Journal: Frontiers in Endocrinology

Article Title: Toll-like receptor signaling pathway triggered by inhibition of serpin A1 stimulates production of inflammatory cytokines by endometrial stromal cells

doi: 10.3389/fendo.2022.966455

Figure Lengend Snippet: Primers for real-time PCR.

Article Snippet: After blocking with Bullet Blocking One (Nacalai Tesque, Inc., Kyoto, Japan), the membranes were incubated with primary antibodies specific for SERPINA1 (1:2,000; Dako, Tokyo, Japan), TLR3 or TLR4 (1:2,000; Proteintech, Tokyo, Japan), MYD88, IRAK1, IRAK4, phosphorylated (p)-IRAK4, IκB, p-IκB, NF-κB, p-NF-κB, IL-1β, COX2, STAT3, JNK, p-JNK, p38, p-p38, ERK1/2, p-ERK1/2 (1:2,000; Cell Signaling Technology, Beverly, MA, USA), MX1 (1:2,000; Sigma-Aldrich), ISG15 (1:2,000; Origene Technologies, Rockville MD, USA), and GAPDH (1:5,000, Fujifilm Wako Pure Chemical Corp.).

Techniques: Sequencing

Journal: Frontiers in Endocrinology

Article Title: Toll-like receptor signaling pathway triggered by inhibition of serpin A1 stimulates production of inflammatory cytokines by endometrial stromal cells

doi: 10.3389/fendo.2022.966455

Figure Lengend Snippet: TLR3 upregulates expression of TLR- or inflammatory-related genes in ESCs. (A) Expression of TLR3 , TLR4, MYD88, IRAK4, IRF3, IRF7, IL1B, IL6 , CXCL8, and IFNB1 in primary ESCs pre-treated with PIC (10 μg/ml) or TLR3i (10 μM) and then transfected for 24 h with si SERPINA1 (n=3). GAPDH was used as the reference gene. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. si SERPINA alone. (B, C) Primary ESCs transfected for 24 h with si SERPINA1 and/or si TLR3 . (B) Expression of TLR3 , TLR4, MYD88, IRAK4, IRF3, IRF7, IL1B, IL6 , CXCL8, and IFNB1 in primary ESCs (n=3). GAPDH was used as the reference gene. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. si SERPINA alone. (C) The culture media were subjected to the ELISA to measure the concentration of IL-1β, IL-6, IL-8, or IFN-β secreted from primary ESCs. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. si SERPINA1 alone.

Article Snippet: After blocking with Bullet Blocking One (Nacalai Tesque, Inc., Kyoto, Japan), the membranes were incubated with primary antibodies specific for SERPINA1 (1:2,000; Dako, Tokyo, Japan), TLR3 or TLR4 (1:2,000; Proteintech, Tokyo, Japan), MYD88, IRAK1, IRAK4, phosphorylated (p)-IRAK4, IκB, p-IκB, NF-κB, p-NF-κB, IL-1β, COX2, STAT3, JNK, p-JNK, p38, p-p38, ERK1/2, p-ERK1/2 (1:2,000; Cell Signaling Technology, Beverly, MA, USA), MX1 (1:2,000; Sigma-Aldrich), ISG15 (1:2,000; Origene Technologies, Rockville MD, USA), and GAPDH (1:5,000, Fujifilm Wako Pure Chemical Corp.).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Endocrinology

Article Title: Toll-like receptor signaling pathway triggered by inhibition of serpin A1 stimulates production of inflammatory cytokines by endometrial stromal cells

doi: 10.3389/fendo.2022.966455

Figure Lengend Snippet: TLR4 increases expression of inflammatory-related genes in SERPINA1-silenced ESCs. (A) Expression of TLR3 , TLR4, MYD88, IRAK4, IRF3, IRF7, IL1B, IL6 , CXCL8, and IFNB1 in primary ESCs pre-treated with LPS (0.2 μg/ml) or TAK (10 μM) and then transfected for 24 h with si SERPINA1 (n=3). GAPDH was used as the reference gene. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. si SERPINA1 alone. (B, C) Primary ESCs transfected for 24 h with si SERPINA1 and/or si TLR4 . (B) Expression of TLR3 , TLR4, MYD88, IRAK4, IRF3, IRF7, IL1B, IL6 , CXCL8, and IFNB1 by ESCs (n=3). GAPDH was used as the reference gene. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. si SERPINA1 alone. (C) Culture media were subjected to the ELISA to measure the concentration of IL-1β, IL-6, IL-8, or IFN-β secreted from primary ESCs. Values represent the mean ± SEM of three independent experiments. * P <0.05 vs. si SERPINA1 alone.

