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GenePharma Company sirna the sirna molecules targeting cd200 mrna
<t>CD200</t> restrains p38 signaling upon Staphylococcal infection. ( A – L ) Mouse PEMs were transfected with CD200 <t>siRNA</t> (siCD200) or control siRNA (NC) for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, JNK, p-TAK1, TAK1, p-MMK3/6, MMK3/6, p-CREB, CREB, p-MSK1, MSK, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( M – Q ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( R ) RAW264.7 macrophages were pre-transfected with CD200 siRNA for 48 h and then challenged with S. aureus (MOI = 10) for 2 h. The AP-1 luciferase activity was analyzed by the dual luciferase. Representative blots from three independent experiments are shown ( A , E , J , M , and O ). Changes in the phosphorylation state of different proteins were semi-quantitated using ImageJ software ( B – D , F – I , K – L , N , P – Q ). ND means not detected; * p
Sirna The Sirna Molecules Targeting Cd200 Mrna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20030659

CD200 restrains p38 signaling upon Staphylococcal infection. ( A – L ) Mouse PEMs were transfected with CD200 siRNA (siCD200) or control siRNA (NC) for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, JNK, p-TAK1, TAK1, p-MMK3/6, MMK3/6, p-CREB, CREB, p-MSK1, MSK, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( M – Q ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( R ) RAW264.7 macrophages were pre-transfected with CD200 siRNA for 48 h and then challenged with S. aureus (MOI = 10) for 2 h. The AP-1 luciferase activity was analyzed by the dual luciferase. Representative blots from three independent experiments are shown ( A , E , J , M , and O ). Changes in the phosphorylation state of different proteins were semi-quantitated using ImageJ software ( B – D , F – I , K – L , N , P – Q ). ND means not detected; * p
Figure Legend Snippet: CD200 restrains p38 signaling upon Staphylococcal infection. ( A – L ) Mouse PEMs were transfected with CD200 siRNA (siCD200) or control siRNA (NC) for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, JNK, p-TAK1, TAK1, p-MMK3/6, MMK3/6, p-CREB, CREB, p-MSK1, MSK, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( M – Q ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( R ) RAW264.7 macrophages were pre-transfected with CD200 siRNA for 48 h and then challenged with S. aureus (MOI = 10) for 2 h. The AP-1 luciferase activity was analyzed by the dual luciferase. Representative blots from three independent experiments are shown ( A , E , J , M , and O ). Changes in the phosphorylation state of different proteins were semi-quantitated using ImageJ software ( B – D , F – I , K – L , N , P – Q ). ND means not detected; * p

Techniques Used: Infection, Transfection, Expressing, Western Blot, Luciferase, Activity Assay, Software

The p38 inhibition largely abolished the effects of CD200 on NO synthesis induced by staphylococcal infection. ( A – D ) Mouse PEMs ( A , B ) or BMDMs ( C , D ) were pre-treated with SB239063 (10 μM, 0.5 h) or vehicle (DMSO), and then treated with S. aureus for indicated time periods (0–18 h). The mRNA level of iNOS was detected by qPCR, and the concentration of NO in culture supernatants was determined using Griess reagent. ( E , F ) Mouse PEMs were transfected with CD200 siRNA or NC for 48 h and treated with SB239063 or vehicle (DMSO) for 0.5 h. Cells were then infected with S. aureus for indicated time periods (0–12 h). The mRNA level of iNOS and the concentration of NO in culture supernatants were analyzed. Results are expressed as the mean ± SD of three independent experiments; * p
Figure Legend Snippet: The p38 inhibition largely abolished the effects of CD200 on NO synthesis induced by staphylococcal infection. ( A – D ) Mouse PEMs ( A , B ) or BMDMs ( C , D ) were pre-treated with SB239063 (10 μM, 0.5 h) or vehicle (DMSO), and then treated with S. aureus for indicated time periods (0–18 h). The mRNA level of iNOS was detected by qPCR, and the concentration of NO in culture supernatants was determined using Griess reagent. ( E , F ) Mouse PEMs were transfected with CD200 siRNA or NC for 48 h and treated with SB239063 or vehicle (DMSO) for 0.5 h. Cells were then infected with S. aureus for indicated time periods (0–12 h). The mRNA level of iNOS and the concentration of NO in culture supernatants were analyzed. Results are expressed as the mean ± SD of three independent experiments; * p

