Structured Review

InvivoGen pam3csk4
Ezh2 regulates TRAF6 degradation and its downstream signaling. (A and B) Analysis of TRAF6 Lys48-linked ubiquitination in WT and Ezh2 -deficient macrophages (A) or in Ezh2- KO BV2 cell that infected with EV or lentiviral vector encoding WT Ezh2 (B) that was stimulated with LPS at the indicated time points. (C–E) IB analysis of TRAF6 protein levels in WT and Ezh2 -deficient macrophages (C), in microglia (D), or in Ezh2- KO BV2 cells that were infected with EV or lentiviral vector encoding WT Ezh2 ( E ) that was stimulated without or with LPS at the indicated time points. The relative expression of Traf6 compared with loading controls (Actin or Hsp60) was quantified and presented below the Traf6 IB panels. (F and G) qRT-PCR analysis of Traf6 mRNA expression in WT and Ezh2 -deficient macrophages (F) or microglia (G) that was stimulated with LPS at the indicated time points. (H–K) Immunoblot analysis of phosphorylated (P-) and total NF-κB and MAPKs signaling proteins, H3K27me3, H3, Ezh2, or Hsp60 (loading control) in whole-cell lysates of WT and Ezh2 -deficient macrophages that were left unstimulated or stimulated with LPS (100 ng/ml; H) or <t>Pam3CSK4</t> (100 ng/ml; I) or poly I:C (pI:C, 20 µg/ml; J), and IB of the indicated signal protein in the whole-cell lysates of WT and Ezh2 -deficient primary microglia that were left unstimulated or stimulated with LPS (100 ng/ml; K) at the indicated time points. The qRT-PCR data were normalized to a reference gene, Actb (β-actin), and other data are shown as mean ± SD based on three independent experiments.
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Images

1) Product Images from "Macrophage/microglial Ezh2 facilitates autoimmune inflammation through inhibition of Socs3"

Article Title: Macrophage/microglial Ezh2 facilitates autoimmune inflammation through inhibition of Socs3

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20171417

Ezh2 regulates TRAF6 degradation and its downstream signaling. (A and B) Analysis of TRAF6 Lys48-linked ubiquitination in WT and Ezh2 -deficient macrophages (A) or in Ezh2- KO BV2 cell that infected with EV or lentiviral vector encoding WT Ezh2 (B) that was stimulated with LPS at the indicated time points. (C–E) IB analysis of TRAF6 protein levels in WT and Ezh2 -deficient macrophages (C), in microglia (D), or in Ezh2- KO BV2 cells that were infected with EV or lentiviral vector encoding WT Ezh2 ( E ) that was stimulated without or with LPS at the indicated time points. The relative expression of Traf6 compared with loading controls (Actin or Hsp60) was quantified and presented below the Traf6 IB panels. (F and G) qRT-PCR analysis of Traf6 mRNA expression in WT and Ezh2 -deficient macrophages (F) or microglia (G) that was stimulated with LPS at the indicated time points. (H–K) Immunoblot analysis of phosphorylated (P-) and total NF-κB and MAPKs signaling proteins, H3K27me3, H3, Ezh2, or Hsp60 (loading control) in whole-cell lysates of WT and Ezh2 -deficient macrophages that were left unstimulated or stimulated with LPS (100 ng/ml; H) or Pam3CSK4 (100 ng/ml; I) or poly I:C (pI:C, 20 µg/ml; J), and IB of the indicated signal protein in the whole-cell lysates of WT and Ezh2 -deficient primary microglia that were left unstimulated or stimulated with LPS (100 ng/ml; K) at the indicated time points. The qRT-PCR data were normalized to a reference gene, Actb (β-actin), and other data are shown as mean ± SD based on three independent experiments.
Figure Legend Snippet: Ezh2 regulates TRAF6 degradation and its downstream signaling. (A and B) Analysis of TRAF6 Lys48-linked ubiquitination in WT and Ezh2 -deficient macrophages (A) or in Ezh2- KO BV2 cell that infected with EV or lentiviral vector encoding WT Ezh2 (B) that was stimulated with LPS at the indicated time points. (C–E) IB analysis of TRAF6 protein levels in WT and Ezh2 -deficient macrophages (C), in microglia (D), or in Ezh2- KO BV2 cells that were infected with EV or lentiviral vector encoding WT Ezh2 ( E ) that was stimulated without or with LPS at the indicated time points. The relative expression of Traf6 compared with loading controls (Actin or Hsp60) was quantified and presented below the Traf6 IB panels. (F and G) qRT-PCR analysis of Traf6 mRNA expression in WT and Ezh2 -deficient macrophages (F) or microglia (G) that was stimulated with LPS at the indicated time points. (H–K) Immunoblot analysis of phosphorylated (P-) and total NF-κB and MAPKs signaling proteins, H3K27me3, H3, Ezh2, or Hsp60 (loading control) in whole-cell lysates of WT and Ezh2 -deficient macrophages that were left unstimulated or stimulated with LPS (100 ng/ml; H) or Pam3CSK4 (100 ng/ml; I) or poly I:C (pI:C, 20 µg/ml; J), and IB of the indicated signal protein in the whole-cell lysates of WT and Ezh2 -deficient primary microglia that were left unstimulated or stimulated with LPS (100 ng/ml; K) at the indicated time points. The qRT-PCR data were normalized to a reference gene, Actb (β-actin), and other data are shown as mean ± SD based on three independent experiments.

Techniques Used: Infection, Plasmid Preparation, Expressing, Quantitative RT-PCR

2) Product Images from "FcαRI co-stimulation converts human intestinal CD103+ dendritic cells into pro-inflammatory cells through glycolytic reprogramming"

Article Title: FcαRI co-stimulation converts human intestinal CD103+ dendritic cells into pro-inflammatory cells through glycolytic reprogramming

Journal: Nature Communications

doi: 10.1038/s41467-018-03318-5

FcαRI stimulation promotes cytokine gene translation and induces caspase-1 activation. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3), IgA immune complexes (IgA-IC), or a combination and analysed for mRNA expression of indicated genes using qPCR (normalized to GAPDH expression, fold increase compared to unstimulated control). b mRNA stability of TNF of CD103 + DCs which were unstimulated or treated with Pam3CSK4 or co-stimulation with IgA-IC was determined after addition of 10 µg/mL actinomycin D (Act D) to prevent de novo synthesis of mRNA. TNF expression was analysed using qPCR (normalized to GAPDH ). c Lysates of CD103 + DCs stimulated for 3 h with Pam3CSK4 or Pam3CSK4 with IgA-IC were loaded on sucrose gradients to measure mRNA translation of TNF (normalized to GAPDH , qPCR was performed in duplicate, mean + s.e.m.). d Induction of caspase-1 activation in CD103 + DCs after stimulation with Pam3CSK4, IgA-IC, or a combination was measured using caspase-1 binding compound FAM-YVAD-FMK (FLICA) by flow cytometry. Representative example ( a – d ) of three independent experiments
Figure Legend Snippet: FcαRI stimulation promotes cytokine gene translation and induces caspase-1 activation. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3), IgA immune complexes (IgA-IC), or a combination and analysed for mRNA expression of indicated genes using qPCR (normalized to GAPDH expression, fold increase compared to unstimulated control). b mRNA stability of TNF of CD103 + DCs which were unstimulated or treated with Pam3CSK4 or co-stimulation with IgA-IC was determined after addition of 10 µg/mL actinomycin D (Act D) to prevent de novo synthesis of mRNA. TNF expression was analysed using qPCR (normalized to GAPDH ). c Lysates of CD103 + DCs stimulated for 3 h with Pam3CSK4 or Pam3CSK4 with IgA-IC were loaded on sucrose gradients to measure mRNA translation of TNF (normalized to GAPDH , qPCR was performed in duplicate, mean + s.e.m.). d Induction of caspase-1 activation in CD103 + DCs after stimulation with Pam3CSK4, IgA-IC, or a combination was measured using caspase-1 binding compound FAM-YVAD-FMK (FLICA) by flow cytometry. Representative example ( a – d ) of three independent experiments

Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Activated Clotting Time Assay, Binding Assay, Flow Cytometry, Cytometry

FcαRI-induced upregulation of cytokine production is dependent on glycolytic reprogramming through Syk, PI3K, and TBK1-IKKε. a Real-time changes in the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of CD103 + dendritic cells (DC) left unstimulated (open circles), stimulated with Pam3CSK4 (Pam3) alone (grey circles), or Pam3CSK4 with IgA immune complexes (IgA-IC) (black circles). IgA-IC are present from the start of the experiment. The dotted line indicates initiation of further treatment. Experiments were performed in quadruplicate, mean + s.e.m. b Relative lactate production of CD103 + DCs after 24 h of treatment. Pooled data from five experiments. * p
Figure Legend Snippet: FcαRI-induced upregulation of cytokine production is dependent on glycolytic reprogramming through Syk, PI3K, and TBK1-IKKε. a Real-time changes in the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of CD103 + dendritic cells (DC) left unstimulated (open circles), stimulated with Pam3CSK4 (Pam3) alone (grey circles), or Pam3CSK4 with IgA immune complexes (IgA-IC) (black circles). IgA-IC are present from the start of the experiment. The dotted line indicates initiation of further treatment. Experiments were performed in quadruplicate, mean + s.e.m. b Relative lactate production of CD103 + DCs after 24 h of treatment. Pooled data from five experiments. * p

Techniques Used:

IgA-IC promote proinflammatory cytokine production through synergy with various PRRs. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3), IgA immune complexes (IgA-IC), or a combination. b Cytokine production by CD103 + DCs stimulated with Pam3CSK4 or Pam3CSK4 combined with IgA-IC. Each pair of dots represents one donor. * p
Figure Legend Snippet: IgA-IC promote proinflammatory cytokine production through synergy with various PRRs. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3), IgA immune complexes (IgA-IC), or a combination. b Cytokine production by CD103 + DCs stimulated with Pam3CSK4 or Pam3CSK4 combined with IgA-IC. Each pair of dots represents one donor. * p

Techniques Used:

Co-stimulation of CD103 + DCs with IgA-IC promotes Th17 responses and activates intestinal ILC3. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3) alone or Pam3CSK4 together with IgA immune complexes (IgA-IC) and co-cultured with CD4 + T Cells. After T-cell outgrowth, resting cells were re-stimulated and after 24 h supernatant was assayed with ELISA, mean + s.e.m. Experiments were performed in triplicate. b , c Supernatant from CD103 + DCs which were stimulated with Pam3CSK4 or Pam3CSK4 with IgA-IC was transferred onto human intestinal ILC3 and cultured for 7 days and analysed for mRNA expression ( b ) or protein expression ( c ). ND, not detected. Experiments were performed in triplicate. mRNA expression of indicated genes were assayed using qPCR (normalized to GAPDH expression) mean + s.e.m. Supernatant was analysed using ELISA, mean + s.e.m. Representative example of three ( a , b ) or two ( c ) experiments using different donors
Figure Legend Snippet: Co-stimulation of CD103 + DCs with IgA-IC promotes Th17 responses and activates intestinal ILC3. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3) alone or Pam3CSK4 together with IgA immune complexes (IgA-IC) and co-cultured with CD4 + T Cells. After T-cell outgrowth, resting cells were re-stimulated and after 24 h supernatant was assayed with ELISA, mean + s.e.m. Experiments were performed in triplicate. b , c Supernatant from CD103 + DCs which were stimulated with Pam3CSK4 or Pam3CSK4 with IgA-IC was transferred onto human intestinal ILC3 and cultured for 7 days and analysed for mRNA expression ( b ) or protein expression ( c ). ND, not detected. Experiments were performed in triplicate. mRNA expression of indicated genes were assayed using qPCR (normalized to GAPDH expression) mean + s.e.m. Supernatant was analysed using ELISA, mean + s.e.m. Representative example of three ( a , b ) or two ( c ) experiments using different donors

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

Complexed IgA1 and IgA2 amplify proinflammatory cytokine production through FcαRI. a FcαRI expression on unstimulated CD103 + dendritic cells (DC) was analysed using flow cytometry. Light grey histogram indicates background staining. b Prior to stimulation, CD103 + DCs were incubated with a blocking antibody against FcαRI and stimulated with Pam3CSK4 (Pam3) alone or Pam3CSK4 together with IgA immune complexes (IgA-IC). c Cytokine production by CD103 + DCs stimulated with Pam3CSK4 alone or in combination with IgA1-IC or IgA2-IC. Experiments b and c were performed in triplicate. After 24 h cytokine levels were analysed using ELISA, mean + s.e.m. Representative example ( a – c ) of three experiments using different donors
Figure Legend Snippet: Complexed IgA1 and IgA2 amplify proinflammatory cytokine production through FcαRI. a FcαRI expression on unstimulated CD103 + dendritic cells (DC) was analysed using flow cytometry. Light grey histogram indicates background staining. b Prior to stimulation, CD103 + DCs were incubated with a blocking antibody against FcαRI and stimulated with Pam3CSK4 (Pam3) alone or Pam3CSK4 together with IgA immune complexes (IgA-IC). c Cytokine production by CD103 + DCs stimulated with Pam3CSK4 alone or in combination with IgA1-IC or IgA2-IC. Experiments b and c were performed in triplicate. After 24 h cytokine levels were analysed using ELISA, mean + s.e.m. Representative example ( a – c ) of three experiments using different donors

Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay

3) Product Images from "Potentiation and tolerance of toll-like receptor priming in human endothelial cells"

Article Title: Potentiation and tolerance of toll-like receptor priming in human endothelial cells

Journal: Translational research : the journal of laboratory and clinical medicine

doi: 10.1016/j.trsl.2016.08.001

The effect of Pam3Csk4 is differentially regulated between HMVECs and HUVECs. HMVECs (left) or HUVECs (right) were primed with the TLR2 agonist, Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 24 hours (HUVECs) or 32 hours (HMVECs). Afterward, cells were exposed to Pam3Csk4 (10 μ g/ml) for 6 hours (HUVECs) or 16 hours (HMVECs). Culture supernatants were collected at the completion of the experiments and examined for IL-6 ( A ) or IP-10 ( B ) by ELISA. Data are expressed as means ± standard error of replicated, individual experiments (n = 4). * indicates P
Figure Legend Snippet: The effect of Pam3Csk4 is differentially regulated between HMVECs and HUVECs. HMVECs (left) or HUVECs (right) were primed with the TLR2 agonist, Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 24 hours (HUVECs) or 32 hours (HMVECs). Afterward, cells were exposed to Pam3Csk4 (10 μ g/ml) for 6 hours (HUVECs) or 16 hours (HMVECs). Culture supernatants were collected at the completion of the experiments and examined for IL-6 ( A ) or IP-10 ( B ) by ELISA. Data are expressed as means ± standard error of replicated, individual experiments (n = 4). * indicates P

Techniques Used: Enzyme-linked Immunosorbent Assay

IRF7 increased while RIP1 decreased in LPS-treated cells primed with Poly I:C. HMVECs were primed with Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 32 hours. Afterward, cells were exposed to LPS (100 ng/ml) for 1 hour. Cell lysates were collected and run for Western blot analysis. Blots were probed for total and phosphorylated proteins. ( A ) Representative images showing phosphorylated (p-RIP1, p-IKK) and total proteins (IRF7, RIP1, IKK, and tubulin). ( B ) Bar graphs showing the relative abundance of phosphorylated or total proteins after normalization to respective total protein or tubulin expression. Data are expressed as means ± standard error of individual experiments (n = 4) after normalization to control quantities. * indicates P
Figure Legend Snippet: IRF7 increased while RIP1 decreased in LPS-treated cells primed with Poly I:C. HMVECs were primed with Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 32 hours. Afterward, cells were exposed to LPS (100 ng/ml) for 1 hour. Cell lysates were collected and run for Western blot analysis. Blots were probed for total and phosphorylated proteins. ( A ) Representative images showing phosphorylated (p-RIP1, p-IKK) and total proteins (IRF7, RIP1, IKK, and tubulin). ( B ) Bar graphs showing the relative abundance of phosphorylated or total proteins after normalization to respective total protein or tubulin expression. Data are expressed as means ± standard error of individual experiments (n = 4) after normalization to control quantities. * indicates P