Article Snippet: After blocking with Bullet Blocking One (Nacalai Tesque, Inc., Kyoto, Japan), the membranes were incubated with primary antibodies specific for SERPINA1 (1:2,000; Dako, Tokyo, Japan), TLR3 or TLR4 (1:2,000; Proteintech, Tokyo, Japan), MYD88, IRAK1, IRAK4, phosphorylated (p)-IRAK4, IκB, p-IκB, NF-κB, p-NF-κB, IL-1β, COX2, STAT3, JNK, p-JNK, p38, p-p38, ERK1/2, p-ERK1/2 (1:2,000; Cell Signaling Technology, Beverly, MA, USA), MX1 (1:2,000; Sigma-Aldrich), ISG15 (1:2,000; Origene Technologies, Rockville MD, USA), and GAPDH (1:5,000, Fujifilm Wako Pure Chemical Corp.).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Endocrinology

Article Title: Toll-like receptor signaling pathway triggered by inhibition of serpin A1 stimulates production of inflammatory cytokines by endometrial stromal cells

doi: 10.3389/fendo.2022.966455

Figure Lengend Snippet: IL-1β or IFN-β increases expression of inflammatory-related genes. (A, B) Expression of TLR3 , TLR4, MYD88, IRAK4, IRF3, IRF7, IL1B, IL6 , CXCL8, and IFNB1 in primary ESCs transfected for 24 h with si SERPINA1 and then treated with IL-1β ( A ; 10 ng/ml) or IFN-β ( B ; 500 ng/ml) (n=3). GAPDH was used as the reference gene. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. Ctrl. # P <0.05, ## P <0.01 vs. si SERPINA1 alone.

Article Snippet: After blocking with Bullet Blocking One (Nacalai Tesque, Inc., Kyoto, Japan), the membranes were incubated with primary antibodies specific for SERPINA1 (1:2,000; Dako, Tokyo, Japan), TLR3 or TLR4 (1:2,000; Proteintech, Tokyo, Japan), MYD88, IRAK1, IRAK4, phosphorylated (p)-IRAK4, IκB, p-IκB, NF-κB, p-NF-κB, IL-1β, COX2, STAT3, JNK, p-JNK, p38, p-p38, ERK1/2, p-ERK1/2 (1:2,000; Cell Signaling Technology, Beverly, MA, USA), MX1 (1:2,000; Sigma-Aldrich), ISG15 (1:2,000; Origene Technologies, Rockville MD, USA), and GAPDH (1:5,000, Fujifilm Wako Pure Chemical Corp.).

Techniques: Expressing, Transfection

Journal: Mediators of Inflammation

Article Title: Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells

doi: 10.1155/2017/9891673

Figure Lengend Snippet: Sheep gene specific primers for qRT-PCR.

Article Snippet: Antibodies used in this study included rabbit anti-Toll-like receptor 4, rabbit anti-AP-1, rabbit anti-p-AP-1 (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-MyD88, rabbit anti-IRAK1, rabbit anti-IRAK4, rabbit anti-TRAF6, rabbit anti-TAB1, rabbit anti-TRIF, rabbit anti-TRAF3, rabbit anti-IRF3, rabbit anti-IRF5, rabbit anti- β -actin (Proteintech Group, Chicago, USA), rabbit anti-IRAK2, rabbit anti-TBK1 (ABGENT, San Diego, USA), and rabbit anti-p-NFkB (Signalway Antibody, Maryland, USA).