Techniques Used: Inhibition, Infection, Real-time Polymerase Chain Reaction, Concentration Assay, Transfection

CD200 inhibits the production of inflammatory cytokines induced by S. aureus . Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–12 h). ( A – F ) Relative mRNA expression levels of IL-1β ( A ), IL-6 ( B ), TNF-α ( C ), IL-12p40 ( D ), IL-10 ( E ), or CXCL1 ( F ) were detected by qPCR, with β-actin as an internal control. ( G – L ) The amount of IL-1β ( G ), IL-6 ( H ), TNF-α ( I ), IL-12 ( J ), IL-10 ( K ), or CXCL1 ( L ) in the cell culture supernatant was determined by ELISA. Results are expressed as the mean ± SD of three independent experiments; * p
Figure Legend Snippet: CD200 inhibits the production of inflammatory cytokines induced by S. aureus . Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–12 h). ( A – F ) Relative mRNA expression levels of IL-1β ( A ), IL-6 ( B ), TNF-α ( C ), IL-12p40 ( D ), IL-10 ( E ), or CXCL1 ( F ) were detected by qPCR, with β-actin as an internal control. ( G – L ) The amount of IL-1β ( G ), IL-6 ( H ), TNF-α ( I ), IL-12 ( J ), IL-10 ( K ), or CXCL1 ( L ) in the cell culture supernatant was determined by ELISA. Results are expressed as the mean ± SD of three independent experiments; * p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

Knockdown of CD200 enhances S. aureus -induced inflammatory responses. Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–18 h). ( A – F ) Relative mRNA levels of IL-1β ( A ), IL-6 ( B ), TNF-α ( C ), IL-12p40 ( D ), IL-10 ( E ), or CXCL1 ( F ) were detected by qPCR, with β-actin as an internal control. ( G – L ) The amount of IL-1β ( G ), IL-6 ( H ), TNF-α ( I ), IL-12 ( J ), IL-10 ( K ), or CXCL1 ( L ) in the cell culture supernatant was determined by ELISA. ( M ) The CD200 knockdown efficiency was detected by qPCR. Results are expressed as the mean ± SD of three independent experiments; * p
Figure Legend Snippet: Knockdown of CD200 enhances S. aureus -induced inflammatory responses. Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–18 h). ( A – F ) Relative mRNA levels of IL-1β ( A ), IL-6 ( B ), TNF-α ( C ), IL-12p40 ( D ), IL-10 ( E ), or CXCL1 ( F ) were detected by qPCR, with β-actin as an internal control. ( G – L ) The amount of IL-1β ( G ), IL-6 ( H ), TNF-α ( I ), IL-12 ( J ), IL-10 ( K ), or CXCL1 ( L ) in the cell culture supernatant was determined by ELISA. ( M ) The CD200 knockdown efficiency was detected by qPCR. Results are expressed as the mean ± SD of three independent experiments; * p

Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

CD200 signaling inhibits NO synthesis and bactericidal activity of S. aureus -infected macrophages. ( A – C ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–12 h). Relative mRNA levels of Arg1 ( A ) or iNOS ( B ) were detected by qPCR. NO release was determined using Griess reagent system ( C ). ( D – F ) Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–18 h). Arg1 ( D ) and iNOS ( E ) mRNA levels and NO release ( F ) were determined. ( G ) PEMs transfected with siCD200 or NC siRNA were challenged with CFSE-labeled S. aureus (MOI = 10) for indicated time periods (0–18 h). Cells were collected, fixed, and stained with DAPI. Intracellular bacterial were observed using a fluorescence microscope. A partially enlarged view was shown in the lower left corner. Scale bars: 10 µm. Shown are representative images from three independent experiments. ( H ) Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then challenged with S. aureus (MOI = 10) for indicated time periods (0–18 h). Cells were lysed and the intracellular bacterial burden was determined by CFU counting. Results are expressed as the mean ± SD of three independent experiments; * p
Figure Legend Snippet: CD200 signaling inhibits NO synthesis and bactericidal activity of S. aureus -infected macrophages. ( A – C ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–12 h). Relative mRNA levels of Arg1 ( A ) or iNOS ( B ) were detected by qPCR. NO release was determined using Griess reagent system ( C ). ( D – F ) Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–18 h). Arg1 ( D ) and iNOS ( E ) mRNA levels and NO release ( F ) were determined. ( G ) PEMs transfected with siCD200 or NC siRNA were challenged with CFSE-labeled S. aureus (MOI = 10) for indicated time periods (0–18 h). Cells were collected, fixed, and stained with DAPI. Intracellular bacterial were observed using a fluorescence microscope. A partially enlarged view was shown in the lower left corner. Scale bars: 10 µm. Shown are representative images from three independent experiments. ( H ) Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then challenged with S. aureus (MOI = 10) for indicated time periods (0–18 h). Cells were lysed and the intracellular bacterial burden was determined by CFU counting. Results are expressed as the mean ± SD of three independent experiments; * p

Techniques Used: Activity Assay, Infection, Real-time Polymerase Chain Reaction, Transfection, Labeling, Staining, Fluorescence, Microscopy

S. aureus infection induces CD200 expression in murine macrophages. Mouse BMDMs ( A , D ), PEMs ( B , E ), or RAW264.7 cells ( C , F ) were challenged with S. aureus (MOI = 1) for the indicated time periods (0–18 h), or with S. aureus at the indicated MOIs (0–20) for 6 h. Cells were then collected and detected for CD200 mRNA level by qPCR. Results are expressed as the mean ± SD of three independent experiments; * p
Figure Legend Snippet: S. aureus infection induces CD200 expression in murine macrophages. Mouse BMDMs ( A , D ), PEMs ( B , E ), or RAW264.7 cells ( C , F ) were challenged with S. aureus (MOI = 1) for the indicated time periods (0–18 h), or with S. aureus at the indicated MOIs (0–20) for 6 h. Cells were then collected and detected for CD200 mRNA level by qPCR. Results are expressed as the mean ± SD of three independent experiments; * p

Techniques Used: Infection, Expressing, Real-time Polymerase Chain Reaction

CD200 mildly affects NF-κB activation triggered by Staphylococcal infection. Mouse PEMs were transfected with CD200 siRNA or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–2 h). ( A ) Cells were lysed, and the protein level of p-p65, p65, p-IκBα, or IκBα was detected by western blotting. β-Actin served as a loading control. ( B ) Immunofluorescence was performed to visualize p65 nuclear translocation. Scale bars: 10 µm. Representative images of three independent experiments are shown. The phosphorylation state of p65 was semi-quantitated using ImageJ software. ND means not detected; * p
Figure Legend Snippet: CD200 mildly affects NF-κB activation triggered by Staphylococcal infection. Mouse PEMs were transfected with CD200 siRNA or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–2 h). ( A ) Cells were lysed, and the protein level of p-p65, p65, p-IκBα, or IκBα was detected by western blotting. β-Actin served as a loading control. ( B ) Immunofluorescence was performed to visualize p65 nuclear translocation. Scale bars: 10 µm. Representative images of three independent experiments are shown. The phosphorylation state of p65 was semi-quantitated using ImageJ software. ND means not detected; * p

Techniques Used: Activation Assay, Infection, Transfection, Western Blot, Immunofluorescence, Translocation Assay, Software

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Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling
Article Snippet: .. Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions. .. RNA Isolation, Reverse Transcription, and qRT-PCR Total RNA was isolated from BMDMs, PEMs, or RAW264.7 cells with the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

