole-cell extracts were prepared 24 h after transfection and the expression of target proteins and mRNAs was examined. As analyzed by western blot assay, topoisomerase IIα levels were decreased after transfection with JWA plasmid ( c ) Also, overexpression of topoisomerase IIα does dependently inhibited JWA protein expression ( d ) β-actin was used for the protein loading control. Total RNAs were isolated from NCI-H460 cells transfected with Flag-JWA or Flag- topoisomerase IIα plasmid (4 μg) and subjected to real-time PCR. JWA transfection suppressed topoisomerase IIα mRNA expression ( e ) The level of JWA mRNA was down-regulated in topoisomerase IIα overexpressed NCI-H460 cells ( f ) GAPDH was used as an internal control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Expressing, Control, Western Blot, Transfection, Plasmid Preparation, Over Expression, Isolation, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Transfection, Plasmid Preparation, Incubation, Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: ( a ) Schematic showed the domains of full length and indicated fragments JWA protein. “Dark grey” represented the TMD of JWA. Amino acid sequences ranged from 1 to 60 for JWA-1, 1 to 90 for JWA-2, 1 to 140 for JWA-3 and 141–188 for JWA-4. ( b ) NCI-H460 cells were transiently transfected with full-length Flag-JWA plasmid and four different fragments of JWA plasmid respectively. Whole-cell extracts were prepared 24 h after transfection, and topoisomerase IIα expression was measured by western blotting. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: NCI-H460 cells were plated on 60 mm plates in DMEM (10% fetal bovine serum), which was changed to starving DMEM (without serum) for 24 h or 30 h. ( a ) Flow cytometric analysed the change of cell cycle. And the protein levels of JWA and topoisomerase IIα were examined by Western blotting. ( c ) and ( d ) Then NCI-H460 cells were treated with 200 nM nocodazole, respectively for 12 h, 18 h and 24 h. ( b ) Flow cytometric analysed the cell cycle. ( e ) and ( f )The expression of JWA and topoisomerase IIα were examined by Western blotting. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01 and ***p < 0.001.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: ( a ) NCI-H460 cells were transfected with siRNA-topoisomerase IIα (100 pmol), shRNA-JWA, JWA and topoisomerase IIα plasmids (4 μg) as well as the control vector. Migration ability of the cells at various time points after transfection (24 h, 48 h) was assessed by scratch migration assay. ( b ) The JWA or topoisomerase IIα plasmid (4 μg) was transiently transfected into NCI-H460 cells. 24 hours later, target proteins in cell lysates were detected by immunoblotting using antibodies against MMP-2/9, N- cadherin, ZEB1, slug, snail and E-cadherin. β-actin expression served as a loading control. ( c ) and ( d ) NCI-H460 cells were transfected with Flag-JWA , Flag- topoisomerase IIα and Flag-vector plasmids, (4 μg) in the presence or absence of EGCG (40 μM) for 24 h. The cells were harvested and lysed for the detection expression of JWA or topoisomerase by Western blot analysis. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Transfection, shRNA, Control, Plasmid Preparation, Migration, Western Blot, Expressing

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Interference efficiency of mGluR4 siRNA in bone marrow-derived dendritic cells (BMDC). Three mGluR4-targeted siRNAs were predesigned. Mouse BMDC were transfected with negative control siRNA (NC) or one of the three mGluR4 siRNAs (siRNA). Twenty-four hours later, the efficiency of silencing was detected by qRT-PCR ( A ) and western blotting analysis ( B ). C , Quantitative mGluR4/β-actin expression levels. Data are reported as means±SD. *P<0.05, ***P<0.001 vs NC (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Derivative Assay, Transfection, Negative Control, Quantitative RT-PCR, Western Blot, Expressing

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Th17 cell differentiation induced by mGluR4 knockdown in bone marrow-derived dendritic cells (BMDC). The relative mRNA expressions of interleukin (IL)-6 ( A ) and IL-23 ( B ) were evaluated by qRT-PCR in immature (Control), mature (lipopolysaccharide, LPS), and mGluR4 siRNA transfected (siRNA) BMDC. CD4 + T cells were co-cultured with the BMDC for 24 h at the ratio of 1:1. The relative mRNA expressions of IL-17A ( C ) and RORγt ( D ) in the co-culture environment were determined by qRT-PCR. Data are reported as means±SD. ***P<0.001 vs Control (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Cell Differentiation, Derivative Assay, Quantitative RT-PCR, Transfection, Cell Culture, Co-Culture Assay