Techniques: Sequencing

Journal: Mediators of Inflammation

Article Title: Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells

doi: 10.1155/2017/9891673

Figure Lengend Snippet: The impact of CPS on the expression of MyD88-dependent TLR signaling pathway in ALI cultures of sheep bronchial epithelial cells. 4-week old ALI cultures of sheep bronchial epithelial cells were exposed to indicated conditions for 48 h. (a) The expression of several key components of MyD88-dependent signaling cascade, including the MyD88, IRAK1, IRAK2, IRAK4, TRAF6, and TAB1 transcripts were determined by qRT-PCR assays. In comparison with the controls, all tested transcripts showed a significant increase in the cells treated with CPS or M. ovipneumoniae . (b) Immunoblotting assay also showed an evoked expression of all MyD88-dependent signaling-associated proteins in ALI cultures upon CPS/ MO. ovipneumoniae stimulation. (c) Densitometric analysis of western blot showed MyD88, IRAK1, IRAK2, IRAK4, TRAF6, and TAB1 expression over β -actin. Values are mean ± SD for at least three independent experiments performed in triplicate. ∗∗ p < 0.01 versus that of the control. Compared between indicated groups, ▲▲ p < 0.01.

Article Snippet: Antibodies used in this study included rabbit anti-Toll-like receptor 4, rabbit anti-AP-1, rabbit anti-p-AP-1 (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-MyD88, rabbit anti-IRAK1, rabbit anti-IRAK4, rabbit anti-TRAF6, rabbit anti-TAB1, rabbit anti-TRIF, rabbit anti-TRAF3, rabbit anti-IRF3, rabbit anti-IRF5, rabbit anti- β -actin (Proteintech Group, Chicago, USA), rabbit anti-IRAK2, rabbit anti-TBK1 (ABGENT, San Diego, USA), and rabbit anti-p-NFkB (Signalway Antibody, Maryland, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Endocrinology

Article Title: Toll-like receptor signaling pathway triggered by inhibition of serpin A1 stimulates production of inflammatory cytokines by endometrial stromal cells

doi: 10.3389/fendo.2022.966455

Figure Lengend Snippet: Primers for real-time PCR.

Article Snippet: After blocking with Bullet Blocking One (Nacalai Tesque, Inc., Kyoto, Japan), the membranes were incubated with primary antibodies specific for SERPINA1 (1:2,000; Dako, Tokyo, Japan), TLR3 or TLR4 (1:2,000; Proteintech, Tokyo, Japan), MYD88, IRAK1, IRAK4, phosphorylated (p)-IRAK4, IκB, p-IκB, NF-κB, p-NF-κB, IL-1β, COX2, STAT3, JNK, p-JNK, p38, p-p38, ERK1/2, p-ERK1/2 (1:2,000; Cell Signaling Technology, Beverly, MA, USA), MX1 (1:2,000; Sigma-Aldrich), ISG15 (1:2,000; Origene Technologies, Rockville MD, USA), and GAPDH (1:5,000, Fujifilm Wako Pure Chemical Corp.).

Techniques: Sequencing

Journal: Frontiers in Endocrinology

Article Title: Toll-like receptor signaling pathway triggered by inhibition of serpin A1 stimulates production of inflammatory cytokines by endometrial stromal cells

doi: 10.3389/fendo.2022.966455

Figure Lengend Snippet: TLR3 upregulates expression of TLR- or inflammatory-related genes in ESCs. (A) Expression of TLR3 , TLR4, MYD88, IRAK4, IRF3, IRF7, IL1B, IL6 , CXCL8, and IFNB1 in primary ESCs pre-treated with PIC (10 μg/ml) or TLR3i (10 μM) and then transfected for 24 h with si SERPINA1 (n=3). GAPDH was used as the reference gene. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. si SERPINA alone. (B, C) Primary ESCs transfected for 24 h with si SERPINA1 and/or si TLR3 . (B) Expression of TLR3 , TLR4, MYD88, IRAK4, IRF3, IRF7, IL1B, IL6 , CXCL8, and IFNB1 in primary ESCs (n=3). GAPDH was used as the reference gene. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. si SERPINA alone. (C) The culture media were subjected to the ELISA to measure the concentration of IL-1β, IL-6, IL-8, or IFN-β secreted from primary ESCs. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. si SERPINA1 alone.