Techniques Used: Western Blot, Expressing

Pam3Csk4 and LPS priming induced tolerance, while Poly I:C potentiated. HMVECs (left) or HUVECs (right) were primed with the Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 24 hours (HUVECs) or 32 hours (HMVECs). Afterward, cells were exposed to LPS (100 ng/ml) for 6 hours (HUVECs) or 16 hours (HMVECs). Culture supernatants were collected at the completion of the experiments and examined for IL-6 ( A ) or IP-10 ( B ) by ELISA. ( C ) HMVECs (left) and HUVECs (right) were exposed to Poly I:C (10 μ g/ml) or medium (control) for 16 hours, washed in fresh media for 24 hours and then exposed to LPS (100 ng/ml) or medium (control). Supernatants were collected at the end of the 16 hours and examined for IP-10 via ELISA. Data are expressed as means ± standard error of replicated, individual experiments (n = 4). * indicates P
Figure Legend Snippet: Pam3Csk4 and LPS priming induced tolerance, while Poly I:C potentiated. HMVECs (left) or HUVECs (right) were primed with the Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 24 hours (HUVECs) or 32 hours (HMVECs). Afterward, cells were exposed to LPS (100 ng/ml) for 6 hours (HUVECs) or 16 hours (HMVECs). Culture supernatants were collected at the completion of the experiments and examined for IL-6 ( A ) or IP-10 ( B ) by ELISA. ( C ) HMVECs (left) and HUVECs (right) were exposed to Poly I:C (10 μ g/ml) or medium (control) for 16 hours, washed in fresh media for 24 hours and then exposed to LPS (100 ng/ml) or medium (control). Supernatants were collected at the end of the 16 hours and examined for IP-10 via ELISA. Data are expressed as means ± standard error of replicated, individual experiments (n = 4). * indicates P

Techniques Used: Enzyme-linked Immunosorbent Assay

MAPKs are increased in Poly I:C primed cells challenged with LPS. HMVECs were primed with Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 32 hours. Afterward, cells were exposed to LPS (100 ng/ml) for 1 hour. Cell lysates were collected and run for Western blot analysis. Blots were probed for total and phosphorylated proteins. ( A ) Representative images showing phosphorylated (p-p38, p-JNK, p-ERK 1/2) and total proteins (p38, JNK, ERK 1/2). ( B ) Bar graphs showing the relative abundance of phosphorylated after normalization to respective total protein. Data are expressed as means ± standard error of individual experiments (n = 4) after normalization to control quantities. * indicates P
Figure Legend Snippet: MAPKs are increased in Poly I:C primed cells challenged with LPS. HMVECs were primed with Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 32 hours. Afterward, cells were exposed to LPS (100 ng/ml) for 1 hour. Cell lysates were collected and run for Western blot analysis. Blots were probed for total and phosphorylated proteins. ( A ) Representative images showing phosphorylated (p-p38, p-JNK, p-ERK 1/2) and total proteins (p38, JNK, ERK 1/2). ( B ) Bar graphs showing the relative abundance of phosphorylated after normalization to respective total protein. Data are expressed as means ± standard error of individual experiments (n = 4) after normalization to control quantities. * indicates P

Techniques Used: Western Blot

4) Product Images from "Bacterial Lipoproteins Induce BAFF Production via TLR2/MyD88/JNK Signaling Pathways in Dendritic Cells"

Article Title: Bacterial Lipoproteins Induce BAFF Production via TLR2/MyD88/JNK Signaling Pathways in Dendritic Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.564699

Pam3CSK4 increases BAFF expression in a dose- and time-dependent manner. (A, C) For time experiments, the DCs were stimulated with 10 μg/ml of Pam3CSK4 for 0–72 h. (B, D) For dose experiments, the cells were stimulated with 0-10 μg/ml of Pam3CSK4 for 72 h. (A, B) After stimulation, the cells were stained with antibodies for mouse CD11c conjugated with APC and BAFF conjugated with FITC, and subjected to flow cytometry. The histograms are representative results from flow cytometry for mBAFF, and the value given in each histogram is the percentage of mBAFF-positive cells among 10,000 events of CD11c-positive cells. (C, D) At the end the stimulation, culture media were collected and soluble BAFF (sBAFF) levels were measured by ELISA. Values are the mean ± standard deviation of three separate experiments. Asterisk (*) indicates statistical significance at p
Figure Legend Snippet: Pam3CSK4 increases BAFF expression in a dose- and time-dependent manner. (A, C) For time experiments, the DCs were stimulated with 10 μg/ml of Pam3CSK4 for 0–72 h. (B, D) For dose experiments, the cells were stimulated with 0-10 μg/ml of Pam3CSK4 for 72 h. (A, B) After stimulation, the cells were stained with antibodies for mouse CD11c conjugated with APC and BAFF conjugated with FITC, and subjected to flow cytometry. The histograms are representative results from flow cytometry for mBAFF, and the value given in each histogram is the percentage of mBAFF-positive cells among 10,000 events of CD11c-positive cells. (C, D) At the end the stimulation, culture media were collected and soluble BAFF (sBAFF) levels were measured by ELISA. Values are the mean ± standard deviation of three separate experiments. Asterisk (*) indicates statistical significance at p

Techniques Used: Expressing, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

NF-κB and Sp1 are major transcription factors involved in the Pam3CSK4-induced BAFF expression. (A) Nuclear extracts were prepared from DCs stimulated with 10 μg/ml of Pam3CSK4 for 0–8 h and incubated with 32 P-labeled oligonucleotides containing NF-κB, Sp1, or C/EBP consensus sequences. One picomole of an unlabeled probe (Cold) was used in a competition assay to confirm specific binding. Reaction products were separated on a polyacrylamide gel and visualized by autoradiography. (B) The 293/TLR1-TLR2 or 293/Null cells transiently transfected with the mouse BAFF promoter luciferase reporter construct together with pRL-TK vector were stimulated with 10 μg/ml of Pam3CSK4 for 24 h. (C, D) The cells were pre-treated with indicated inhibitor (BAY11-7082 for NF-κB inhibition or mithramycin A for Sp1 inhibition) for 1 h followed by stimulation with 10 μg/ml of Pam3CSK4 for an additional 24 h. DMSO (0.1%) was used as a vehicle control (VC) for each inhibitor. After the stimulation, cytosolic extracts were subjected to the luciferase activity assay. Values are the mean ± standard deviation of triplicates. Asterisk (*) indicates a significant difference at p
Figure Legend Snippet: NF-κB and Sp1 are major transcription factors involved in the Pam3CSK4-induced BAFF expression. (A) Nuclear extracts were prepared from DCs stimulated with 10 μg/ml of Pam3CSK4 for 0–8 h and incubated with 32 P-labeled oligonucleotides containing NF-κB, Sp1, or C/EBP consensus sequences. One picomole of an unlabeled probe (Cold) was used in a competition assay to confirm specific binding. Reaction products were separated on a polyacrylamide gel and visualized by autoradiography. (B) The 293/TLR1-TLR2 or 293/Null cells transiently transfected with the mouse BAFF promoter luciferase reporter construct together with pRL-TK vector were stimulated with 10 μg/ml of Pam3CSK4 for 24 h. (C, D) The cells were pre-treated with indicated inhibitor (BAY11-7082 for NF-κB inhibition or mithramycin A for Sp1 inhibition) for 1 h followed by stimulation with 10 μg/ml of Pam3CSK4 for an additional 24 h. DMSO (0.1%) was used as a vehicle control (VC) for each inhibitor. After the stimulation, cytosolic extracts were subjected to the luciferase activity assay. Values are the mean ± standard deviation of triplicates. Asterisk (*) indicates a significant difference at p

Techniques Used: Expressing, Incubation, Labeling, Competitive Binding Assay, Binding Assay, Autoradiography, Transfection, Luciferase, Construct, Plasmid Preparation, Inhibition, Activity Assay, Standard Deviation

Lipopeptides mimicking bacterial lipoproteins induce higher BAFF expression compared with other microbial products in DCs. (A) DCs were stimulated with 0-10 μg/ml of Pam3CSK4, Pam2CSK4, standard LPS (stdLPS), or ultra-pure LPS (upLPS) for 48 h. (B) DCs were stimulated with Pam3CSK4 (10 μg/ml), poly(I:C) (25 μg/ml), peptidoglycan (10 μg/ml), or flagellin (100 ng/ml) for 48 h. (C) DCs were stimulated with 0–10 μg/ml of Pam3CSK4, LTA from S. aureus , or zymosan for 48 h. After stimulation, the cells were stained with CD11c antibodies conjugated with APC and BAFF antibodies conjugated with FITC, and subjected to flow cytometry. The histograms are representative results from flow cytometry for mBAFF from each treatment group, and the value given in each histogram is the percentage of mBAFF-positive cells among 10,000 events of CD11c-positive cells.
Figure Legend Snippet: Lipopeptides mimicking bacterial lipoproteins induce higher BAFF expression compared with other microbial products in DCs. (A) DCs were stimulated with 0-10 μg/ml of Pam3CSK4, Pam2CSK4, standard LPS (stdLPS), or ultra-pure LPS (upLPS) for 48 h. (B) DCs were stimulated with Pam3CSK4 (10 μg/ml), poly(I:C) (25 μg/ml), peptidoglycan (10 μg/ml), or flagellin (100 ng/ml) for 48 h. (C) DCs were stimulated with 0–10 μg/ml of Pam3CSK4, LTA from S. aureus , or zymosan for 48 h. After stimulation, the cells were stained with CD11c antibodies conjugated with APC and BAFF antibodies conjugated with FITC, and subjected to flow cytometry. The histograms are representative results from flow cytometry for mBAFF from each treatment group, and the value given in each histogram is the percentage of mBAFF-positive cells among 10,000 events of CD11c-positive cells.

Techniques Used: Expressing, Staining, Flow Cytometry

Pam3CSK4 enhances TLR1 expression. (A) Total RNA was isolated from DCs and subjected to RT-PCR to determine TLR1 to TLR9 mRNA expression levels. Each TLR mRNA expression normalized with that of β-actin is presented as the percentage change ± standard deviation of three separate experiments against TLR1 mRNA expression, set as 100% by densitometric analysis. Asterisk (*) indicates statistical significance at p
Figure Legend Snippet: Pam3CSK4 enhances TLR1 expression. (A) Total RNA was isolated from DCs and subjected to RT-PCR to determine TLR1 to TLR9 mRNA expression levels. Each TLR mRNA expression normalized with that of β-actin is presented as the percentage change ± standard deviation of three separate experiments against TLR1 mRNA expression, set as 100% by densitometric analysis. Asterisk (*) indicates statistical significance at p

Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Pam3CSK4 induces an increase in BAFF mRNA levels. (A) DCs were stimulated with 10 μg/ml of Pam3CSK4 for 0–72 h, or (B) with Pam3CSK4 at 0-10 μg/ml for 72 h. Total RNA was isolated and cDNA was synthesized as described in the Materials and Methods. The cDNA was subjected to RT-PCR to determine expression levels of BAFF mRNA. Upper , β-actin–normalized BAFF mRNA expression is presented as the percentage change ± standard deviation of three separate experiments against the group showing the highest ratio [72 h and 10 μg/ml treatment group in (A) and (B) , respectively], set as 100% by densitometric analysis. Lower , a representative BAFF mRNA expression determined by RT-PCR. Asterisk (*) indicates statistical significance at p
Figure Legend Snippet: Pam3CSK4 induces an increase in BAFF mRNA levels. (A) DCs were stimulated with 10 μg/ml of Pam3CSK4 for 0–72 h, or (B) with Pam3CSK4 at 0-10 μg/ml for 72 h. Total RNA was isolated and cDNA was synthesized as described in the Materials and Methods. The cDNA was subjected to RT-PCR to determine expression levels of BAFF mRNA. Upper , β-actin–normalized BAFF mRNA expression is presented as the percentage change ± standard deviation of three separate experiments against the group showing the highest ratio [72 h and 10 μg/ml treatment group in (A) and (B) , respectively], set as 100% by densitometric analysis. Lower , a representative BAFF mRNA expression determined by RT-PCR. Asterisk (*) indicates statistical significance at p

Techniques Used: Isolation, Synthesized, Reverse Transcription Polymerase Chain Reaction, Expressing, Standard Deviation

Pam3CSK4-induced BAFF expression is mediated through TLR2 and MyD88. (A) DCs from wild-type, TLR2- or MyD88-deficient mice were stimulated with Pam3CSK4 at 0-10 μg/ml for 72 h. The cells were then stained with antibodies for mouse CD11c conjugated with APC and BAFF conjugated with FITC, and subjected to flow cytometry. The histograms are representative results from flow cytometry for mBAFF, and the value in each histogram is the percentage of mBAFF-positive cells among 10,000 events of CD11c-positive cells. (B) Under the same culture condition, sBAFF levels in culture media were measured by ELISA. The values are the mean ± standard deviation of three separate experiments. Asterisk (*) indicates a significant difference at p
Figure Legend Snippet: Pam3CSK4-induced BAFF expression is mediated through TLR2 and MyD88. (A) DCs from wild-type, TLR2- or MyD88-deficient mice were stimulated with Pam3CSK4 at 0-10 μg/ml for 72 h. The cells were then stained with antibodies for mouse CD11c conjugated with APC and BAFF conjugated with FITC, and subjected to flow cytometry. The histograms are representative results from flow cytometry for mBAFF, and the value in each histogram is the percentage of mBAFF-positive cells among 10,000 events of CD11c-positive cells. (B) Under the same culture condition, sBAFF levels in culture media were measured by ELISA. The values are the mean ± standard deviation of three separate experiments. Asterisk (*) indicates a significant difference at p

Techniques Used: Expressing, Mouse Assay, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

JNK MAP kinase is essential for Pam3CSK4-induced BAFF expression at the transcriptional level. (A–D) DCs were pre-treated with 0–20 μM of the indicated MAP kinase inhibitor (SP600125 and JNK V inhibitor for JNK inhibition, SB202190 for p38 inhibition, or PD98059 for ERK inhibition) for 1 h followed by stimulation with 10 μg/ml of Pam3CSK4 for an additional 72 h. Dimethyl sulfoxide (DMSO; 0.1%) was used as a vehicle control (VC) for each MAP kinase inhibitor. Total RNA was isolated and subjected to RT-PCR to measure BAFF mRNA levels. Upper , BAFF mRNA expression normalized with that of β-actin is presented as the percentage change ± standard deviation of three separate experiments against the Pam3CSK4 treatment group, set as 100% by densitometric analysis. Lower , a representative BAFF mRNA expression determined by RT-PCR. Asterisk (*) indicates statistical significance at p
Figure Legend Snippet: JNK MAP kinase is essential for Pam3CSK4-induced BAFF expression at the transcriptional level. (A–D) DCs were pre-treated with 0–20 μM of the indicated MAP kinase inhibitor (SP600125 and JNK V inhibitor for JNK inhibition, SB202190 for p38 inhibition, or PD98059 for ERK inhibition) for 1 h followed by stimulation with 10 μg/ml of Pam3CSK4 for an additional 72 h. Dimethyl sulfoxide (DMSO; 0.1%) was used as a vehicle control (VC) for each MAP kinase inhibitor. Total RNA was isolated and subjected to RT-PCR to measure BAFF mRNA levels. Upper , BAFF mRNA expression normalized with that of β-actin is presented as the percentage change ± standard deviation of three separate experiments against the Pam3CSK4 treatment group, set as 100% by densitometric analysis. Lower , a representative BAFF mRNA expression determined by RT-PCR. Asterisk (*) indicates statistical significance at p

Techniques Used: Expressing, Inhibition, Isolation, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

5) Product Images from "First Insight into the Modulation of Noncanonical NF-κB Signaling Components by Poxviruses in Established Immune-Derived Cell Lines: An In Vitro Model of Ectromelia Virus Infection"

Article Title: First Insight into the Modulation of Noncanonical NF-κB Signaling Components by Poxviruses in Established Immune-Derived Cell Lines: An In Vitro Model of Ectromelia Virus Infection

