Journal: Cells

Article Title: MyD88 Contributes to TLR3-Mediated NF-κB Activation and Cytokine Production in Macrophages

doi: 10.3390/cells14191507

Figure Lengend Snippet: MyD88 contributes to NF-κB activation in response to poly(I:C). ( A ) Western blot analysis of MyD88, TRIF, and TLR3 basal expression levels in unstimulated cells. β-actin was used as a loading control. ( B ) Quantification of basal protein expression levels shown in ( A ). Data represent the mean ± SEM of three independent experiments. Data are presented as mean ± SD (N = 3, biological replicates). ( C ) Cells were stimulated with 1 µg/mL poly(I:C), and total protein lysates were collected at the indicated time points (0, 30, 60, and 120 min). Western blot analysis was performed to detect p65, phosphorylated p65 (p-p65), IκB, and phosphorylated IκB (p-IκB), with β-actin used as a loading control. ( D – G ) Quantification of protein band intensities from ( C ) was performed using ImageJ (1.54g)software. Relative intensities of ( D ) p65, ( F ) IκB, and ( G ) p-IκB were normalized to β-actin and presented as fold changes compared to controls (CTRL), while ( E ) p-p65 was normalized to the total p65 ( D ). Data are presented as mean ± SD (N = 5, biological replicates). Statistical analysis for comparisons among multiple groups was performed using two-way ANOVA followed by Sidak’s multiple comparisons testing. ns, not significant,* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001.

Article Snippet: The full-length human TLR3 coding sequence was obtained from Sino Biological, Beijing, China (Cat# MG50161-CF) and cloned into the pCMV3 expression vector between the KpnI and NotI restriction sites to create pCMV3-TLR3-Flag.

Techniques: Activation Assay, Western Blot, Expressing, Control, Software

Journal: Cells

Article Title: MyD88 Contributes to TLR3-Mediated NF-κB Activation and Cytokine Production in Macrophages

doi: 10.3390/cells14191507

Figure Lengend Snippet: MyD88 contributes to NF-κB-dependent cytokine transcription in response to poly(I:C) stimulation. ( A – C ) Primary macrophages from WT, MyD88 -/- , TRIF -/- , and TLR3 -/- mice were stimulated with 1 µg/mL Poly(I:C) for 2 h. ( D – F ) WT macrophages or ( G – I ) MyD88 -/- macrophages were pretreated with the NF-κB inhibitor SC75741 for 1 h before stimulation with 1 µg/mL Poly(I:C) for 2 h. Total RNA was extracted, and mRNA levels of cytokines were analyzed by quantitative RT-PCR (qRT-PCR). Relative expression levels of ( A , D , G ) IFN-β, ( B , E , H ) IL-6, and ( C , F , I ) TNF-α were normalized to a reference gene and presented as fold changes compared to untreated controls (CTRL). Data are presented as mean ± SD (N = 7 for ( A – F ) or 6 for ( G – I ), biological replicates). Statistical analysis for comparisons among multiple groups was performed using two-way ANOVA followed by Sidak’s multiple comparisons testing. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001.

Article Snippet: The full-length human TLR3 coding sequence was obtained from Sino Biological, Beijing, China (Cat# MG50161-CF) and cloned into the pCMV3 expression vector between the KpnI and NotI restriction sites to create pCMV3-TLR3-Flag.

Techniques: Quantitative RT-PCR, Expressing

Journal: Cells

Article Title: MyD88 Contributes to TLR3-Mediated NF-κB Activation and Cytokine Production in Macrophages

doi: 10.3390/cells14191507

Figure Lengend Snippet: MyD88 contributes to cytokine production in response to poly(A:U). ( A – C ) Primary macrophages from WT, MyD88 -/- , TRIF -/- , and TLR3 -/- mice were transfected with 250 µg/mL poly(A:U) using Lipofectamine 3000 for 24 h. Cytokine levels in the supernatants were measured by ELISA. ( A ) IFN-β, ( B ) TNF-α, and ( C ) IL-6 levels are shown. Data are presented as mean ± SD (N = 6, biological replicates). Statistical analysis for comparisons among multiple groups was performed using two-way ANOVA followed by Sidak’s multiple comparisons testing. ns, not significant, *** p < 0.005, **** p < 0.0001.

Article Snippet: The full-length human TLR3 coding sequence was obtained from Sino Biological, Beijing, China (Cat# MG50161-CF) and cloned into the pCMV3 expression vector between the KpnI and NotI restriction sites to create pCMV3-TLR3-Flag.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: MyD88 Contributes to TLR3-Mediated NF-κB Activation and Cytokine Production in Macrophages

doi: 10.3390/cells14191507

Figure Lengend Snippet: Poly(I:C) stimulation induces TLR3 and MyD88 interaction and MyD88 oligomerization. ( A ) CHO and CHO cells transfected with FLAG-tagged TLR3 (CHO-TLR3) were stimulated with 1 µg/mL poly(I:C) for 2 h. Cell lysates were subjected to co-immunoprecipitation using anti-FLAG beads, followed by immunoblotting with anti-FLAG and anti-MyD88 antibodies to detect the interaction between TLR3 and MyD88. ( B ) BMDMs from WT, MyD88 -/- , and TLR3 -/- mice were stimulated with 1 µg/mL poly(I:C) for the indicated times. Cell lysates were treated with DSS for 2 h, then analyzed by SDS-PAGE and immunoblotted with anti-MyD88 antibody to assess oligomerization status. β-actin was used as a loading control.

Article Snippet: The full-length human TLR3 coding sequence was obtained from Sino Biological, Beijing, China (Cat# MG50161-CF) and cloned into the pCMV3 expression vector between the KpnI and NotI restriction sites to create pCMV3-TLR3-Flag.

Techniques: Transfection, Immunoprecipitation, Western Blot, SDS Page, Control