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    GenePharma Company sirna the sirna molecules targeting cd200 mrna
    <t>CD200</t> restrains p38 signaling upon Staphylococcal infection. ( A – L ) Mouse PEMs were transfected with CD200 <t>siRNA</t> (siCD200) or control siRNA (NC) for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, JNK, p-TAK1, TAK1, p-MMK3/6, MMK3/6, p-CREB, CREB, p-MSK1, MSK, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( M – Q ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( R ) RAW264.7 macrophages were pre-transfected with CD200 siRNA for 48 h and then challenged with S. aureus (MOI = 10) for 2 h. The AP-1 luciferase activity was analyzed by the dual luciferase. Representative blots from three independent experiments are shown ( A , E , J , M , and O ). Changes in the phosphorylation state of different proteins were semi-quantitated using ImageJ software ( B – D , F – I , K – L , N , P – Q ). ND means not detected; * p
    Sirna The Sirna Molecules Targeting Cd200 Mrna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna the sirna molecules targeting cd200 mrna/product/GenePharma Company
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sirna the sirna molecules targeting cd200 mrna - by Bioz Stars, 2020-09
    93/100 stars
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    CD200 restrains p38 signaling upon Staphylococcal infection. ( A – L ) Mouse PEMs were transfected with CD200 siRNA (siCD200) or control siRNA (NC) for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, JNK, p-TAK1, TAK1, p-MMK3/6, MMK3/6, p-CREB, CREB, p-MSK1, MSK, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( M – Q ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( R ) RAW264.7 macrophages were pre-transfected with CD200 siRNA for 48 h and then challenged with S. aureus (MOI = 10) for 2 h. The AP-1 luciferase activity was analyzed by the dual luciferase. Representative blots from three independent experiments are shown ( A , E , J , M , and O ). Changes in the phosphorylation state of different proteins were semi-quantitated using ImageJ software ( B – D , F – I , K – L , N , P – Q ). ND means not detected; * p

    Journal: International Journal of Molecular Sciences

    Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

    doi: 10.3390/ijms20030659

    Figure Lengend Snippet: CD200 restrains p38 signaling upon Staphylococcal infection. ( A – L ) Mouse PEMs were transfected with CD200 siRNA (siCD200) or control siRNA (NC) for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, JNK, p-TAK1, TAK1, p-MMK3/6, MMK3/6, p-CREB, CREB, p-MSK1, MSK, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( M – Q ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( R ) RAW264.7 macrophages were pre-transfected with CD200 siRNA for 48 h and then challenged with S. aureus (MOI = 10) for 2 h. The AP-1 luciferase activity was analyzed by the dual luciferase. Representative blots from three independent experiments are shown ( A , E , J , M , and O ). Changes in the phosphorylation state of different proteins were semi-quantitated using ImageJ software ( B – D , F – I , K – L , N , P – Q ). ND means not detected; * p

    Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

    Techniques: Infection, Transfection, Expressing, Western Blot, Luciferase, Activity Assay, Software

    The p38 inhibition largely abolished the effects of CD200 on NO synthesis induced by staphylococcal infection. ( A – D ) Mouse PEMs ( A , B ) or BMDMs ( C , D ) were pre-treated with SB239063 (10 μM, 0.5 h) or vehicle (DMSO), and then treated with S. aureus for indicated time periods (0–18 h). The mRNA level of iNOS was detected by qPCR, and the concentration of NO in culture supernatants was determined using Griess reagent. ( E , F ) Mouse PEMs were transfected with CD200 siRNA or NC for 48 h and treated with SB239063 or vehicle (DMSO) for 0.5 h. Cells were then infected with S. aureus for indicated time periods (0–12 h). The mRNA level of iNOS and the concentration of NO in culture supernatants were analyzed. Results are expressed as the mean ± SD of three independent experiments; * p

    Journal: International Journal of Molecular Sciences

    Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

    doi: 10.3390/ijms20030659

    Figure Lengend Snippet: The p38 inhibition largely abolished the effects of CD200 on NO synthesis induced by staphylococcal infection. ( A – D ) Mouse PEMs ( A , B ) or BMDMs ( C , D ) were pre-treated with SB239063 (10 μM, 0.5 h) or vehicle (DMSO), and then treated with S. aureus for indicated time periods (0–18 h). The mRNA level of iNOS was detected by qPCR, and the concentration of NO in culture supernatants was determined using Griess reagent. ( E , F ) Mouse PEMs were transfected with CD200 siRNA or NC for 48 h and treated with SB239063 or vehicle (DMSO) for 0.5 h. Cells were then infected with S. aureus for indicated time periods (0–12 h). The mRNA level of iNOS and the concentration of NO in culture supernatants were analyzed. Results are expressed as the mean ± SD of three independent experiments; * p

    Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

    Techniques: Inhibition, Infection, Real-time Polymerase Chain Reaction, Concentration Assay, Transfection

    CD200 inhibits the production of inflammatory cytokines induced by S. aureus . Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–12 h). ( A – F ) Relative mRNA expression levels of IL-1β ( A ), IL-6 ( B ), TNF-α ( C ), IL-12p40 ( D ), IL-10 ( E ), or CXCL1 ( F ) were detected by qPCR, with β-actin as an internal control. ( G – L ) The amount of IL-1β ( G ), IL-6 ( H ), TNF-α ( I ), IL-12 ( J ), IL-10 ( K ), or CXCL1 ( L ) in the cell culture supernatant was determined by ELISA. Results are expressed as the mean ± SD of three independent experiments; * p

    Journal: International Journal of Molecular Sciences

    Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

    doi: 10.3390/ijms20030659

    Figure Lengend Snippet: CD200 inhibits the production of inflammatory cytokines induced by S. aureus . Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–12 h). ( A – F ) Relative mRNA expression levels of IL-1β ( A ), IL-6 ( B ), TNF-α ( C ), IL-12p40 ( D ), IL-10 ( E ), or CXCL1 ( F ) were detected by qPCR, with β-actin as an internal control. ( G – L ) The amount of IL-1β ( G ), IL-6 ( H ), TNF-α ( I ), IL-12 ( J ), IL-10 ( K ), or CXCL1 ( L ) in the cell culture supernatant was determined by ELISA. Results are expressed as the mean ± SD of three independent experiments; * p

    Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    Knockdown of CD200 enhances S. aureus -induced inflammatory responses. Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–18 h). ( A – F ) Relative mRNA levels of IL-1β ( A ), IL-6 ( B ), TNF-α ( C ), IL-12p40 ( D ), IL-10 ( E ), or CXCL1 ( F ) were detected by qPCR, with β-actin as an internal control. ( G – L ) The amount of IL-1β ( G ), IL-6 ( H ), TNF-α ( I ), IL-12 ( J ), IL-10 ( K ), or CXCL1 ( L ) in the cell culture supernatant was determined by ELISA. ( M ) The CD200 knockdown efficiency was detected by qPCR. Results are expressed as the mean ± SD of three independent experiments; * p

    Journal: International Journal of Molecular Sciences

    Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

    doi: 10.3390/ijms20030659

    Figure Lengend Snippet: Knockdown of CD200 enhances S. aureus -induced inflammatory responses. Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–18 h). ( A – F ) Relative mRNA levels of IL-1β ( A ), IL-6 ( B ), TNF-α ( C ), IL-12p40 ( D ), IL-10 ( E ), or CXCL1 ( F ) were detected by qPCR, with β-actin as an internal control. ( G – L ) The amount of IL-1β ( G ), IL-6 ( H ), TNF-α ( I ), IL-12 ( J ), IL-10 ( K ), or CXCL1 ( L ) in the cell culture supernatant was determined by ELISA. ( M ) The CD200 knockdown efficiency was detected by qPCR. Results are expressed as the mean ± SD of three independent experiments; * p

    Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    CD200 signaling inhibits NO synthesis and bactericidal activity of S. aureus -infected macrophages. ( A – C ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–12 h). Relative mRNA levels of Arg1 ( A ) or iNOS ( B ) were detected by qPCR. NO release was determined using Griess reagent system ( C ). ( D – F ) Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–18 h). Arg1 ( D ) and iNOS ( E ) mRNA levels and NO release ( F ) were determined. ( G ) PEMs transfected with siCD200 or NC siRNA were challenged with CFSE-labeled S. aureus (MOI = 10) for indicated time periods (0–18 h). Cells were collected, fixed, and stained with DAPI. Intracellular bacterial were observed using a fluorescence microscope. A partially enlarged view was shown in the lower left corner. Scale bars: 10 µm. Shown are representative images from three independent experiments. ( H ) Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then challenged with S. aureus (MOI = 10) for indicated time periods (0–18 h). Cells were lysed and the intracellular bacterial burden was determined by CFU counting. Results are expressed as the mean ± SD of three independent experiments; * p