Journal: Pathogens

doi: 10.3390/pathogens9100814

ECTV inhibits phosphorylation of p100 and RelB and affects p100 processing in JAWS II and RAW 264.7 cells. ( A ) Immunoblot analysis of phosphorylation of RelB in unstimulated or PMA + Io-stimulated mock-, uvi-ECTV-, and ECTV-infected JAWS II cells. ( B ) Immunoblot analysis of the content of p-RelB and p-p100 in unstimulated or rmIFN-γ + Escherichia coli O111:B4 LPS-stimulated mock- and ECTV-infected JAWS II cells. ( C ) Immunoblot analysis of p-RelB and p-p100 in unstimulated and Pam3CSK4- or rmIFN-γ + LPS O111:B4-stimulated mock- and ECTV-infected RAW 264.7 cells. ( D ) Immunoblot analysis of p100/p52 content in mock- and ECTV-infected JAWS II cells treated with poly(I:C) or rmIFN-γ + LPS O111:B4. ( E ) Densitometric analysis of the p100/p52 ratio and p52 content in JAWS II cells. Data obtained from two independent experiments are shown on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control. ( F ) Immunoblot analysis of p100/p52 proteins in mock- and ECTV-infected RAW 264.7 cells at 4 hpi. The cells were left untreated or treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( G ) Densitometric analysis of the p100/p52 ratio and p52 content in RAW 264.7 cells based on the data obtained from two independent experiments. Data are presented on histograms with a logarithmic scale. GAPDH—loading control. ( H ) Immunoblot analysis of p100/p52 proteins in mock- and ECTV-infected RAW 264.7 cells at 18 hpi. The cells were left untreated or treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( I ) Densitometric analysis of the p100/p52 ratio and p52 content in RAW 264.7 cells based on the data obtained from two independent experiments. Data are presented on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control.
Figure Legend Snippet: ECTV inhibits phosphorylation of p100 and RelB and affects p100 processing in JAWS II and RAW 264.7 cells. ( A ) Immunoblot analysis of phosphorylation of RelB in unstimulated or PMA + Io-stimulated mock-, uvi-ECTV-, and ECTV-infected JAWS II cells. ( B ) Immunoblot analysis of the content of p-RelB and p-p100 in unstimulated or rmIFN-γ + Escherichia coli O111:B4 LPS-stimulated mock- and ECTV-infected JAWS II cells. ( C ) Immunoblot analysis of p-RelB and p-p100 in unstimulated and Pam3CSK4- or rmIFN-γ + LPS O111:B4-stimulated mock- and ECTV-infected RAW 264.7 cells. ( D ) Immunoblot analysis of p100/p52 content in mock- and ECTV-infected JAWS II cells treated with poly(I:C) or rmIFN-γ + LPS O111:B4. ( E ) Densitometric analysis of the p100/p52 ratio and p52 content in JAWS II cells. Data obtained from two independent experiments are shown on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control. ( F ) Immunoblot analysis of p100/p52 proteins in mock- and ECTV-infected RAW 264.7 cells at 4 hpi. The cells were left untreated or treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( G ) Densitometric analysis of the p100/p52 ratio and p52 content in RAW 264.7 cells based on the data obtained from two independent experiments. Data are presented on histograms with a logarithmic scale. GAPDH—loading control. ( H ) Immunoblot analysis of p100/p52 proteins in mock- and ECTV-infected RAW 264.7 cells at 18 hpi. The cells were left untreated or treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( I ) Densitometric analysis of the p100/p52 ratio and p52 content in RAW 264.7 cells based on the data obtained from two independent experiments. Data are presented on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control.

Techniques Used: Infection

ECTV influences the expression of components of the noncanonical NF-κB signaling pathway in JAWS II and RAW 264.7 cells. ( A ) Immunoblots of mock- and ECTV-infected JAWS II cells untreated or treated with poly(I:C) or rmIFN-γ + Escherichia coli LPS O111:B4. ( B ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in JAWS II cells. The analysis was based on the data of two independent biological experiments. The data are shown on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control. ( C ) Immunoblots of mock- and ECTV-infected RAW 264.7 cells at 4 hpi. Cells were left untreated or were treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( D ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in RAW 264.7 cells. The analysis was based on the data of two independent biological experiments and is plotted on histograms with a logarithmic scale. GAPDH—loading control. ( E ) Immunoblots of mock- and ECTV-infected RAW 264.7 cells at 18 hpi. Cells were left untreated or were treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( F ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in RAW 264.7 cells. The analysis was based on the data of two independent biological experiments and is plotted on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control.
Figure Legend Snippet: ECTV influences the expression of components of the noncanonical NF-κB signaling pathway in JAWS II and RAW 264.7 cells. ( A ) Immunoblots of mock- and ECTV-infected JAWS II cells untreated or treated with poly(I:C) or rmIFN-γ + Escherichia coli LPS O111:B4. ( B ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in JAWS II cells. The analysis was based on the data of two independent biological experiments. The data are shown on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control. ( C ) Immunoblots of mock- and ECTV-infected RAW 264.7 cells at 4 hpi. Cells were left untreated or were treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( D ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in RAW 264.7 cells. The analysis was based on the data of two independent biological experiments and is plotted on histograms with a logarithmic scale. GAPDH—loading control. ( E ) Immunoblots of mock- and ECTV-infected RAW 264.7 cells at 18 hpi. Cells were left untreated or were treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( F ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in RAW 264.7 cells. The analysis was based on the data of two independent biological experiments and is plotted on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control.

Techniques Used: Expressing, Western Blot, Infection

6) Product Images from "Tissue‐resident macrophages actively suppress IL‐1beta release via a reactive prostanoid/IL‐10 pathway"

Article Title: Tissue‐resident macrophages actively suppress IL‐1beta release via a reactive prostanoid/IL‐10 pathway

Journal: The EMBO Journal

doi: 10.15252/embj.2019103454

Aberrant cytokine release from LPS‐stimulated Gata6‐deficient resident peritoneal macrophages Gata6‐WT and Gata6‐KO mye peritoneal macrophages (pMФ) were unstimulated (–) or stimulated with TLR2L Pam3CSK4 (500 ng/ml), TLR3L Poly(I:C) (1 μg/ml), TLR4L ultrapure LPS (100 ng/ml), TLR5L flagellin (100 ng/ml), TLR7 and 8L R848 (1 μg/ml) or TLR9L CpG ODN1826 (5 μM). Culture supernatants were collected 24 h after the start of stimulation and IL‐1β and TNF ELISA were performed. n = 5, two‐way ANOVA analysis with Tukey's multiple comparison post‐test. IL‐1β (C) and TNF (D) ELISA of Gata6‐WT and Gata6‐KO mye pMФ stimulated for 24 h with the indicated LPS concentrations. n = 3, two‐way ANOVA analysis with Sidak's multiple comparison post‐test. IL‐1β (E) and TNF (F) ELISA from Gata6‐WT and Gata6‐KO mye pMФ stimulated with LPS (100 ng/ml) for the indicated times. n = 3, two‐way ANOVA analysis with Sidak's multiple comparison post‐test. IL‐1β ELISA of Gata6‐WT and Gata6‐KO mye pMФ stimulated for 18 h with 100 ng/ml LPS or recombinant IL‐1 receptor antagonist (rIL‐1ra), n = 4–5, two‐way ANOVA analysis with Tukey's multiple comparison post‐test. IL‐1β ELISA of Gata6‐WT and Gata6‐KO mye pMФ stimulated for 18 h with 100 ng/ml LPS or 100 ng/ml recombinant TNF (recTNF), n = 4–5, two‐way ANOVA analysis with Tukey's multiple comparison post‐test. Data information: Data are expressed as mean ± SEM. * P
Figure Legend Snippet: Aberrant cytokine release from LPS‐stimulated Gata6‐deficient resident peritoneal macrophages Gata6‐WT and Gata6‐KO mye peritoneal macrophages (pMФ) were unstimulated (–) or stimulated with TLR2L Pam3CSK4 (500 ng/ml), TLR3L Poly(I:C) (1 μg/ml), TLR4L ultrapure LPS (100 ng/ml), TLR5L flagellin (100 ng/ml), TLR7 and 8L R848 (1 μg/ml) or TLR9L CpG ODN1826 (5 μM). Culture supernatants were collected 24 h after the start of stimulation and IL‐1β and TNF ELISA were performed. n = 5, two‐way ANOVA analysis with Tukey's multiple comparison post‐test. IL‐1β (C) and TNF (D) ELISA of Gata6‐WT and Gata6‐KO mye pMФ stimulated for 24 h with the indicated LPS concentrations. n = 3, two‐way ANOVA analysis with Sidak's multiple comparison post‐test. IL‐1β (E) and TNF (F) ELISA from Gata6‐WT and Gata6‐KO mye pMФ stimulated with LPS (100 ng/ml) for the indicated times. n = 3, two‐way ANOVA analysis with Sidak's multiple comparison post‐test. IL‐1β ELISA of Gata6‐WT and Gata6‐KO mye pMФ stimulated for 18 h with 100 ng/ml LPS or recombinant IL‐1 receptor antagonist (rIL‐1ra), n = 4–5, two‐way ANOVA analysis with Tukey's multiple comparison post‐test. IL‐1β ELISA of Gata6‐WT and Gata6‐KO mye pMФ stimulated for 18 h with 100 ng/ml LPS or 100 ng/ml recombinant TNF (recTNF), n = 4–5, two‐way ANOVA analysis with Tukey's multiple comparison post‐test. Data information: Data are expressed as mean ± SEM. * P

Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant

7) Product Images from "Human Innate Immune Cells Respond Differentially to Poly-γ-Glutamic Acid Polymers from Bacillus anthracis and Nonpathogenic Bacillus Species"

Article Title: Human Innate Immune Cells Respond Differentially to Poly-γ-Glutamic Acid Polymers from Bacillus anthracis and Nonpathogenic Bacillus Species

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1901066

B. subtilis PGA is a human TLR2/6 agonist. 500 μg/ml B. anthracis PGA, B. licheniformis PGA, or B. subtilis PGA was tested in a TLR2 assay with neutralizing polyclonal Abs against TLR1 or TLR6 or normal rat IgG. Pam3CSK4 and FSL-1 were run as TLR1/2-and TLR2/6-positive controls, respectively. Mean data for three experiments are presented as ligand/vehicle ratios ± SEM ( n = 3). Statistical significance of signal for each PGA and positive control ligand above its vehicle was determined by t tests comparing the mean treatment/background ratios. Statistical significance of inhibition by the Abs was determined by t tests comparing the mean ligand/vehicle ratios for normal rat IgG treatment versus anti-TLR1 or anti-TLR6 Ab treatment. * p
Figure Legend Snippet: B. subtilis PGA is a human TLR2/6 agonist. 500 μg/ml B. anthracis PGA, B. licheniformis PGA, or B. subtilis PGA was tested in a TLR2 assay with neutralizing polyclonal Abs against TLR1 or TLR6 or normal rat IgG. Pam3CSK4 and FSL-1 were run as TLR1/2-and TLR2/6-positive controls, respectively. Mean data for three experiments are presented as ligand/vehicle ratios ± SEM ( n = 3). Statistical significance of signal for each PGA and positive control ligand above its vehicle was determined by t tests comparing the mean treatment/background ratios. Statistical significance of inhibition by the Abs was determined by t tests comparing the mean ligand/vehicle ratios for normal rat IgG treatment versus anti-TLR1 or anti-TLR6 Ab treatment. * p

Techniques Used: Positive Control, Inhibition

8) Product Images from "Increased CD40 Expression Enhances Early STING-Mediated Type I Interferon Response and Host Survival in a Rodent Malaria Model"

Article Title: Increased CD40 Expression Enhances Early STING-Mediated Type I Interferon Response and Host Survival in a Rodent Malaria Model

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005930

Stimulation of CD40 expression and ISRE driven luciferase signals by infected red blood cells (iRBCs), parasite DNA/RNA, or parasite GPI. ( A ) Flow cytometry counts of RAW Lucia cells containing N67 iRBCs 1 h and 8 h post incubation. The top two panels are RBCs, and the bottom panels are iRBCs. SSC, side-scattered light; CF-SE, signals of carboxyfluorescein succinimidyl ester (CF-SE) labeled RBCs or iRBCs ( B ) CD40 protein expression in RAW Lucia cells after incubation with RBCs or iRBCs. The antibody was against an epitope at C-terminus of the CD40 protein. Anti-β-actin was used as protein loading control. ( C ) The same as ( B ) but in DC2.4 cells. ( D ) ISRE promoter driven luciferase signals in RAW Lucia cells after ingestion with RBCs or iRBCs. ( E ) CD40 protein expression in RAW Lucia cells stimulated with parasite genomic DNA or RNA with or without the presence of lipofectamine. ( F ) Luciferase activities driven by ISRE promoter after stimulation with parasite DNA or RNA as in ( E ). ( G ) CD40 protein expression in RAW Lucia cells stimulated with parasite DNA or RNA with or without DNase or RNase treatment, respectively. EDTA and/or lipofectamine (Lipo) were used as buffer controls. ( H ) Luciferase activities driven by ISRE promoter after stimulation with parasite DNA or RNA with or without DNase or RNase treatment, respectively, as in ( G ). ( I ) CD40 protein expression in RAW Lucia cells stimulated with LPS, Pam3CSK4, and Plasmodium falciparum GPI (glycophosphatidylinositol) dissolved in ethanol (marked with EH) or H 2 O. ( J ) Protein band intensities in ( I ) relative to non-stimulated in ethanol (NoneEH = 1). ( K ) Luciferase activities driven by ISRE promoter in RAW Lucia cells stimulated with LPS, Pam3CSK4, and P . falciparum GPI as in ( I ). For ( D ), ( F ), ( H ), and ( K ), data are means+s.d. from three experiments; t -test, * P
Figure Legend Snippet: Stimulation of CD40 expression and ISRE driven luciferase signals by infected red blood cells (iRBCs), parasite DNA/RNA, or parasite GPI. ( A ) Flow cytometry counts of RAW Lucia cells containing N67 iRBCs 1 h and 8 h post incubation. The top two panels are RBCs, and the bottom panels are iRBCs. SSC, side-scattered light; CF-SE, signals of carboxyfluorescein succinimidyl ester (CF-SE) labeled RBCs or iRBCs ( B ) CD40 protein expression in RAW Lucia cells after incubation with RBCs or iRBCs. The antibody was against an epitope at C-terminus of the CD40 protein. Anti-β-actin was used as protein loading control. ( C ) The same as ( B ) but in DC2.4 cells. ( D ) ISRE promoter driven luciferase signals in RAW Lucia cells after ingestion with RBCs or iRBCs. ( E ) CD40 protein expression in RAW Lucia cells stimulated with parasite genomic DNA or RNA with or without the presence of lipofectamine. ( F ) Luciferase activities driven by ISRE promoter after stimulation with parasite DNA or RNA as in ( E ). ( G ) CD40 protein expression in RAW Lucia cells stimulated with parasite DNA or RNA with or without DNase or RNase treatment, respectively. EDTA and/or lipofectamine (Lipo) were used as buffer controls. ( H ) Luciferase activities driven by ISRE promoter after stimulation with parasite DNA or RNA with or without DNase or RNase treatment, respectively, as in ( G ). ( I ) CD40 protein expression in RAW Lucia cells stimulated with LPS, Pam3CSK4, and Plasmodium falciparum GPI (glycophosphatidylinositol) dissolved in ethanol (marked with EH) or H 2 O. ( J ) Protein band intensities in ( I ) relative to non-stimulated in ethanol (NoneEH = 1). ( K ) Luciferase activities driven by ISRE promoter in RAW Lucia cells stimulated with LPS, Pam3CSK4, and P . falciparum GPI as in ( I ). For ( D ), ( F ), ( H ), and ( K ), data are means+s.d. from three experiments; t -test, * P

Techniques Used: Expressing, Luciferase, Infection, Flow Cytometry, Cytometry, Incubation, Labeling

Stimulation of CD40 and STING expression and ISRE driven luciferase activities by TLR ligands and infected red blood cells (iRBCs). ( A ) RAW Lucia cells (2 × 10 5 ) were stimulated with different ligands for indicated times, and protein levels of CD40 and STING were detected using an antibody against C-terminus of CD40 and anti-STING, respectively. Pam is Pam3CSK4. ( B ) Plots of protein band intensities of CD40 and STING and ISRE-luciferase signals 1, 8, and 24 h after infected red blood cell (iRBC) stimulation. All data for ISRE-luciferase signals are averaged values from three experiments. ( C ) The same plots as in ( B ) after infected poly(I:C) stimulation. ( D ) The same plots as in ( B ) except stimulation with LPS. ( E ) Western blot detection of CD40 protein levels in 293T cells (2 × 10 5 ) after TLR ligand stimulations for 12 h. ( F ) Expression of CD40 in 293T cells (2 × 10 5 ) transfected with various TLRs for 18 h and with or without ligand stimulations for another 12 h before harvesting for proteins and Western blot analysis. The three TLR3 and TLR4 experiments, respectively, were conducted using plasmids containing the genes from different sources.
Figure Legend Snippet: Stimulation of CD40 and STING expression and ISRE driven luciferase activities by TLR ligands and infected red blood cells (iRBCs). ( A ) RAW Lucia cells (2 × 10 5 ) were stimulated with different ligands for indicated times, and protein levels of CD40 and STING were detected using an antibody against C-terminus of CD40 and anti-STING, respectively. Pam is Pam3CSK4. ( B ) Plots of protein band intensities of CD40 and STING and ISRE-luciferase signals 1, 8, and 24 h after infected red blood cell (iRBC) stimulation. All data for ISRE-luciferase signals are averaged values from three experiments. ( C ) The same plots as in ( B ) after infected poly(I:C) stimulation. ( D ) The same plots as in ( B ) except stimulation with LPS. ( E ) Western blot detection of CD40 protein levels in 293T cells (2 × 10 5 ) after TLR ligand stimulations for 12 h. ( F ) Expression of CD40 in 293T cells (2 × 10 5 ) transfected with various TLRs for 18 h and with or without ligand stimulations for another 12 h before harvesting for proteins and Western blot analysis. The three TLR3 and TLR4 experiments, respectively, were conducted using plasmids containing the genes from different sources.