Article Snippet: After blocking with Bullet Blocking One (Nacalai Tesque, Inc., Kyoto, Japan), the membranes were incubated with primary antibodies specific for SERPINA1 (1:2,000; Dako, Tokyo, Japan), TLR3 or TLR4 (1:2,000; Proteintech, Tokyo, Japan), MYD88, IRAK1, IRAK4, phosphorylated (p)-IRAK4, IκB, p-IκB, NF-κB, p-NF-κB, IL-1β, COX2, STAT3, JNK, p-JNK, p38, p-p38, ERK1/2, p-ERK1/2 (1:2,000; Cell Signaling Technology, Beverly, MA, USA), MX1 (1:2,000; Sigma-Aldrich), ISG15 (1:2,000; Origene Technologies, Rockville MD, USA), and GAPDH (1:5,000, Fujifilm Wako Pure Chemical Corp.).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Endocrinology

Article Title: Toll-like receptor signaling pathway triggered by inhibition of serpin A1 stimulates production of inflammatory cytokines by endometrial stromal cells

doi: 10.3389/fendo.2022.966455

Figure Lengend Snippet: TLR4 increases expression of inflammatory-related genes in SERPINA1-silenced ESCs. (A) Expression of TLR3 , TLR4, MYD88, IRAK4, IRF3, IRF7, IL1B, IL6 , CXCL8, and IFNB1 in primary ESCs pre-treated with LPS (0.2 μg/ml) or TAK (10 μM) and then transfected for 24 h with si SERPINA1 (n=3). GAPDH was used as the reference gene. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. si SERPINA1 alone. (B, C) Primary ESCs transfected for 24 h with si SERPINA1 and/or si TLR4 . (B) Expression of TLR3 , TLR4, MYD88, IRAK4, IRF3, IRF7, IL1B, IL6 , CXCL8, and IFNB1 by ESCs (n=3). GAPDH was used as the reference gene. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. si SERPINA1 alone. (C) Culture media were subjected to the ELISA to measure the concentration of IL-1β, IL-6, IL-8, or IFN-β secreted from primary ESCs. Values represent the mean ± SEM of three independent experiments. * P <0.05 vs. si SERPINA1 alone.

Article Snippet: After blocking with Bullet Blocking One (Nacalai Tesque, Inc., Kyoto, Japan), the membranes were incubated with primary antibodies specific for SERPINA1 (1:2,000; Dako, Tokyo, Japan), TLR3 or TLR4 (1:2,000; Proteintech, Tokyo, Japan), MYD88, IRAK1, IRAK4, phosphorylated (p)-IRAK4, IκB, p-IκB, NF-κB, p-NF-κB, IL-1β, COX2, STAT3, JNK, p-JNK, p38, p-p38, ERK1/2, p-ERK1/2 (1:2,000; Cell Signaling Technology, Beverly, MA, USA), MX1 (1:2,000; Sigma-Aldrich), ISG15 (1:2,000; Origene Technologies, Rockville MD, USA), and GAPDH (1:5,000, Fujifilm Wako Pure Chemical Corp.).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Endocrinology

Article Title: Toll-like receptor signaling pathway triggered by inhibition of serpin A1 stimulates production of inflammatory cytokines by endometrial stromal cells

doi: 10.3389/fendo.2022.966455

Figure Lengend Snippet: IL-1β or IFN-β increases expression of inflammatory-related genes. (A, B) Expression of TLR3 , TLR4, MYD88, IRAK4, IRF3, IRF7, IL1B, IL6 , CXCL8, and IFNB1 in primary ESCs transfected for 24 h with si SERPINA1 and then treated with IL-1β ( A ; 10 ng/ml) or IFN-β ( B ; 500 ng/ml) (n=3). GAPDH was used as the reference gene. Values represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01 vs. Ctrl. # P <0.05, ## P <0.01 vs. si SERPINA1 alone.

Article Snippet: After blocking with Bullet Blocking One (Nacalai Tesque, Inc., Kyoto, Japan), the membranes were incubated with primary antibodies specific for SERPINA1 (1:2,000; Dako, Tokyo, Japan), TLR3 or TLR4 (1:2,000; Proteintech, Tokyo, Japan), MYD88, IRAK1, IRAK4, phosphorylated (p)-IRAK4, IκB, p-IκB, NF-κB, p-NF-κB, IL-1β, COX2, STAT3, JNK, p-JNK, p38, p-p38, ERK1/2, p-ERK1/2 (1:2,000; Cell Signaling Technology, Beverly, MA, USA), MX1 (1:2,000; Sigma-Aldrich), ISG15 (1:2,000; Origene Technologies, Rockville MD, USA), and GAPDH (1:5,000, Fujifilm Wako Pure Chemical Corp.).

Techniques: Expressing, Transfection