    Journal: International Journal of Molecular Sciences

    Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

    doi: 10.3390/ijms20030659

    Figure Lengend Snippet: CD200 signaling inhibits NO synthesis and bactericidal activity of S. aureus -infected macrophages. ( A – C ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–12 h). Relative mRNA levels of Arg1 ( A ) or iNOS ( B ) were detected by qPCR. NO release was determined using Griess reagent system ( C ). ( D – F ) Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–18 h). Arg1 ( D ) and iNOS ( E ) mRNA levels and NO release ( F ) were determined. ( G ) PEMs transfected with siCD200 or NC siRNA were challenged with CFSE-labeled S. aureus (MOI = 10) for indicated time periods (0–18 h). Cells were collected, fixed, and stained with DAPI. Intracellular bacterial were observed using a fluorescence microscope. A partially enlarged view was shown in the lower left corner. Scale bars: 10 µm. Shown are representative images from three independent experiments. ( H ) Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then challenged with S. aureus (MOI = 10) for indicated time periods (0–18 h). Cells were lysed and the intracellular bacterial burden was determined by CFU counting. Results are expressed as the mean ± SD of three independent experiments; * p

    Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

    Techniques: Activity Assay, Infection, Real-time Polymerase Chain Reaction, Transfection, Labeling, Staining, Fluorescence, Microscopy

    S. aureus infection induces CD200 expression in murine macrophages. Mouse BMDMs ( A , D ), PEMs ( B , E ), or RAW264.7 cells ( C , F ) were challenged with S. aureus (MOI = 1) for the indicated time periods (0–18 h), or with S. aureus at the indicated MOIs (0–20) for 6 h. Cells were then collected and detected for CD200 mRNA level by qPCR. Results are expressed as the mean ± SD of three independent experiments; * p

    Journal: International Journal of Molecular Sciences

    Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

    doi: 10.3390/ijms20030659

    Figure Lengend Snippet: S. aureus infection induces CD200 expression in murine macrophages. Mouse BMDMs ( A , D ), PEMs ( B , E ), or RAW264.7 cells ( C , F ) were challenged with S. aureus (MOI = 1) for the indicated time periods (0–18 h), or with S. aureus at the indicated MOIs (0–20) for 6 h. Cells were then collected and detected for CD200 mRNA level by qPCR. Results are expressed as the mean ± SD of three independent experiments; * p

    Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

    Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction

    CD200 mildly affects NF-κB activation triggered by Staphylococcal infection. Mouse PEMs were transfected with CD200 siRNA or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–2 h). ( A ) Cells were lysed, and the protein level of p-p65, p65, p-IκBα, or IκBα was detected by western blotting. β-Actin served as a loading control. ( B ) Immunofluorescence was performed to visualize p65 nuclear translocation. Scale bars: 10 µm. Representative images of three independent experiments are shown. The phosphorylation state of p65 was semi-quantitated using ImageJ software. ND means not detected; * p

    Journal: International Journal of Molecular Sciences

    Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

    doi: 10.3390/ijms20030659

    Figure Lengend Snippet: CD200 mildly affects NF-κB activation triggered by Staphylococcal infection. Mouse PEMs were transfected with CD200 siRNA or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–2 h). ( A ) Cells were lysed, and the protein level of p-p65, p65, p-IκBα, or IκBα was detected by western blotting. β-Actin served as a loading control. ( B ) Immunofluorescence was performed to visualize p65 nuclear translocation. Scale bars: 10 µm. Representative images of three independent experiments are shown. The phosphorylation state of p65 was semi-quantitated using ImageJ software. ND means not detected; * p

    Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

    Techniques: Activation Assay, Infection, Transfection, Western Blot, Immunofluorescence, Translocation Assay, Software