Techniques Used: Expressing, Luciferase, Infection, Western Blot, Transfection

9) Product Images from "Babaodan controls excessive immune responses and may represent a cytokine-targeted agent suitable for COVID-19 treatment"

Article Title: Babaodan controls excessive immune responses and may represent a cytokine-targeted agent suitable for COVID-19 treatment

Journal: Biomedicine & Pharmacotherapy

doi: 10.1016/j.biopha.2021.111586

BBD abolishes IL-6 and TNF-α production after optimized TLR1/2 and TLR3 dual activation of Raw264.7 macrophages. (A) Raw 264.7 cells were grown on 12-well plates and incubated with different combinations of Poly(I:C) and Pam3CSK4 for 24 h. Supernatants were collected, and TNF-α and IL-6 concentrations were determined using the corresponding ELISA kits. (B) Raw 264.7 cells were grown on 6-well plates and incubated with 1 mg/mL of BBD for 24 h, and this was followed by treatment with a combination of Poly(I:C) (10 μg/mL) and Pam3CSK4 (100 ng/mL) for 8 h. IL-6 and TNF-α mRNA levels were detected by RT-qPCR. *** P
Figure Legend Snippet: BBD abolishes IL-6 and TNF-α production after optimized TLR1/2 and TLR3 dual activation of Raw264.7 macrophages. (A) Raw 264.7 cells were grown on 12-well plates and incubated with different combinations of Poly(I:C) and Pam3CSK4 for 24 h. Supernatants were collected, and TNF-α and IL-6 concentrations were determined using the corresponding ELISA kits. (B) Raw 264.7 cells were grown on 6-well plates and incubated with 1 mg/mL of BBD for 24 h, and this was followed by treatment with a combination of Poly(I:C) (10 μg/mL) and Pam3CSK4 (100 ng/mL) for 8 h. IL-6 and TNF-α mRNA levels were detected by RT-qPCR. *** P

Techniques Used: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Gene expression profiling by RNA-seq. Raw 264.7 cells grown on 6-well plates were treated with a combination of 10 μg/mL Poly(I:C) and 100 ng/mL Pam3CSK4 (model) for 8 h with or without pre-treatment with 1 mg/mL of Babaodan (BBD) for 24 h. RNA samples were collected for RNA-seq analysis. Two samples were analyzed per group. Differentially expressed genes (DEG) are identified by absolute log 2 (FC) > 2 and P
Figure Legend Snippet: Gene expression profiling by RNA-seq. Raw 264.7 cells grown on 6-well plates were treated with a combination of 10 μg/mL Poly(I:C) and 100 ng/mL Pam3CSK4 (model) for 8 h with or without pre-treatment with 1 mg/mL of Babaodan (BBD) for 24 h. RNA samples were collected for RNA-seq analysis. Two samples were analyzed per group. Differentially expressed genes (DEG) are identified by absolute log 2 (FC) > 2 and P

Techniques Used: Expressing, RNA Sequencing Assay

Babaodan (BBD) inhibits TLR1/2 and TLR3 dual stimulation-induced cytokine expression in Raw 264.7 macrophages. Raw 264.7 cells were grown on 12-well plates and incubated with different doses of BBD for 24 h, and this was followed by treatment with a combination of Poly(I:C) (10 μg/mL) and Pam3CSK4 (100 ng/mL) for 24 h. Supernatants were collected, and TNF-α and IL-6 concentrations were analyzed using the corresponding ELISA kits. * P
Figure Legend Snippet: Babaodan (BBD) inhibits TLR1/2 and TLR3 dual stimulation-induced cytokine expression in Raw 264.7 macrophages. Raw 264.7 cells were grown on 12-well plates and incubated with different doses of BBD for 24 h, and this was followed by treatment with a combination of Poly(I:C) (10 μg/mL) and Pam3CSK4 (100 ng/mL) for 24 h. Supernatants were collected, and TNF-α and IL-6 concentrations were analyzed using the corresponding ELISA kits. * P

Techniques Used: Expressing, Incubation, Enzyme-linked Immunosorbent Assay

BBD inhibits NF-κB and MAPK signaling in macrophages promoting excessive inflammatory processes. Raw 264.7 cells were grown on 6-well plates and incubated with 1 mg/mL BBD for 24 h, and this was followed by treatment with a combination of Poly(I:C) (10 μg/mL) and Pam3CSK4 (100 ng/mL) (P2P) for 15, 30, 60, and 120 min. The cells were harvested at the indicated time points after stimulation. Cell lysates were resolved by SDS-PAGE, blotted, and analyzed using the respective antibodies. One representative experiment of three is shown.
Figure Legend Snippet: BBD inhibits NF-κB and MAPK signaling in macrophages promoting excessive inflammatory processes. Raw 264.7 cells were grown on 6-well plates and incubated with 1 mg/mL BBD for 24 h, and this was followed by treatment with a combination of Poly(I:C) (10 μg/mL) and Pam3CSK4 (100 ng/mL) (P2P) for 15, 30, 60, and 120 min. The cells were harvested at the indicated time points after stimulation. Cell lysates were resolved by SDS-PAGE, blotted, and analyzed using the respective antibodies. One representative experiment of three is shown.

Techniques Used: Incubation, SDS Page

10) Product Images from "Genotoxic stress modulates the release of exosomes from multiple myeloma cells capable of activating NK cell cytokine production: Role of HSP70/TLR2/NF-kB axis"

Article Title: Genotoxic stress modulates the release of exosomes from multiple myeloma cells capable of activating NK cell cytokine production: Role of HSP70/TLR2/NF-kB axis

Journal: Oncoimmunology

doi: 10.1080/2162402X.2017.1279372

MM exosomes stimulate IFNγ production in a TLR2-dependent manner. (A) NK cells were incubated with increasing doses of Pam3CSK4 or SKO-007(J3) cell-derived exosomes, as indicated. After 24 h, BFA (5 µg/mL) was added and left for additional 24 h. Intracellular IFNγ expression was evaluated by immunofluorescence and FACS analysis. The gating strategy used consists in separating CD56high cells from CD56low NK cells. Numbers indicate the percentage of IFNγ+ cells. A representative experiment is shown. (B) NK cells were incubated with exosomes (20 µg/mL) as described in panel (A). The mean values of four independent experiments ± SEM is shown. (C) NK cells were FACs sorted based on CD56 expression levels, and incubated for 48 h with SKO-007(J3)-derived exosomes (20 µg/mL) or Pam3CSK4 (1 μM). Real-time PCR analysis of IFNγ mRNA was performed and the data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells considered as calibrator. The mean values of five independent experiments ± SEM are shown. (D) Cell surface expression of TLR2 was evaluated on CD56 + CD3 − total NK cells, on CD56 high CD3 − and CD56 low CD3 − NK cell subsets of total PBMC derived from 10 different healthy donors. Values represent the mean fluorescence intensity (MFI) of TLR2 substracted from the MFI value of the isotype control Ig. (E) CD56high NK cells were pretreated for 20 min with anti-TLR2 neutralizing antibody (1μg/106 cells), and then incubated with both TLR2 agonist and exosomes as described in panel (B). Real-time PCR analysis of IFNγ mRNA was performed and the data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells considered as calibrator. Results relative to two representative donors are shown.
Figure Legend Snippet: MM exosomes stimulate IFNγ production in a TLR2-dependent manner. (A) NK cells were incubated with increasing doses of Pam3CSK4 or SKO-007(J3) cell-derived exosomes, as indicated. After 24 h, BFA (5 µg/mL) was added and left for additional 24 h. Intracellular IFNγ expression was evaluated by immunofluorescence and FACS analysis. The gating strategy used consists in separating CD56high cells from CD56low NK cells. Numbers indicate the percentage of IFNγ+ cells. A representative experiment is shown. (B) NK cells were incubated with exosomes (20 µg/mL) as described in panel (A). The mean values of four independent experiments ± SEM is shown. (C) NK cells were FACs sorted based on CD56 expression levels, and incubated for 48 h with SKO-007(J3)-derived exosomes (20 µg/mL) or Pam3CSK4 (1 μM). Real-time PCR analysis of IFNγ mRNA was performed and the data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells considered as calibrator. The mean values of five independent experiments ± SEM are shown. (D) Cell surface expression of TLR2 was evaluated on CD56 + CD3 − total NK cells, on CD56 high CD3 − and CD56 low CD3 − NK cell subsets of total PBMC derived from 10 different healthy donors. Values represent the mean fluorescence intensity (MFI) of TLR2 substracted from the MFI value of the isotype control Ig. (E) CD56high NK cells were pretreated for 20 min with anti-TLR2 neutralizing antibody (1μg/106 cells), and then incubated with both TLR2 agonist and exosomes as described in panel (B). Real-time PCR analysis of IFNγ mRNA was performed and the data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells considered as calibrator. Results relative to two representative donors are shown.

Techniques Used: Incubation, Derivative Assay, Expressing, Immunofluorescence, FACS, Real-time Polymerase Chain Reaction, Fluorescence

11) Product Images from "Interleukin-19 Impairment in Active Crohn's Disease Patients"

Article Title: Interleukin-19 Impairment in Active Crohn's Disease Patients

Journal: PLoS ONE

doi: 10.1371/journal.pone.0093910

IL-19 production after TLR activation in PBMC cultures from active CD patients (n = 3) and HC (n = 6). PBMC were cultured 24(A) and 48 h (B) with different TLR ligands (LPS, Pam3CSK4 or FSL-1) and IL-19 production was determined by ELISA as described in Materials and Methods. The viability of PBMC was determined by Trypan Blue ( > 99%). *p
Figure Legend Snippet: IL-19 production after TLR activation in PBMC cultures from active CD patients (n = 3) and HC (n = 6). PBMC were cultured 24(A) and 48 h (B) with different TLR ligands (LPS, Pam3CSK4 or FSL-1) and IL-19 production was determined by ELISA as described in Materials and Methods. The viability of PBMC was determined by Trypan Blue ( > 99%). *p

Techniques Used: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

rhIL-19 down-regulates TNFα production in cultures from HC after LPS activation. PBMC from HC and active CD patients were cultured with LPS (HC n = 10 and CD n = 5), Pam3CSK4 (HC n = 10 and CD n = 5) and FSL-1 (HC n = 5 and CD n = 3) for 24 h with or without rhIL-19 (400 ng/ml). A and B) TNFα and IL-10 production in cultures from HC after TLR activation with or without rhIL-19. D and E) TNFα and IL-10 production in cultures from active CD patients after TLR activation with or without rhIL-19. C and F) TNFα production from HC and active CD patients cultured with rhIL-10 (200 ng/ml). *p
Figure Legend Snippet: rhIL-19 down-regulates TNFα production in cultures from HC after LPS activation. PBMC from HC and active CD patients were cultured with LPS (HC n = 10 and CD n = 5), Pam3CSK4 (HC n = 10 and CD n = 5) and FSL-1 (HC n = 5 and CD n = 3) for 24 h with or without rhIL-19 (400 ng/ml). A and B) TNFα and IL-10 production in cultures from HC after TLR activation with or without rhIL-19. D and E) TNFα and IL-10 production in cultures from active CD patients after TLR activation with or without rhIL-19. C and F) TNFα production from HC and active CD patients cultured with rhIL-10 (200 ng/ml). *p

Techniques Used: Activation Assay, Cell Culture

Heat map image of IL-10 family cytokines. Monocytes mRNA was extracted from HC (n = 3) and active CD patients (n = 3) after 6 h of culture with medium in the absence or presence of Pam3CSK4 and FSL-1. A) Green denoted down-regulated and red up-regulated genes. B) logFC of IL-19 mRNA expression between CD and HC are shown. **p
Figure Legend Snippet: Heat map image of IL-10 family cytokines. Monocytes mRNA was extracted from HC (n = 3) and active CD patients (n = 3) after 6 h of culture with medium in the absence or presence of Pam3CSK4 and FSL-1. A) Green denoted down-regulated and red up-regulated genes. B) logFC of IL-19 mRNA expression between CD and HC are shown. **p

Techniques Used: Expressing

IL-10 regulates IL-19 expression. PBMC from A) HC (n = 3) and B) active CD patients (n = 3) were activated 24 h with TLR ligands (LPS, Pam3CSK4 and FSL-1) in the presence of rhIL-10 (200 ng/ml) and blocking anti-IL10 (500 ng/ml). Results are expressed as: (IL19 production with TLR+rhIL-10 or anti-IL10-IL-19 production with TLR)/IL-19 production with TLR.
Figure Legend Snippet: IL-10 regulates IL-19 expression. PBMC from A) HC (n = 3) and B) active CD patients (n = 3) were activated 24 h with TLR ligands (LPS, Pam3CSK4 and FSL-1) in the presence of rhIL-10 (200 ng/ml) and blocking anti-IL10 (500 ng/ml). Results are expressed as: (IL19 production with TLR+rhIL-10 or anti-IL10-IL-19 production with TLR)/IL-19 production with TLR.