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Effect of increased production of Th17-related cytokines on melanocyte function. B16 cells were treated with co-culture medium of CD4 + T cells and the negative control siRNA transduced dendritic cells (DC), mGluR4 siRNA transduced DC (siRNA), or only RIPM-1640 medium (Control). Twenty-four hours later, the mRNA expressions of MITF ( A ), TYR ( B ), and TRP-1 ( C ) were measured by qRT-PCR. D , Forty-eight hours later, B16 cells exposed to the different interventions were treated with 1 M NaOH and processed for absorbance (OD) at 405 nm. Data are reported as means±SD. ***P<0.001 vs Control (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Co-Culture Assay, Negative Control, Quantitative RT-PCR

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Effect of increased production of Th17-related cytokines on morphology of B16 cells. B16 cells were exposed to the co-culture medium of CD4 + T cells and the negative control siRNA transduced dendritic cells (DC), mGluR4 siRNA transduced DC (siRNA), or only RIPM-1640 medium (Control). Twenty-four hours later, melanocyte morphology was observed under a microscope with 100 and 200× magnification (scale bars: upper panels 20 µm; lower panels 10 µm). Representative images are shown.

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Co-Culture Assay, Negative Control, Microscopy

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: mGluR4 over-expression in bone marrow-derived dentritic cells (BMDC). BMDC were transfected with empty vector (negative control, NC), plasmid expressing mGluR4 (mGluR4), or untreated (Control). Twenty-four hours later, the expression of mGluR4 was determined by qRT-PCR ( A ) and western blotting analysis ( B ). C , Quantitative mGluR4/β-actin expression levels. Data are reported as means±SD. **P<0.01, ***P<0.001 vs Control (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Over Expression, Derivative Assay, Transfection, Plasmid Preparation, Negative Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Effect of mGluR4 over-expression on surface marker expression of costimulatory molecules CD80 and CD86 in dendritic cells (DC). DC were transfected with empty vector (negative control, NC), plasmid expressing mGluR4 (mGluR4), or untreated (Control). Twenty-four hours later, cells were collected and the percent of DC expressing CD80 ( A ) and CD86 ( B ) was quantified by flow cytometry. Data are reported as means±SD. *P<0.05, ***P< 0.001 vs Control (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Over Expression, Marker, Expressing, Transfection, Plasmid Preparation, Negative Control, Flow Cytometry

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Effect of mGluR4 over-expression on Th17 differentiation and melanogenesis of B16 cells. Dendritic cells (DC) were transfected with empty vector (negative control, NC), plasmid expressing mGluR4 (mGluR4), or untreated (Control). Twenty-four hours later, the relative mRNA expressions of interleukin (IL)-6 ( A ) and IL-23 ( B ) were evaluated by qRT-PCR. CD4 + T cells were co-cultured with the above DC for 24 h at the ratio of 1:1. The relative mRNA expressions of IL-17A ( C ) and RORγt ( D ) in co-culture environment were determined by qRT-PCR. B16 cells were treated with co-culture medium of CD4 + T cells and empty vector transfected DC (DC), pcDNA3.1 mGluR4 transfected DC (mGluR4), or only RIPM-1640 medium (Control) for 24 h ( E ). mRNA expression of MITF of B16 cells was measured by qRT-PCR. Data are reported as means±SD. ***P<0.001 vs Control (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Over Expression, Transfection, Plasmid Preparation, Negative Control, Expressing, Quantitative RT-PCR, Cell Culture, Co-Culture Assay

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Effect of decreased production of Th17-related cytokines on morphology of B16 cells. B16 cells were exposed to the co-culture medium of CD4 + T cells and empty vector transfected dendritic cells (DC), pcDNA3.1 mGluR4 transfected DC (mGluR4), or only RIPM-1640 medium (Control). Twenty-four hours later, melanocyte morphology was observed under the microscope with 100 and 200× magnification (scale bars: upper panels 20 µm; lower panels 10 µm). Representative images are shown.

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Co-Culture Assay, Plasmid Preparation, Transfection, Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Upregulation of IRF3 in Mϕ-LPS/IFNγ cells and HCFs under inflammatory stimulation. ( A ) Alteration of the relative mRNA expression of IRF3 in human LPS- and IFNγ-stimulated macrophages (Mϕ-LPS/IFNγ cells). The mRNA expression was normalized to GAPDH ( n = 8 samples per group). ( B ) Schematic illustration of the experimental procedure for primary culture of HCFs from donor corneas and stimulation with LPS for IRF3 analysis. ( C ) Comparison of relative mRNA expression of IRF3 between LPS-stimulated and untreated control HCFs ( n = 6 per group). ( D, E ) Schematic illustration of the in vitro coculture model using HCFs and Mϕ-LPS/IFNγ cells D and relative mRNA expression of IRF3 in HCFs cocultured with or without Mϕ-LPS/IFNγ cells E ( n = 3 samples per group). Statistical analyses were performed using the t -test. *** P < 0.001 and **** P < 0.0001.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Expressing, Comparison, Control, In Vitro