Techniques Used: Expressing, Blocking Assay

12) Product Images from "Effect of vitamin D3 on the osteogenic differentiation of human periodontal ligament stromal cells under inflammatory conditions"

Article Title: Effect of vitamin D3 on the osteogenic differentiation of human periodontal ligament stromal cells under inflammatory conditions

Journal: Journal of Periodontal Research

doi: 10.1111/jre.12858

1,25(OH) 2 D 3 ‐induced gene expression levels of osteocalcin under inflammatory and osteogenic conditions. Primary hPDLSCs (n = 5) were stimulated with osteogenic induction medium containing 1,25(OH) 2 D 3 (0, 0.1, 10 nM) in the presence and absence of P. gingivalis LPS (1 µg/ml) or Pam3CSK4 (1 µg/ml) for 7 (A), 14 (B) or 21 (C) days. Resulting gene expression levels of osteocalcin were analysed with qPCR. Y‐axes represent the n‐fold expression of the target gene in relation with untreated hPDLSCs. Data are presented as mean ±standard error of the mean of five independent experiments. *Significant difference between groups, p
Figure Legend Snippet: 1,25(OH) 2 D 3 ‐induced gene expression levels of osteocalcin under inflammatory and osteogenic conditions. Primary hPDLSCs (n = 5) were stimulated with osteogenic induction medium containing 1,25(OH) 2 D 3 (0, 0.1, 10 nM) in the presence and absence of P. gingivalis LPS (1 µg/ml) or Pam3CSK4 (1 µg/ml) for 7 (A), 14 (B) or 21 (C) days. Resulting gene expression levels of osteocalcin were analysed with qPCR. Y‐axes represent the n‐fold expression of the target gene in relation with untreated hPDLSCs. Data are presented as mean ±standard error of the mean of five independent experiments. *Significant difference between groups, p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

1,25(OH) 2 D 3 ‐induced osteogenic differentiation under inflammatory conditions. Primary hPDLSCs (n = 5) were stimulated with osteogenic induction medium containing 1,25(OH) 2 D 3 (0, 0.1, 10 nM) in the presence and absence of P. gingivalis LPS (1 µg/ml) or Pam3CSK4 (1 µg/ml) for 21 days. Resulting calcium deposition was analysed by Alizarin Red S staining (A: representative picture of one donor). Quantification of calcium deposits was performed photometrically. Y‐axis represents the optical density measured at 405 nm. Data are presented as mean ±standard error of the mean of five independent experiments. *Significant difference between groups, p
Figure Legend Snippet: 1,25(OH) 2 D 3 ‐induced osteogenic differentiation under inflammatory conditions. Primary hPDLSCs (n = 5) were stimulated with osteogenic induction medium containing 1,25(OH) 2 D 3 (0, 0.1, 10 nM) in the presence and absence of P. gingivalis LPS (1 µg/ml) or Pam3CSK4 (1 µg/ml) for 21 days. Resulting calcium deposition was analysed by Alizarin Red S staining (A: representative picture of one donor). Quantification of calcium deposits was performed photometrically. Y‐axis represents the optical density measured at 405 nm. Data are presented as mean ±standard error of the mean of five independent experiments. *Significant difference between groups, p

Techniques Used: Staining

1,25(OH) 2 D 3 ‐induced gene expression levels of osteopontin under inflammatory and osteogenic conditions. Primary hPDLSCs (n = 5) were stimulated with osteogenic induction medium containing 1,25(OH) 2 D 3 (0, 0.1, 10 nM) in the presence and absence of P. gingivalis LPS (1 µg/ml) or Pam3CSK4 (1 µg/ml) for 7 (A), 14 (B) or 21 (C) days. Resulting gene expression levels of osteopontin were analysed with qPCR. Y‐axes represent the n‐fold expression of the target gene in relation to untreated hPDLSCs. Data are presented as mean ±standard error of the mean of five independent experiments. *Significant difference between groups, p
Figure Legend Snippet: 1,25(OH) 2 D 3 ‐induced gene expression levels of osteopontin under inflammatory and osteogenic conditions. Primary hPDLSCs (n = 5) were stimulated with osteogenic induction medium containing 1,25(OH) 2 D 3 (0, 0.1, 10 nM) in the presence and absence of P. gingivalis LPS (1 µg/ml) or Pam3CSK4 (1 µg/ml) for 7 (A), 14 (B) or 21 (C) days. Resulting gene expression levels of osteopontin were analysed with qPCR. Y‐axes represent the n‐fold expression of the target gene in relation to untreated hPDLSCs. Data are presented as mean ±standard error of the mean of five independent experiments. *Significant difference between groups, p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

1,25(OH) 2 D 3 ‐induced gene expression levels of RUNX2 under inflammatory and osteogenic conditions. Primary hPDLSCs (n = 5) were stimulated with osteogenic induction medium containing 1,25(OH) 2 D 3 (0, 0.1, 10 nM) in the presence and absence of P. gingivalis LPS (1 µg/ml) or Pam3CSK4 (1 µg/ml) for 7 (A), 14 (B) or 21 (C) days. Resulting gene expression levels of RUNX2 were analysed with qPCR. Y‐axes represent the n‐fold expression of the target gene in relation to untreated hPDLSCs. Data are presented as mean ±standard error of the mean of five independent experiments
Figure Legend Snippet: 1,25(OH) 2 D 3 ‐induced gene expression levels of RUNX2 under inflammatory and osteogenic conditions. Primary hPDLSCs (n = 5) were stimulated with osteogenic induction medium containing 1,25(OH) 2 D 3 (0, 0.1, 10 nM) in the presence and absence of P. gingivalis LPS (1 µg/ml) or Pam3CSK4 (1 µg/ml) for 7 (A), 14 (B) or 21 (C) days. Resulting gene expression levels of RUNX2 were analysed with qPCR. Y‐axes represent the n‐fold expression of the target gene in relation to untreated hPDLSCs. Data are presented as mean ±standard error of the mean of five independent experiments

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

13) Product Images from "Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome"

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01920

CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO 2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1β levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p
Figure Legend Snippet: CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO 2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1β levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p

Techniques Used: Activation Assay, Incubation, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

14) Product Images from "Platelet-Rich Fibrin Decreases the Inflammatory Response of Mesenchymal Cells"

Article Title: Platelet-Rich Fibrin Decreases the Inflammatory Response of Mesenchymal Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms222111333

PPP, BC, and PRF can reduce inflammation provoked by TLR2 agonist in ST2 cells. The ST2 cells were exposed to 10% PPP, BC, and 30% PRF lysates in the presence of 5 μg/mL Pam3CSK4, agonists of TLR2. ( A , B ) Data show the x-fold changes of IL6 and iNOS gene expression, and ( C ) the concentration of IL6 in the supernatant of ST2 cells. n = 4.
Figure Legend Snippet: PPP, BC, and PRF can reduce inflammation provoked by TLR2 agonist in ST2 cells. The ST2 cells were exposed to 10% PPP, BC, and 30% PRF lysates in the presence of 5 μg/mL Pam3CSK4, agonists of TLR2. ( A , B ) Data show the x-fold changes of IL6 and iNOS gene expression, and ( C ) the concentration of IL6 in the supernatant of ST2 cells. n = 4.

Techniques Used: Expressing, Concentration Assay

15) Product Images from "NK Cell-Dependent Antibody-Mediated Immunotherapy Is Improved In Vitro and In Vivo When Combined with Agonists for Toll-like Receptor 2 in Head and Neck Cancer Models"

Article Title: NK Cell-Dependent Antibody-Mediated Immunotherapy Is Improved In Vitro and In Vivo When Combined with Agonists for Toll-like Receptor 2 in Head and Neck Cancer Models

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms222011057

Treatment of tumor-bearing mice with cetuximab and Pam3CSK4 results in prolonged survival. Nude mice (six/group) were subcutaneously injected with ADCC-insensitive UM-SCC-47 cells in both flanks. When tumors reached a tumor volume of approximately 70 mm 3 , intraperitoneal treatments were started (day 0). Mice were treated with ( A ) PBS (vehicle), ( B ) Pam3CSK4 (50 µg/mouse), ( C ) cetuximab (CTX, 1 mg/mouse) or ( D ) cetuximab with Pam3CSK4. Treatment days are indicated with arrows (day 0, 3, 7, 10, 17 and 24). Tumor volume is depicted as the mean of two tumors (both flanks, mm 3 ) per mouse. Fragmented grey line represents a tumor volume of 100 mm 3 . ( E ) Relative survival rate of mice in the different treatment groups (six mice/group set at 100%).
Figure Legend Snippet: Treatment of tumor-bearing mice with cetuximab and Pam3CSK4 results in prolonged survival. Nude mice (six/group) were subcutaneously injected with ADCC-insensitive UM-SCC-47 cells in both flanks. When tumors reached a tumor volume of approximately 70 mm 3 , intraperitoneal treatments were started (day 0). Mice were treated with ( A ) PBS (vehicle), ( B ) Pam3CSK4 (50 µg/mouse), ( C ) cetuximab (CTX, 1 mg/mouse) or ( D ) cetuximab with Pam3CSK4. Treatment days are indicated with arrows (day 0, 3, 7, 10, 17 and 24). Tumor volume is depicted as the mean of two tumors (both flanks, mm 3 ) per mouse. Fragmented grey line represents a tumor volume of 100 mm 3 . ( E ) Relative survival rate of mice in the different treatment groups (six mice/group set at 100%).

Techniques Used: Mouse Assay, Injection

Treatment with cetuximab and Pam3CSK4 facilitates infiltration of immune cells into tumors. Nude mice (three/group) were subcutaneously injected with ADCC-insensitive UM-SCC-47 cells in both flanks. Tumors were harvested at day 11 after treatment (cetuximab or cetuximab with Pam3CSK4 at day 0 and 4), n = 1. ( A ) Tumor volume is depicted as the mean of two tumors (both flanks, mm 3 ) ± SEM; * p
Figure Legend Snippet: Treatment with cetuximab and Pam3CSK4 facilitates infiltration of immune cells into tumors. Nude mice (three/group) were subcutaneously injected with ADCC-insensitive UM-SCC-47 cells in both flanks. Tumors were harvested at day 11 after treatment (cetuximab or cetuximab with Pam3CSK4 at day 0 and 4), n = 1. ( A ) Tumor volume is depicted as the mean of two tumors (both flanks, mm 3 ) ± SEM; * p

Techniques Used: Mouse Assay, Injection

Combination of cetuximab with TLR2 agonists induces profound pro-inflammatory responses by PBMCs. ADCC experiments with the HNSCC cell lines VU-SCC-096 (black bars) and UM-SCC-47 (grey bars) were performed with PBMCs in the absence or presence of cetuximab (0.5 µg/mL) and/or Pam3CSK4 (5 µg/mL). After 24 h, supernatants were harvested and used for ( A ) cytokine and ( B ) chemokine analysis. Bars represent mean ± SD; n = 2; ** p
Figure Legend Snippet: Combination of cetuximab with TLR2 agonists induces profound pro-inflammatory responses by PBMCs. ADCC experiments with the HNSCC cell lines VU-SCC-096 (black bars) and UM-SCC-47 (grey bars) were performed with PBMCs in the absence or presence of cetuximab (0.5 µg/mL) and/or Pam3CSK4 (5 µg/mL). After 24 h, supernatants were harvested and used for ( A ) cytokine and ( B ) chemokine analysis. Bars represent mean ± SD; n = 2; ** p

Techniques Used:

HNSCC patient PBMCs are able to induce tumor cell killing. PBMCs isolated from ( A ) 8 healthy controls and ( B ) 16 HNSCC patients were used in ADCC experiments with VU-SCC-096 cells. PBMCs were harvested after 4 h incubation and analyzed for percentages (%) of CD69 (activation marker) and CD107a (degranulation marker) double positive NK cells (gated as CD3-CD56+ cells). Graphs depict % CD69+CD107a+ NK cells in the presence of cetuximab (CTX, 0.5 µg/mL) or cetuximab in combination with Pam3CSK4 (5 µg/mL). * p
Figure Legend Snippet: HNSCC patient PBMCs are able to induce tumor cell killing. PBMCs isolated from ( A ) 8 healthy controls and ( B ) 16 HNSCC patients were used in ADCC experiments with VU-SCC-096 cells. PBMCs were harvested after 4 h incubation and analyzed for percentages (%) of CD69 (activation marker) and CD107a (degranulation marker) double positive NK cells (gated as CD3-CD56+ cells). Graphs depict % CD69+CD107a+ NK cells in the presence of cetuximab (CTX, 0.5 µg/mL) or cetuximab in combination with Pam3CSK4 (5 µg/mL). * p

Techniques Used: Isolation, Incubation, Activation Assay, Marker

TLR2 agonists enhance the number of cytotoxic NK cells in combination with cetuximab. NK cells were harvested from ADCC experiments with the HNSCC cell lines ( A ) VU-SCC-096 or ( B ) UM-SCC-47 after 4 h incubation. NK cells (gated as CD3-CD56+ cells) were analyzed for percentages (%) of CD69 (activation marker) and CD107a (degranulation marker) double positive NK cells (upper right square). ADCC conditions included no stimulation (no mAb, no TLR agonist), cetuximab (CTX), Pam2CSK4 or Pam3CSK4. Combined treatments of cetuximab with TLR2 agonists are indicated by CTX + Pam2CSK4 or CTX + Pam3CSK4. Granzyme B release was measured in supernatants of ADCC experiments with NK cells and ( C ) VU-SCC-096 cells or ( D ) or UM-SCC-47 cells. Bars represent mean ± SD; n > 3; *** p
Figure Legend Snippet: TLR2 agonists enhance the number of cytotoxic NK cells in combination with cetuximab. NK cells were harvested from ADCC experiments with the HNSCC cell lines ( A ) VU-SCC-096 or ( B ) UM-SCC-47 after 4 h incubation. NK cells (gated as CD3-CD56+ cells) were analyzed for percentages (%) of CD69 (activation marker) and CD107a (degranulation marker) double positive NK cells (upper right square). ADCC conditions included no stimulation (no mAb, no TLR agonist), cetuximab (CTX), Pam2CSK4 or Pam3CSK4. Combined treatments of cetuximab with TLR2 agonists are indicated by CTX + Pam2CSK4 or CTX + Pam3CSK4. Granzyme B release was measured in supernatants of ADCC experiments with NK cells and ( C ) VU-SCC-096 cells or ( D ) or UM-SCC-47 cells. Bars represent mean ± SD; n > 3; *** p

Techniques Used: Incubation, Activation Assay, Marker

16) Product Images from "Common and specific downstream signaling targets controlled by Tlr2 and Tlr5 innate immune signaling in zebrafish"

Article Title: Common and specific downstream signaling targets controlled by Tlr2 and Tlr5 innate immune signaling in zebrafish

Journal: BMC Genomics

doi: 10.1186/s12864-015-1740-9

Effects on tlr2 and tlr5 knockdown on the expression of Pam3CSK4 and Flagellin responsive genes a , based on RNAseq data, all the 48 Pam3CSK4 specific fold-changes are inhibited or down-regulated in tlr2 morphants ( tlr2 MO) upon this PAMP stimulation. b , based on RNAseq data, all the 42 flagellin specific fold-changes are inhibited or down-regulated in tlr5a morphants ( tlr5a MO) upon this PAMP stimulation. c , d , all the 80 common fold-changes are inhibited or down-regulated in tlr 2 and tlr 5a morphants. FC, fold-change. Panel a , b , c , d : for the quantitative data and accession numbers of the shown genes (or numbers) we refer to Additional file 7 : Table S2 and Additional file 6 : Table S3
Figure Legend Snippet: Effects on tlr2 and tlr5 knockdown on the expression of Pam3CSK4 and Flagellin responsive genes a , based on RNAseq data, all the 48 Pam3CSK4 specific fold-changes are inhibited or down-regulated in tlr2 morphants ( tlr2 MO) upon this PAMP stimulation. b , based on RNAseq data, all the 42 flagellin specific fold-changes are inhibited or down-regulated in tlr5a morphants ( tlr5a MO) upon this PAMP stimulation. c , d , all the 80 common fold-changes are inhibited or down-regulated in tlr 2 and tlr 5a morphants. FC, fold-change. Panel a , b , c , d : for the quantitative data and accession numbers of the shown genes (or numbers) we refer to Additional file 7 : Table S2 and Additional file 6 : Table S3

Techniques Used: Expressing

Specific and common responses to Tlr2 and Tlr5 ligands. The whole organism transcriptome response of zebrafish embryos to treatment with the Tlr2 ligand Pam3CSK4 or the Tlr5 ligand flagellin results in the induction of specific and common transcription factor genes as indicated in the figure. The common transcription factor genes (in cooperation with other non-inducible factors, e.g. of the NF-κB family), likely function upstream of the effector genes commonly induced by Pam3CSK4 and flagellin. The transcription factor genes induced by only one of the two ligands are likely to contribute to further specificity in the transcriptional responses of downstream effector genes
Figure Legend Snippet: Specific and common responses to Tlr2 and Tlr5 ligands. The whole organism transcriptome response of zebrafish embryos to treatment with the Tlr2 ligand Pam3CSK4 or the Tlr5 ligand flagellin results in the induction of specific and common transcription factor genes as indicated in the figure. The common transcription factor genes (in cooperation with other non-inducible factors, e.g. of the NF-κB family), likely function upstream of the effector genes commonly induced by Pam3CSK4 and flagellin. The transcription factor genes induced by only one of the two ligands are likely to contribute to further specificity in the transcriptional responses of downstream effector genes

Techniques Used:

Immune genes expression at different time points upon Pam3CSK4 stimulation. Embryos were injected at 27 hpf with 1 ng Pam3CSK4 and expression levels of il1b ( a ), il8 ( b ), tnfa ( c ), il10 ( d ) and il6 ( e ) were determined at 1, 3 and 6 hpi by qPCR. Data (mean ± SEM) are combined from at least three biological replicates ( n = 15 embryos per group) and expressed relative to their corresponding mock injection (water) control, which is set at 1. Statistical significance of differences between mock and Pam3CSK4 groups was determined by ANOVA analysis and Tukey’s multiple comparisons test, *p
Figure Legend Snippet: Immune genes expression at different time points upon Pam3CSK4 stimulation. Embryos were injected at 27 hpf with 1 ng Pam3CSK4 and expression levels of il1b ( a ), il8 ( b ), tnfa ( c ), il10 ( d ) and il6 ( e ) were determined at 1, 3 and 6 hpi by qPCR. Data (mean ± SEM) are combined from at least three biological replicates ( n = 15 embryos per group) and expressed relative to their corresponding mock injection (water) control, which is set at 1. Statistical significance of differences between mock and Pam3CSK4 groups was determined by ANOVA analysis and Tukey’s multiple comparisons test, *p

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

il1b expression in tlr2 and tlr5 morphants following PAMPs stimulation. Embryos were injected at the 1-2 cells stage with standard control (Sc), tlr2, tlr5a or tlr5b morpholino (MO) and subsequently injected with Pam3CSK4 at 27 hpf, flagellin or water as a mock control. Expression of il1b was determined by qPCR at 1 hpi. a tlr5a and tlr5b knockdown effect on il1b RNA expression in response to flagellin. b C tlr2 knockdown effect on il1b RNA expression in response to Pam3CSK4 ( b ) or flagellin ( c ). Data (mean ± SEM) are combined from at least three biological replicates ( n = 10 embryos per group) and expressed relative to their corresponding water control, which is set at 1. Statistical significance was determined by ANOVA analysis and Tukey’s multiple comparisons test, *p
Figure Legend Snippet: il1b expression in tlr2 and tlr5 morphants following PAMPs stimulation. Embryos were injected at the 1-2 cells stage with standard control (Sc), tlr2, tlr5a or tlr5b morpholino (MO) and subsequently injected with Pam3CSK4 at 27 hpf, flagellin or water as a mock control. Expression of il1b was determined by qPCR at 1 hpi. a tlr5a and tlr5b knockdown effect on il1b RNA expression in response to flagellin. b C tlr2 knockdown effect on il1b RNA expression in response to Pam3CSK4 ( b ) or flagellin ( c ). Data (mean ± SEM) are combined from at least three biological replicates ( n = 10 embryos per group) and expressed relative to their corresponding water control, which is set at 1. Statistical significance was determined by ANOVA analysis and Tukey’s multiple comparisons test, *p

Techniques Used: Expressing, Injection, Real-time Polymerase Chain Reaction, RNA Expression

qPCR analysis of transcription factors genes responsive to PAMPs stimulation. Expression levels of fosl1a, fosb, junbb, cebpeb, egr3 and nr4a1 following Pam3CSK4 and flagellin stimulation are determined by qPCR. Data (mean ± SD) are combined from at least tree biological replicates ( n = 15 embryos per group) and expressed relative to their corresponding water control, which is set at 1. Statistical significance was determined by two-way ANOVA analysis and Tukey’s multiple comparisons test, *p
Figure Legend Snippet: qPCR analysis of transcription factors genes responsive to PAMPs stimulation. Expression levels of fosl1a, fosb, junbb, cebpeb, egr3 and nr4a1 following Pam3CSK4 and flagellin stimulation are determined by qPCR. Data (mean ± SD) are combined from at least tree biological replicates ( n = 15 embryos per group) and expressed relative to their corresponding water control, which is set at 1. Statistical significance was determined by two-way ANOVA analysis and Tukey’s multiple comparisons test, *p

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

RNAseq experimental setup and comparison of gene sets responsive to Pam3CSK4 or flagellin stimulation. a , setup of the RNAseq experiment. Zebrafish embryos received a 1 nl injection of 1 mg/ml Pam3CSK4 and 100 μg/ml flagellin respectively into the caudal vein at 27hpf. Control embryos were injected with water. Samples for RNAseq were taken at 1hpi. The numbers of differentially expressed genes were assessed by two criteria: 1) p
Figure Legend Snippet: RNAseq experimental setup and comparison of gene sets responsive to Pam3CSK4 or flagellin stimulation. a , setup of the RNAseq experiment. Zebrafish embryos received a 1 nl injection of 1 mg/ml Pam3CSK4 and 100 μg/ml flagellin respectively into the caudal vein at 27hpf. Control embryos were injected with water. Samples for RNAseq were taken at 1hpi. The numbers of differentially expressed genes were assessed by two criteria: 1) p

Techniques Used: Injection

17) Product Images from "HIV-1 Tat protein binds to TLR4-MD2 and signals to induce TNF-α and IL-10"

Article Title: HIV-1 Tat protein binds to TLR4-MD2 and signals to induce TNF-α and IL-10

Journal: Retrovirology

doi: 10.1186/1742-4690-10-123

Tat protein fails to stimulate TNF-α  and IL-10 in macrophages from TLR4 −/−  MD2 −/− , and CD14 −/−  mice. A-B)  Tat induces mTNFα and mIL-10 production in wt C57Bl/6 murine macrophages .  Peritoneal macrophages (5.10 5 /well) from wild type C57BL/6 mice were stimulated for 24 h with increasing concentration of GST-Tat 1–101, GST-Tat 1–45, GST-Tat 30–72 or GST as control. LPS was used as a positive control.  C-F)  TLR4, CD14 and MD2 cell surface membrane proteins are essential for TNF-α and IL-10 production by Tat. Macrophages were isolated from wt mice or mice KO for TLR4, TLR2  (C-D)  or CD14 or MD2  (E-F) . The cells were stimulated with increasing concentrations of GST-Tat 1–101, GST-Tat 1–45 or GST as control. Positive control experiments were performed by using the following TLR ligands: LPS (TLR4-CD14-MD2) and Pam 3 CsK 4  (TLR2-CD14).  G-H)  Human monocytes were pretreated with blocking antibodies, anti-CD14 or anti-MD2 (0.1 - 1 μg/ml) for 60 min. Cells were then stimulated with GST or GST-Tat 1–101 (100 nM).  I-J)  TNF-α and IL-10 production in peritoneal washes from wt  (I)  or TLR4 KO mice  (J)  injected with Tat protein. The data represent means +/− SD of three independent experiments.
Figure Legend Snippet: Tat protein fails to stimulate TNF-α and IL-10 in macrophages from TLR4 −/− MD2 −/− , and CD14 −/− mice. A-B) Tat induces mTNFα and mIL-10 production in wt C57Bl/6 murine macrophages . Peritoneal macrophages (5.10 5 /well) from wild type C57BL/6 mice were stimulated for 24 h with increasing concentration of GST-Tat 1–101, GST-Tat 1–45, GST-Tat 30–72 or GST as control. LPS was used as a positive control. C-F) TLR4, CD14 and MD2 cell surface membrane proteins are essential for TNF-α and IL-10 production by Tat. Macrophages were isolated from wt mice or mice KO for TLR4, TLR2 (C-D) or CD14 or MD2 (E-F) . The cells were stimulated with increasing concentrations of GST-Tat 1–101, GST-Tat 1–45 or GST as control. Positive control experiments were performed by using the following TLR ligands: LPS (TLR4-CD14-MD2) and Pam 3 CsK 4 (TLR2-CD14). G-H) Human monocytes were pretreated with blocking antibodies, anti-CD14 or anti-MD2 (0.1 - 1 μg/ml) for 60 min. Cells were then stimulated with GST or GST-Tat 1–101 (100 nM). I-J) TNF-α and IL-10 production in peritoneal washes from wt (I) or TLR4 KO mice (J) injected with Tat protein. The data represent means +/− SD of three independent experiments.

Techniques Used: Mouse Assay, Concentration Assay, Positive Control, Isolation, Blocking Assay, Injection

18) Product Images from "Type I IFN Induces IL-10 Production in an IL-27–Independent Manner and Blocks Responsiveness to IFN-γ for Production of IL-12 and Bacterial Killing in Mycobacterium tuberculosis–Infected Macrophages"

Article Title: Type I IFN Induces IL-10 Production in an IL-27–Independent Manner and Blocks Responsiveness to IFN-γ for Production of IL-12 and Bacterial Killing in Mycobacterium tuberculosis–Infected Macrophages

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1401088

Type I IFN regulates IL-10 production in M. tuberculosis –infected macrophages independently of IL-27 signaling. ( A ) WT macrophages were infected with M. tuberculosis in the presence of increasing concentrations of IFN-β, added at the time of infection, and levels of IL-10 in culture supernatant were determined by ELISA at 24 h postinfection. ( B ) WT macrophages were infected with M. tuberculosis in the presence or absence of 2 ng/ml IFN-β, added at the time of infection, and levels of Il10 mRNA were determined by quantitative RT-PCR (qRT-PCR) at the time points indicated after infection. ( C ) WT and Ifnar1 −/− macrophages were infected with M. tuberculosis and levels of IL-10 in culture supernatant were determined by ELISA at 24 h postinfection. ( D ) WT and Ifnar1 −/− macrophages were infected with M. tuberculosis and levels of Il10 mRNA determined by qRT-PCR at the time points indicated after infection. ( E ) WT myeloid cells taken ex vivo from lungs ( i ) and BM ( ii ) were infected with M. tuberculosis in the presence or absence of 2 ng/ml IFN-β, added at the time of infection, and levels of IL-10 in culture supernatant were determined by Luminex bead array at 24 h postinfection. ( F ) WT, Ifnar1 −/− , and WT treated with IFN-β macrophages were infected with M. tuberculosis , and at 1 h postinfection ActD was added. mRNA was then taken at the time points indicated and Il10 mRNA levels were determined by qRT-PCR. ( G ) WT macrophages treated (or not) with 2 ng/ml IFN-β at the time of infection ( i ) and WT and Ifnar1 −/− macrophages ( ii ) were infected with M. tuberculosis , and levels of IL-27 in culture supernatant were determined by ELISA at 24 h postinfection. ( H and I ) WT and Tccr −/− (IL-27Rα −/− ) macrophages were infected with M. tuberculosis in the presence or absence of 2 ng/ml IFN-β, added at the time of infection, and levels of IL-10 (H) or TNF-α (I) in culture supernatant were determined by ELISA at 24 h postinfection. ( J ) WT and Tccr −/− (IL-27Rα −/− ) macrophages were treated for 20 min with rIL-27 (50 ng/ml), rIFN-γ (10 ng/ml), or rIL-10 (10 ng/ml) and whole-cell extracts were then analyzed by immunoblotting with the indicated Abs. ( K ) WT and Tccr −/− (IL-27Rα −/− ) macrophages were stimulated for 0, 3, or 6 h with Pam3CSK4 (200 ng/ml) and then treated (or not) for 20 min with rIL-27 (50 ng/ml). WT macrophages treated with IFN-γ (10 ng/ml) or IL-10 (10 ng/ml) were included as positive controls for STAT-1 and STAT-3 phosphorylation, respectively. Whole-cell extracts were then analyzed by immunoblotting with the indicated Abs. ( L ) WT and Tccr −/− splenocytes were treated for the indicated times with rIL-27 (50 ng/ml) or rIFN-γ (10 ng/ml). Whole-cell extracts were then analyzed by immunoblotting with the indicated Abs. Graphs show means ± SEM of triplicate samples, except for (E), which shows duplicates. For ELISA and Luminex bead array results, uninfected control samples were below the detection limit (20 and 5 pg/ml, respectively) for the cytokines measured (data not shown). Significance was determined using an unpaired t test (A, C, E, and G), a one-way ANOVA with a Bonferroni post hoc test (H and I), or a two-way ANOVA, with significance relative to WT (F). Data are representative of at least two independent experiments. * p
Figure Legend Snippet: Type I IFN regulates IL-10 production in M. tuberculosis –infected macrophages independently of IL-27 signaling. ( A ) WT macrophages were infected with M. tuberculosis in the presence of increasing concentrations of IFN-β, added at the time of infection, and levels of IL-10 in culture supernatant were determined by ELISA at 24 h postinfection. ( B ) WT macrophages were infected with M. tuberculosis in the presence or absence of 2 ng/ml IFN-β, added at the time of infection, and levels of Il10 mRNA were determined by quantitative RT-PCR (qRT-PCR) at the time points indicated after infection. ( C ) WT and Ifnar1 −/− macrophages were infected with M. tuberculosis and levels of IL-10 in culture supernatant were determined by ELISA at 24 h postinfection. ( D ) WT and Ifnar1 −/− macrophages were infected with M. tuberculosis and levels of Il10 mRNA determined by qRT-PCR at the time points indicated after infection. ( E ) WT myeloid cells taken ex vivo from lungs ( i ) and BM ( ii ) were infected with M. tuberculosis in the presence or absence of 2 ng/ml IFN-β, added at the time of infection, and levels of IL-10 in culture supernatant were determined by Luminex bead array at 24 h postinfection. ( F ) WT, Ifnar1 −/− , and WT treated with IFN-β macrophages were infected with M. tuberculosis , and at 1 h postinfection ActD was added. mRNA was then taken at the time points indicated and Il10 mRNA levels were determined by qRT-PCR. ( G ) WT macrophages treated (or not) with 2 ng/ml IFN-β at the time of infection ( i ) and WT and Ifnar1 −/− macrophages ( ii ) were infected with M. tuberculosis , and levels of IL-27 in culture supernatant were determined by ELISA at 24 h postinfection. ( H and I ) WT and Tccr −/− (IL-27Rα −/− ) macrophages were infected with M. tuberculosis in the presence or absence of 2 ng/ml IFN-β, added at the time of infection, and levels of IL-10 (H) or TNF-α (I) in culture supernatant were determined by ELISA at 24 h postinfection. ( J ) WT and Tccr −/− (IL-27Rα −/− ) macrophages were treated for 20 min with rIL-27 (50 ng/ml), rIFN-γ (10 ng/ml), or rIL-10 (10 ng/ml) and whole-cell extracts were then analyzed by immunoblotting with the indicated Abs. ( K ) WT and Tccr −/− (IL-27Rα −/− ) macrophages were stimulated for 0, 3, or 6 h with Pam3CSK4 (200 ng/ml) and then treated (or not) for 20 min with rIL-27 (50 ng/ml). WT macrophages treated with IFN-γ (10 ng/ml) or IL-10 (10 ng/ml) were included as positive controls for STAT-1 and STAT-3 phosphorylation, respectively. Whole-cell extracts were then analyzed by immunoblotting with the indicated Abs. ( L ) WT and Tccr −/− splenocytes were treated for the indicated times with rIL-27 (50 ng/ml) or rIFN-γ (10 ng/ml). Whole-cell extracts were then analyzed by immunoblotting with the indicated Abs. Graphs show means ± SEM of triplicate samples, except for (E), which shows duplicates. For ELISA and Luminex bead array results, uninfected control samples were below the detection limit (20 and 5 pg/ml, respectively) for the cytokines measured (data not shown). Significance was determined using an unpaired t test (A, C, E, and G), a one-way ANOVA with a Bonferroni post hoc test (H and I), or a two-way ANOVA, with significance relative to WT (F). Data are representative of at least two independent experiments. * p

Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Ex Vivo, Luminex

19) Product Images from "A Critical Role for Human Caspase-4 in Endotoxin Sensitivity"

Article Title: A Critical Role for Human Caspase-4 in Endotoxin Sensitivity

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1303424

Caspase-4 mediates IL-1β and IL-18 secretion after stimulation with TLR ligands. ( A ) BMDMs from WT or caspase-4 transgenic (CASP4) mice were stimulated with the indicated concentration of LPS or Pam3CSK4 for 24 h, and the levels of IL-1β,
Figure Legend Snippet: Caspase-4 mediates IL-1β and IL-18 secretion after stimulation with TLR ligands. ( A ) BMDMs from WT or caspase-4 transgenic (CASP4) mice were stimulated with the indicated concentration of LPS or Pam3CSK4 for 24 h, and the levels of IL-1β,