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: IRF3 expression in Mϕ-LPS/IFNγ cells and the alteration of inflammatory and myeloid markers by IRF3 knockdown. ( A, B ) Relative mRNA A and protein B expression of IRF3 in Mϕ-LPS/IFNγ cells transfected with small interfering RNA targeting IRF3 (si IRF3 ) or negative control siRNA (si Con ). The mRNA levels were normalized to GAPDH ( n = 6 samples per group). ( C ) Schematic illustration of the preparation of si IRF3 -Mϕ-LPS/IFNγ cells and si Con -Mϕ-LPS/IFNγ cells used for subsequent analyses. ( D–L ) Relative mRNA expression (normalized to GAPDH ) of IL1B D , IL6 E , TNF F , PTGS2 G , ITGAM H , CD68 I , CD80 J , CD86 K , and MRC1 L in si Con -transfected Mϕs as control and Mϕ-LPS/IFNγ cells transfected with si Con or si IRF3 ( n = 3 samples per group). Statistical analyses were performed using the t -test A and 1-way ANOVA followed by Tukey's post hoc test D to L . * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Expressing, Knockdown, Transfection, Small Interfering RNA, Negative Control, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: IRF3 expression in the LPS-stimulated inflammatory HCFs and the alteration of downstream signals elicited by TLR4/LPS complex by IRF3 knockdown. ( A ) Relative mRNA expression of IRF3 in HCFs transfected with small interfering RNA targeting IRF3 (si IRF3 ) or negative control siRNA (si Con ; n = 8 samples per group). ( B ) Schematic illustration of the preparation of LPS-stimulated cells in untreated HCFs and si IRF3 -transfected HCFs (si IRF3 -HCF) used for subsequent analyses. ( C–I ) Relative mRNA expression (normalized to GAPDH ) of IL1B D , IL6 E , TNF F , CXCL10 G , TLR4 H , and MYD88 I in untreated control HCFs, LPS-HCFs and LPS-stimulated si IRF3 -HCFs ( n = 3 samples per group). ( J ) Illustration showing the intracellular signal pathway elicited by TLR4 binding of LPS to activate MyD88-NF-κB pathway and IRF3 pathway to produce IL6 , IL1B , TNF , and CXCL10 transcripts in HCFs. Statistical analyses were performed using the t -test A and 1-way ANOVA followed by Tukey's post hoc test C to I . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Expressing, Knockdown, Transfection, Small Interfering RNA, Negative Control, Control, Binding Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Suppression of TLR4-associated inflammatory activation in HCFs by IRF3 -silenced macrophages in the coculture model. ( A ) Schematic illustration of the in vitro coculture system using human corneal stromal fibroblasts (HCFs) and Mϕ-LPS/IFNγ cells transfected with small interfering RNA targeting IRF3 (si IRF3 ) or negative control siRNA (si Con ) for subsequent analyses. ( B–H ) Relative mRNA expression (normalized to GAPDH ) of IRF3 B , IL1B C , IL6 D , TNF E , CXCL10 F , TLR4 G , and MYD88 H in HCFs without coculture or cocultured with si Con -Mϕ-LPS/IFNγ cells or si IRF3 -Mϕ-LPS/IFNγ cells ( n = 3 samples per group). Statistical analyses were performed using 1-way ANOVA followed by Tukey's post hoc test. ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Activation Assay, In Vitro, Transfection, Small Interfering RNA, Negative Control, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Suppression of TLR4-IRF3 signaling-mediated corneal inflammation and opacity in Irf3 -deficient mice. ( A ) Schematic illustration of the experimental design using wild-type (WT) and homozygous Irf3 -deficient ( Irf3 ⁻/⁻; B6.Cg- Irf3 < tm2.2Ttg >) mice. After mechanical corneal epithelial removal, phosphate-buffered saline (PBS; vehicle control) or lipopolysaccharide (LPS) eye drops were topically instilled 6 times, and corneas were harvested 48 hours post-injury for analysis. ( B ) Relative mRNA expression of Irf3 (normalized to Gapdh ) in excised corneas from WT mice treated with PBS (vehicle) or LPS and from Irf3⁻/⁻ mice treated with LPS ( n = 10 corneas from 5 mice per group). ( C ) Representative photographs of the corneas before (0 hours) and after (48 hours) six topical LPS instillations in WT and Irf3 ⁻/⁻ mice. ( D–F ) Corneal opacity grades D and relative expression of Vim E (epithelial–mesenchymal transition marker) and Fap F (fibroblast activation marker) in each group ( n = 10 corneas from 5 mice per group). ( G–J ) Relative mRNA expression (normalized to Gapdh ) of Il1b G , Il6 H , Tnf I , and Cxcl10 J in the corneas ( n = 10 corneas from 5 mice per group). Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test B, D–H, and J or Kruskal-Wallis test followed by Dunn's post hoc test I . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Saline, Control, Eye Drops, Expressing, Marker, Activation Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Attenuation of monocyte recruitment and activation of inflammatory macrophages by Irf3 deficiency in the LPS-induced mouse corneal inflammation model. ( A ) Relative mRNA expression of Ccl2 (MCP-1) (normalized to Gapdh ) in excised corneas from WT mice treated with PBS (vehicle) or LPS and from Irf3 ⁻/⁻ mice treated with LPS ( n = 10 corneas from 5 mice per group). ( B ) Flow cytometric gating strategy for identifying myeloid cells, including neutrophils and monocytes, in the mouse cornea. Four corneas were pooled into one sample for each analysis. ( C–E ) Comparison of monocyte (CD45⁺CD11b⁺Ly6C⁺Ly6G⁻) and neutrophil (CD45⁺CD11b⁺Ly6C int Ly6G⁺) populations in WT and Irf3 ⁻/⁻ corneas after LPS instillation ( n = 4 pooled samples, prepared from 16 corneas from 8 mice, per group). ( F–I ) Relative mRNA expression (normalized to Gapdh ) of Itgam (CD11b) F , Adgre1 (F4/80) G , Cd80 H , and Mrc1 (CD206) I in excised corneas from WT mice treated with PBS (vehicle) or LPS and from Irf3 ⁻/⁻ mice treated with LPS ( n = 10 corneas from 5 mice per group). Statistical analyses were performed using t -test D and E 1-way ANOVA followed by Tukey's post hoc test F to I or Kruskal-Wallis test followed by Dunn's post hoc test A . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Activation Assay, Expressing, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Suppression of TBK1-independent IRF3 signaling and downstream inflammation by piceatannol in LPS-stimulated HCFs. ( A–D ) Relative mRNA expression of IRF3 A , IL1B B , IL6 C , and CXCL10 D in untreated HCFs and LPS-stimulated HCFs treated with or without piceatannol (PIC; 10 µM, 24 hours,( n = 3 samples per group). ( E–G ) Relative mRNA expression of IL1B E , IL6 F , and CXCL10 G in HCFs transfected with si IRF3 (si IRF3 -HCFs) with or without LPS stimulation and/or PIC treatment ( n = 3 samples per group). ( H–K ) Phosphorylated (p-) and total TBK1 and IRF3 protein expression in LPS-stimulated HCFs treated with or without PIC as shown by Western blot analysis ( n = 3 samples per group). Statistical analyses were performed using one-way ANOVA followed by Tukey's post-hoc test ( A–G ) or t -test ( I, J ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Expressing, Transfection, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity

doi: 10.1167/iovs.67.3.49

Figure Lengend Snippet: Suppression of TLR4-IRF3 signaling-mediated corneal inflammation by topical PIC treatment in the LPS-induced mouse model. ( A ) Schematic illustration of the experimental protocol using PIC eye drops in the LPS-induced inflammatory corneal model. After mechanical corneal epithelial removal in naïve mouse corneas, LPS was instilled twice as pretreatment, followed by four co-instillations of LPS and PIC, and finally two additional PIC-only treatments. Corneas were harvested 58 hours post-injury for subsequent analyses. ( B ) Relative mRNA expression of Irf3 (normalized to Gapdh ) in excised corneas treated with PBS (vehicle) or LPS and with or without topical PIC at different concentrations (2, 20, or 200 µg/µL, n = 5 to 10 corneas from 4 to 5 mice per group). ( C–L ) Relative mRNA expression (normalized to Gapdh ) of TLR4-IRF3 signaling–related factors, including Il1b C , Il6 D , Tnf E , Cxcl10 F , and Myd88 G , and myeloid/macrophage markers, including Itgam (CD11b) H , Cd68 I , Adgre1 (F4/80) J , Cd86 K , and Mrc1 (CD206) L , in excised corneas ( n = 7 to 8 corneas from 4 mice per group). Statistical analyses were performed using 1-way ANOVA followed by Tukey's post hoc test B to K or Kruskal-Wallis test followed by Dunn's post hoc test L . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant.

Article Snippet: The AccuTarget Negative Control and AccuTarget Predesigned IRF3 siRNAs were obtained from Bioneer (Daejeon, Korea).

Techniques: Eye Drops, Expressing