Techniques Used: Transgenic Assay, Mouse Assay, Concentration Assay

20) Product Images from "Nucleic acid scavenging microfiber mesh inhibits trauma-induced inflammation and thrombosis"

Article Title: Nucleic acid scavenging microfiber mesh inhibits trauma-induced inflammation and thrombosis

Journal: Biomaterials

doi: 10.1016/j.biomaterials.2016.12.024

NABP-immobilized PSMA/polystyrene microfiber mesh scavenges multiple TLR agonists without cytotoxicity. A. scanning electron microscope (SEM) image of surface of PSMA/polystyrene microfiber mesh. B. Complete culture media (1 ml) supplemented with TLR agonists Pam3CSK4, LPS, Heparan sulfate (HS), polyI:C or CpG ODN, were incubated for 1 min with or without either PEI- or PAMAM-G3-immobilized microfiber mesh (2.9 cm 2 ) at room temperature. The treatment was repeated once. TLR reporter cells were incubated for 3 days in either untreated or treated complete culture media. For the treatment with free NABPs, TLR reporter cells in the complete culture media containing fetal bovine serum and TLR agonist were directly treated with either PEI (20 µg/ml) or PAMAM-G3 (25 µg/ml). The activation of NF-κB in TLR signaling pathway was determined by a colorimetric enzyme assay. C-F. Human primary fibroblasts were cultured in either complete culture media containing free (C) PEI or free (D) PAMAM-G3 at various concentrations or complete culture media pre-treated with (E) PEI-immobilized mesh or (F) PAMAM-G3-immobilized mesh at various surface areas (0.288 mg/cm 2 PEI on mesh; 0.128 mg/cm 2 PAMAM-G3 on mesh). After 3 days incubation, cell proliferation was determined by a MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H–tetrazolium) assay. Error bars are S.D.. * Significant different ( P
Figure Legend Snippet: NABP-immobilized PSMA/polystyrene microfiber mesh scavenges multiple TLR agonists without cytotoxicity. A. scanning electron microscope (SEM) image of surface of PSMA/polystyrene microfiber mesh. B. Complete culture media (1 ml) supplemented with TLR agonists Pam3CSK4, LPS, Heparan sulfate (HS), polyI:C or CpG ODN, were incubated for 1 min with or without either PEI- or PAMAM-G3-immobilized microfiber mesh (2.9 cm 2 ) at room temperature. The treatment was repeated once. TLR reporter cells were incubated for 3 days in either untreated or treated complete culture media. For the treatment with free NABPs, TLR reporter cells in the complete culture media containing fetal bovine serum and TLR agonist were directly treated with either PEI (20 µg/ml) or PAMAM-G3 (25 µg/ml). The activation of NF-κB in TLR signaling pathway was determined by a colorimetric enzyme assay. C-F. Human primary fibroblasts were cultured in either complete culture media containing free (C) PEI or free (D) PAMAM-G3 at various concentrations or complete culture media pre-treated with (E) PEI-immobilized mesh or (F) PAMAM-G3-immobilized mesh at various surface areas (0.288 mg/cm 2 PEI on mesh; 0.128 mg/cm 2 PAMAM-G3 on mesh). After 3 days incubation, cell proliferation was determined by a MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H–tetrazolium) assay. Error bars are S.D.. * Significant different ( P

Techniques Used: Microscopy, Incubation, Activation Assay, Enzymatic Assay, Cell Culture

21) Product Images from "Regulatory effects of TLR2 on megakaryocytic cell function"

Article Title: Regulatory effects of TLR2 on megakaryocytic cell function

Journal: Blood

doi: 10.1182/blood-2010-09-304949

TLR2 increases polyploidization in mouse megakaryocytes. WT (A) and TLR2 −/− (B) mouse bone marrow was treated for 3 days with 50 ng/mL mTPO or 1μg/mL Pam3CSK4 (PAM). (C) WT mouse bone marrow was treated for 3 days with 1 μg/mL
Figure Legend Snippet: TLR2 increases polyploidization in mouse megakaryocytes. WT (A) and TLR2 −/− (B) mouse bone marrow was treated for 3 days with 50 ng/mL mTPO or 1μg/mL Pam3CSK4 (PAM). (C) WT mouse bone marrow was treated for 3 days with 1 μg/mL

Techniques Used:

TLR2 ligand increases inflammatory-related gene and protein expressions. (A-D) Meg-01 cells were pretreated for 45 minutes with 50μM LY294002, U0126, BAY11-7082, or DMSO, and then treated for 3 hours with 1 μg/mL of Pam3CSK4 (PAM). MCP-1
Figure Legend Snippet: TLR2 ligand increases inflammatory-related gene and protein expressions. (A-D) Meg-01 cells were pretreated for 45 minutes with 50μM LY294002, U0126, BAY11-7082, or DMSO, and then treated for 3 hours with 1 μg/mL of Pam3CSK4 (PAM). MCP-1

Techniques Used:

Pam3CSK4 treatment in WT mice affects platelet counts and megakaryocyte maturation. WT and TLR2 −/− mice were injected once intraperitoneally with saline or 4 mg/kg of Pam3CSK4, and monitored for 1 week. (A) Platelet counts were done on
Figure Legend Snippet: Pam3CSK4 treatment in WT mice affects platelet counts and megakaryocyte maturation. WT and TLR2 −/− mice were injected once intraperitoneally with saline or 4 mg/kg of Pam3CSK4, and monitored for 1 week. (A) Platelet counts were done on

Techniques Used: Mouse Assay, Injection

TLR2 activates the NFκB, PI3K/Akt, and ERK pathways in Meg-01 cells. (A) Meg-01 cells were treated for up to 1 hour with 1μg/mL of Pam3CSK4 (PAM). Western blots were run with samples at each time point and probed for phospho-NFκBp65,
Figure Legend Snippet: TLR2 activates the NFκB, PI3K/Akt, and ERK pathways in Meg-01 cells. (A) Meg-01 cells were treated for up to 1 hour with 1μg/mL of Pam3CSK4 (PAM). Western blots were run with samples at each time point and probed for phospho-NFκBp65,

Techniques Used: Western Blot

22) Product Images from "Regulatory effects of TLR2 on megakaryocytic cell function"

Article Title: Regulatory effects of TLR2 on megakaryocytic cell function

Journal: Blood

doi: 10.1182/blood-2010-09-304949

TLR2 increases polyploidization in mouse megakaryocytes. WT (A) and TLR2 −/− (B) mouse bone marrow was treated for 3 days with 50 ng/mL mTPO or 1μg/mL Pam3CSK4 (PAM). (C) WT mouse bone marrow was treated for 3 days with 1 μg/mL
Figure Legend Snippet: TLR2 increases polyploidization in mouse megakaryocytes. WT (A) and TLR2 −/− (B) mouse bone marrow was treated for 3 days with 50 ng/mL mTPO or 1μg/mL Pam3CSK4 (PAM). (C) WT mouse bone marrow was treated for 3 days with 1 μg/mL

Techniques Used:

TLR2 ligand increases inflammatory-related gene and protein expressions. (A-D) Meg-01 cells were pretreated for 45 minutes with 50μM LY294002, U0126, BAY11-7082, or DMSO, and then treated for 3 hours with 1 μg/mL of Pam3CSK4 (PAM). MCP-1
Figure Legend Snippet: TLR2 ligand increases inflammatory-related gene and protein expressions. (A-D) Meg-01 cells were pretreated for 45 minutes with 50μM LY294002, U0126, BAY11-7082, or DMSO, and then treated for 3 hours with 1 μg/mL of Pam3CSK4 (PAM). MCP-1

Techniques Used:

Pam3CSK4 treatment in WT mice affects platelet counts and megakaryocyte maturation. WT and TLR2 −/− mice were injected once intraperitoneally with saline or 4 mg/kg of Pam3CSK4, and monitored for 1 week. (A) Platelet counts were done on
Figure Legend Snippet: Pam3CSK4 treatment in WT mice affects platelet counts and megakaryocyte maturation. WT and TLR2 −/− mice were injected once intraperitoneally with saline or 4 mg/kg of Pam3CSK4, and monitored for 1 week. (A) Platelet counts were done on

Techniques Used: Mouse Assay, Injection

TLR2 activates the NFκB, PI3K/Akt, and ERK pathways in Meg-01 cells. (A) Meg-01 cells were treated for up to 1 hour with 1μg/mL of Pam3CSK4 (PAM). Western blots were run with samples at each time point and probed for phospho-NFκBp65,
Figure Legend Snippet: TLR2 activates the NFκB, PI3K/Akt, and ERK pathways in Meg-01 cells. (A) Meg-01 cells were treated for up to 1 hour with 1μg/mL of Pam3CSK4 (PAM). Western blots were run with samples at each time point and probed for phospho-NFκBp65,

Techniques Used: Western Blot

23) Product Images from "The death domain-containing protein Unc5CL is a novel MyD88-independent activator of the pro-inflammatory IRAK signaling cascade"

Article Title: The death domain-containing protein Unc5CL is a novel MyD88-independent activator of the pro-inflammatory IRAK signaling cascade

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2011.147

Unc5CL-mediated NF- κ B activation is IRAK-dependent but MyD88-independent. ( a ) HEK293T cells were transfected with the indicated siRNAs. Forty eight hours later, cells were transfected with NF- κ B luciferase reporter gene plasmids together with an empty vector (EV) and the indicated expression constructs and were analyzed for NF- κ B-dependent luciferase activity 24 h later. TLR2-transfected cells were additionally treated with 200 ng/ml Pam3CSK4. ( b ) Selected protein samples from experiments shown in ( a ) were probed by western blot using the indicated antibodies. ( c and d ) HEK293T cells were transfected with the indicated siRNAs. 48 h later, cells were transfected with the indicated expression constructs and supernatants were analyzed for IL-8 and CXCL1 secretion by ELISA. TLR2-transfected cells were additionally treated with 200 ng/ml Pam3CSK4. ( a , c and d ) Data represent the mean values±S.D. of technical triplicates; results are representative of three independent experiments. ( e ) HEK293T cells were transfected with the indicated expression constructs. Immunoprecipitates and extracts were analyzed by western blot using the indicated antibodies
Figure Legend Snippet: Unc5CL-mediated NF- κ B activation is IRAK-dependent but MyD88-independent. ( a ) HEK293T cells were transfected with the indicated siRNAs. Forty eight hours later, cells were transfected with NF- κ B luciferase reporter gene plasmids together with an empty vector (EV) and the indicated expression constructs and were analyzed for NF- κ B-dependent luciferase activity 24 h later. TLR2-transfected cells were additionally treated with 200 ng/ml Pam3CSK4. ( b ) Selected protein samples from experiments shown in ( a ) were probed by western blot using the indicated antibodies. ( c and d ) HEK293T cells were transfected with the indicated siRNAs. 48 h later, cells were transfected with the indicated expression constructs and supernatants were analyzed for IL-8 and CXCL1 secretion by ELISA. TLR2-transfected cells were additionally treated with 200 ng/ml Pam3CSK4. ( a , c and d ) Data represent the mean values±S.D. of technical triplicates; results are representative of three independent experiments. ( e ) HEK293T cells were transfected with the indicated expression constructs. Immunoprecipitates and extracts were analyzed by western blot using the indicated antibodies

Techniques Used: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Construct, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay

24) Product Images from "β-Glucan attenuates TLR2- and TLR4-mediated cytokine production by microglia"

Article Title: β-Glucan attenuates TLR2- and TLR4-mediated cytokine production by microglia

Journal: Neuroscience letters

doi: 10.1016/j.neulet.2009.04.039

Particulate β-glucan inhibits TLR2-mediated cytokine production by microglia. Primary microglia were left untreated (control) or were stimulated with β-glucan (100 µg/ml), Pam3Csk4 (Pam; 1 µg/ml) or combination of β-glucan
Figure Legend Snippet: Particulate β-glucan inhibits TLR2-mediated cytokine production by microglia. Primary microglia were left untreated (control) or were stimulated with β-glucan (100 µg/ml), Pam3Csk4 (Pam; 1 µg/ml) or combination of β-glucan

Techniques Used:

Particulate β-glucan suppresses activation of NF-κB by TLR ligands. Primary microglia (A and B) were left untreated (control) or were stimulated with LPS (1 µg/ml) or Pam3Csk4 (Pam; 1 µg/ml) for 15 min (A) or 5 min (B).
Figure Legend Snippet: Particulate β-glucan suppresses activation of NF-κB by TLR ligands. Primary microglia (A and B) were left untreated (control) or were stimulated with LPS (1 µg/ml) or Pam3Csk4 (Pam; 1 µg/ml) for 15 min (A) or 5 min (B).

Techniques Used: Activation Assay

25) Product Images from "Sensing of cytosolic LPS through caspy2 pyrin domain mediates noncanonical inflammasome activation in zebrafish"

Article Title: Sensing of cytosolic LPS through caspy2 pyrin domain mediates noncanonical inflammasome activation in zebrafish

Journal: Nature Communications

doi: 10.1038/s41467-018-04984-1

Caspase-5-like activity is essential for pyroptosis in zebrafish fibroblasts. a ZF4 zebrafish fibroblasts were infected with wild-type (EIB202) or 0909I E. piscicida for 2 h at a multiplicity of infection (MOI) of 50, or left uninfected. Supernatants from the indicated ZF4 cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release. b ZF4 cells were infected with 0909I E. piscicida for 2 h at an MOI of 50. Relative caspase activity was then measured by incubating cell lysates with fluorogenic and chromogenic substrates of caspase-1 (YVAD), caspase-2 (VDQQD), caspase-3/7 (DEVD), caspase-4 (LEVD), caspase-5 (WEHD), caspase-8 (IETD), and caspase-9 (LEHD). c , d ZF4 cells were treated with caspase-3/7, pan-caspase, caspase-1, caspase-4, and caspase-5 inhibitors (Ac-DEVD-CHO, Z-VAD-FMK, Z-YVAD-FMK, Ac-LEVD-CHO, and Z-WEHD-FMK, respectively). c LDH release for cell death was measured 2 h after 0909I E. piscicida infection. d Images were taken after 0909I E. piscicida infection for 2 h. Propidium iodide (PI) was added to detect the loss of plasma membrane integrity. Arrows indicate cells exhibiting pyroptotic-like features. Scale bar, 50 µm. e – g ZF4 cells were primed with Pam3CSK4 for 4 h, before being stimulated with cholera toxin B subunit (CTB) plus LPS, LPS, or CTB alone for 12 h. e Supernatants from the indicated ZF4 cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release. f Cytosolic LPS-delivered ZF4 cells were treated with indicated caspase inhibitors as in Fig. 1c, Supernatants from the indicated ZF4 cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release. g Relative caspases activity was measured by incubating cell lysates with indicated fluorogenic and chromogenic substrates as in Fig. 1b. a – g Results are representative of at least three independent experiments, and error bars denote the SD of triplicate wells. * p
Figure Legend Snippet: Caspase-5-like activity is essential for pyroptosis in zebrafish fibroblasts. a ZF4 zebrafish fibroblasts were infected with wild-type (EIB202) or 0909I E. piscicida for 2 h at a multiplicity of infection (MOI) of 50, or left uninfected. Supernatants from the indicated ZF4 cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release. b ZF4 cells were infected with 0909I E. piscicida for 2 h at an MOI of 50. Relative caspase activity was then measured by incubating cell lysates with fluorogenic and chromogenic substrates of caspase-1 (YVAD), caspase-2 (VDQQD), caspase-3/7 (DEVD), caspase-4 (LEVD), caspase-5 (WEHD), caspase-8 (IETD), and caspase-9 (LEHD). c , d ZF4 cells were treated with caspase-3/7, pan-caspase, caspase-1, caspase-4, and caspase-5 inhibitors (Ac-DEVD-CHO, Z-VAD-FMK, Z-YVAD-FMK, Ac-LEVD-CHO, and Z-WEHD-FMK, respectively). c LDH release for cell death was measured 2 h after 0909I E. piscicida infection. d Images were taken after 0909I E. piscicida infection for 2 h. Propidium iodide (PI) was added to detect the loss of plasma membrane integrity. Arrows indicate cells exhibiting pyroptotic-like features. Scale bar, 50 µm. e – g ZF4 cells were primed with Pam3CSK4 for 4 h, before being stimulated with cholera toxin B subunit (CTB) plus LPS, LPS, or CTB alone for 12 h. e Supernatants from the indicated ZF4 cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release. f Cytosolic LPS-delivered ZF4 cells were treated with indicated caspase inhibitors as in Fig. 1c, Supernatants from the indicated ZF4 cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release. g Relative caspases activity was measured by incubating cell lysates with indicated fluorogenic and chromogenic substrates as in Fig. 1b. a – g Results are representative of at least three independent experiments, and error bars denote the SD of triplicate wells. * p

Techniques Used: Activity Assay, Infection, CtB Assay

Intracellular LPS-triggered-caspy2 noncanonical inflammasome activation. a - c ZF4 cells were primed with Pam3CSK4 for 4 h, before being stimulated with cholera toxin B subunit (CTB) plus LPS, LPS, or CTB alone for 12 h. Relative caspase-5-like activity was measured by incubating cell lysates with fluorogenic and chromogenic caspase-5 substrates (WEHD) ( a ). Supernatants from the indicated HeLa cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release ( b ). Mixtures of cell lysates and supernatants were subjected to immunoblotting ( c ). d , e CASP4 −/− HeLa cells were transduced with a vector expressing wild-type caspy2, caspy2 (C296A), caspy2 (ΔPYD), or the empty vector. Cells were primed with LPS for 4 h, before being stimulated with CTB plus LPS for 12 h. Images were taken as in Fig. 1d ( d ). Propidium iodide (PI) was added to detect the loss of plasma membrane integrity. Arrows denote cells with pyroptotic-like features. Scale bar, 50 µm. Relative caspase-5-like activity and LDH release assays were conducted as in a and b ( e ). Immunoblotting for the caspy2 forms indicated is shown. a – e Results are representative of at least three independent experiments, and error bars denote the SD of triplicate wells. * p
Figure Legend Snippet: Intracellular LPS-triggered-caspy2 noncanonical inflammasome activation. a - c ZF4 cells were primed with Pam3CSK4 for 4 h, before being stimulated with cholera toxin B subunit (CTB) plus LPS, LPS, or CTB alone for 12 h. Relative caspase-5-like activity was measured by incubating cell lysates with fluorogenic and chromogenic caspase-5 substrates (WEHD) ( a ). Supernatants from the indicated HeLa cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release ( b ). Mixtures of cell lysates and supernatants were subjected to immunoblotting ( c ). d , e CASP4 −/− HeLa cells were transduced with a vector expressing wild-type caspy2, caspy2 (C296A), caspy2 (ΔPYD), or the empty vector. Cells were primed with LPS for 4 h, before being stimulated with CTB plus LPS for 12 h. Images were taken as in Fig. 1d ( d ). Propidium iodide (PI) was added to detect the loss of plasma membrane integrity. Arrows denote cells with pyroptotic-like features. Scale bar, 50 µm. Relative caspase-5-like activity and LDH release assays were conducted as in a and b ( e ). Immunoblotting for the caspy2 forms indicated is shown. a – e Results are representative of at least three independent experiments, and error bars denote the SD of triplicate wells. * p

Techniques Used: Activation Assay, CtB Assay, Activity Assay, Transduction, Plasmid Preparation, Expressing

Caspy2 directly binds to LPS to induce its oligomerization. a Streptavidin pulldown assays of the binding of biotin-conjugated LPS and Pam3CSK4 to purified HA-tagged caspy2, caspy2 (C296A), caspy2 (ΔPYD), caspy2 PYD, caspy and caspy PYD in transfected HEK293T cells. Shown are anti-HA immunoblots of pulled down proteins and total lysates (input). b Purified HA-tagged caspy2 and caspy2 PYD in transfected HEK293T cells were incubated with LPS or Pam3CSK4. The samples were analyzed by the pore-limited native gel electrophoresis. c Wild-type and c aspy2 -KO ZF4 cells were infected with wild-type (EIB202) or 0909I E. piscicida (with/without 10 mM glycine) for 2 h at a multiplicity of infection (MOI) of 50. Confocal laser scanning microscopic analysis of nuclei (2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), blue) and caspy2 foci (green, white arrowheads). Direct interference contrast (DIC)/phase images are shown. Scale bar, 10 µm. Statistics of the percentages of cells showing signals for caspy2 foci are listed below. (Approximately 200 cells were counted in each sample. Mean ± SD of triplicate samples.) d Wild-type and c aspy2 -KO ZF4 cells were infected with wild-type (EIB202) or 0909I E. piscicida (with/without 10 mM glycine) for 2 h at an MOI of 50, or left untreated (Mock). The samples were analyzed by the pore-limited native gel electrophoresis and immunoblotting (upper panel). Cell lysates and DSS cross-linked pellets from wild-type and c aspy2 -KO ZF4 cells treated as indicated were analyzed by immunoblotting for caspy2 oligomerization (lower panel). WB western blot. Results are representative of at least three independent experiments
Figure Legend Snippet: Caspy2 directly binds to LPS to induce its oligomerization. a Streptavidin pulldown assays of the binding of biotin-conjugated LPS and Pam3CSK4 to purified HA-tagged caspy2, caspy2 (C296A), caspy2 (ΔPYD), caspy2 PYD, caspy and caspy PYD in transfected HEK293T cells. Shown are anti-HA immunoblots of pulled down proteins and total lysates (input). b Purified HA-tagged caspy2 and caspy2 PYD in transfected HEK293T cells were incubated with LPS or Pam3CSK4. The samples were analyzed by the pore-limited native gel electrophoresis. c Wild-type and c aspy2 -KO ZF4 cells were infected with wild-type (EIB202) or 0909I E. piscicida (with/without 10 mM glycine) for 2 h at a multiplicity of infection (MOI) of 50. Confocal laser scanning microscopic analysis of nuclei (2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), blue) and caspy2 foci (green, white arrowheads). Direct interference contrast (DIC)/phase images are shown. Scale bar, 10 µm. Statistics of the percentages of cells showing signals for caspy2 foci are listed below. (Approximately 200 cells were counted in each sample. Mean ± SD of triplicate samples.) d Wild-type and c aspy2 -KO ZF4 cells were infected with wild-type (EIB202) or 0909I E. piscicida (with/without 10 mM glycine) for 2 h at an MOI of 50, or left untreated (Mock). The samples were analyzed by the pore-limited native gel electrophoresis and immunoblotting (upper panel). Cell lysates and DSS cross-linked pellets from wild-type and c aspy2 -KO ZF4 cells treated as indicated were analyzed by immunoblotting for caspy2 oligomerization (lower panel). WB western blot. Results are representative of at least three independent experiments

Techniques Used: Binding Assay, Purification, Transfection, Western Blot, Incubation, Nucleic Acid Electrophoresis, Infection

26) Product Images from "The Toll-Like Receptor 2 Ligand Pam2CSK4 Activates Platelet Nuclear Factor-κB and Bruton’s Tyrosine Kinase Signaling to Promote Platelet-Endothelial Cell Interactions"

Article Title: The Toll-Like Receptor 2 Ligand Pam2CSK4 Activates Platelet Nuclear Factor-κB and Bruton’s Tyrosine Kinase Signaling to Promote Platelet-Endothelial Cell Interactions

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2021.729951

Platelets enhance endothelial cell inflammation and reduce endothelial permeability to BSA in response to TLR2 agonists. (A–C) HUVECs were cultured onto 12-well plates until reaching confluence and stimulated with Pam2CSK4 (10 µg/ml), Pam3CSK4 (10 µg/ml) or TNF-α (1 ng/ml). 250 µl of platelets (2x10 8 /ml) were mixed with 250 µl of SFM and co-cultured with HUVECs for 4 hours. (A) Adhered platelets were thoroughly removed with SFM and HUVECs lysates analyzed for ICAM-1 and VCAM-1 expression by immunoblot; n=4 experiments. (B, C) Supernatants were collected and assayed by ELISA for IL-6 and IL-8 secretion; n=4 experiments. (D) Washed platelets (1x10 9 /ml) were stimulated with Pam2CSK4 or CRP-XL at 10 µg/ml for 45 min at 37°C. Platelets were then pelleted and supernatants assayed for IL-6 and IL-8 secretion. A positive control from TNF-α stimulated HUVECs was used (Control); n=3. (E) HUVECs were seeded to confluence in transwell devices and stimulated for 4 h at 37°C with Pam2CSK4 (10 µg/ml) or TNF-α (1 ng/ml) in the presence or absence of platelets (1x10 8 /ml). Cells were washed 3 times and incubated with media containing Evans Blue dye (0.67 mg/ml) and 4% BSA for 30 min at 37°C. Samples from the lower chamber were collected and absorbance at 650 nm measured; n=4. * indicates statistical significance (p
Figure Legend Snippet: Platelets enhance endothelial cell inflammation and reduce endothelial permeability to BSA in response to TLR2 agonists. (A–C) HUVECs were cultured onto 12-well plates until reaching confluence and stimulated with Pam2CSK4 (10 µg/ml), Pam3CSK4 (10 µg/ml) or TNF-α (1 ng/ml). 250 µl of platelets (2x10 8 /ml) were mixed with 250 µl of SFM and co-cultured with HUVECs for 4 hours. (A) Adhered platelets were thoroughly removed with SFM and HUVECs lysates analyzed for ICAM-1 and VCAM-1 expression by immunoblot; n=4 experiments. (B, C) Supernatants were collected and assayed by ELISA for IL-6 and IL-8 secretion; n=4 experiments. (D) Washed platelets (1x10 9 /ml) were stimulated with Pam2CSK4 or CRP-XL at 10 µg/ml for 45 min at 37°C. Platelets were then pelleted and supernatants assayed for IL-6 and IL-8 secretion. A positive control from TNF-α stimulated HUVECs was used (Control); n=3. (E) HUVECs were seeded to confluence in transwell devices and stimulated for 4 h at 37°C with Pam2CSK4 (10 µg/ml) or TNF-α (1 ng/ml) in the presence or absence of platelets (1x10 8 /ml). Cells were washed 3 times and incubated with media containing Evans Blue dye (0.67 mg/ml) and 4% BSA for 30 min at 37°C. Samples from the lower chamber were collected and absorbance at 650 nm measured; n=4. * indicates statistical significance (p

Techniques Used: Permeability, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Positive Control, Incubation

27) Product Images from "A Novel Role for C5a in B-1 Cell Homeostasis"

Article Title: A Novel Role for C5a in B-1 Cell Homeostasis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00258

Impact of the C5/C5a/C5aR/TLR2 axes on peritoneal CXCL13 production under steady-state and inflammatory conditions. (A) CXCL13 concentrations in the peritoneal lavage fluid of wild-type (wt), C5aR1-, C5aR2-, and C5-deficient mice (C57BL/6J background) under steady-state conditions. (B) CXCL13 concentrations in the peritoneal lavage fluid of wt, C5aR1- and C5aR2-deficient mice (BALB/c background) under steady-state conditions. (C) In vitro CXCL13 production by peritoneal macrophages from wt mice (C57BL/6J background) 24 h after stimulation with IL-10, Pam3CSK4, or Pam3CSK4 + IL-10. (D) CXCL13 production from wt, C5aR1 −/− , C5aR2 −/− , or C5 −/− peritoneal macrophages (all on C57BL/6J background) in response to Pam3CSK4 + IL-10. (E) CXCL13 concentrations in the peritoneal lavage fluid of wt mice (C57BL/6J background) 6 h after i.p. injection with 200 nM C5a or PBS. (F,G) In vitro CXCL13 (F) or IL-10 (G) production by total peritoneal cavity (PerC) cells from wt mice 24 h after stimulation with Pam3CSK4 compared to unstimulated (left panel) or Pam3CSK4-stimulated cells compared to simultaneous stimulation of Pam3CSK4 together with +anti-IL-10R Ab (right panel). (H) CXCL13 production from unstimulated (left panel) or Pam3CSK4 + IL-10-stimulated (right panel) adherent PerC cells from wt mice in the presence or absence of C5aRA (5 µM). (I) CXCL13 concentrations in the peritoneal lavage fluid of wt and TLR2-deficient mice (C57BL/6J background) under steady-state conditions (left). (J) Total B-1, B-1a, and B-1b cell numbers in the peritoneum of TLR2-deficient as compared to wt mice (both C57BL/6J background). Values shown are the mean ± SD (A,B,D,E,I,J) . Statistical differences between wt and knockout mice were determined using an unpaired t -test (A,B,I,J) or Mann–Whitney test (D) ; statistical differences between different stimulation conditions were determined using an unpaired (E) or paired t -test (C,F–H) . * p
Figure Legend Snippet: Impact of the C5/C5a/C5aR/TLR2 axes on peritoneal CXCL13 production under steady-state and inflammatory conditions. (A) CXCL13 concentrations in the peritoneal lavage fluid of wild-type (wt), C5aR1-, C5aR2-, and C5-deficient mice (C57BL/6J background) under steady-state conditions. (B) CXCL13 concentrations in the peritoneal lavage fluid of wt, C5aR1- and C5aR2-deficient mice (BALB/c background) under steady-state conditions. (C) In vitro CXCL13 production by peritoneal macrophages from wt mice (C57BL/6J background) 24 h after stimulation with IL-10, Pam3CSK4, or Pam3CSK4 + IL-10. (D) CXCL13 production from wt, C5aR1 −/− , C5aR2 −/− , or C5 −/− peritoneal macrophages (all on C57BL/6J background) in response to Pam3CSK4 + IL-10. (E) CXCL13 concentrations in the peritoneal lavage fluid of wt mice (C57BL/6J background) 6 h after i.p. injection with 200 nM C5a or PBS. (F,G) In vitro CXCL13 (F) or IL-10 (G) production by total peritoneal cavity (PerC) cells from wt mice 24 h after stimulation with Pam3CSK4 compared to unstimulated (left panel) or Pam3CSK4-stimulated cells compared to simultaneous stimulation of Pam3CSK4 together with +anti-IL-10R Ab (right panel). (H) CXCL13 production from unstimulated (left panel) or Pam3CSK4 + IL-10-stimulated (right panel) adherent PerC cells from wt mice in the presence or absence of C5aRA (5 µM). (I) CXCL13 concentrations in the peritoneal lavage fluid of wt and TLR2-deficient mice (C57BL/6J background) under steady-state conditions (left). (J) Total B-1, B-1a, and B-1b cell numbers in the peritoneum of TLR2-deficient as compared to wt mice (both C57BL/6J background). Values shown are the mean ± SD (A,B,D,E,I,J) . Statistical differences between wt and knockout mice were determined using an unpaired t -test (A,B,I,J) or Mann–Whitney test (D) ; statistical differences between different stimulation conditions were determined using an unpaired (E) or paired t -test (C,F–H) . * p

Techniques Used: Mouse Assay, In Vitro, Injection, Knock-Out, MANN-WHITNEY

Impact of the C5/C5a/C5aR axes on IL-10 production from peritoneal B-1 cells under steady-state and inflammatory conditions. (A) IL-10 production from non-adherent and adherent PerC cells in response to Pam3CSK4. (B) Determination of IL-10 production in peritoneal B-1a, B-1b, and B-2 cells using GFP-IL-10 knockin mice (Vert-X) (grey histogram = wildtype control, blue line = unstimulated Vert-X mouse). The right panel shows the quantification of IL-10 production (shown as MFI of the GFP signal) from B-1a and B-1b cells. (C) Impact of IL-10 deletion in CD19 + B cells on the number of peritoneal B-1 (left), B-1a (middle), and B-1b (right) cells under steady-state conditions. (D) Impact of C5, C5aR1, or C5aR2 deficiency on IL-10 production from non-adherent PerC cells in response to Pam3CSK4 stimulation. (E) Impact of C5aR1/2 blockade (using the C5aRA A8 Δ71–73 ) on Pam3CSK4-induced IL-10 production. (F) GFP-C5aR1 expression (left panel) or C5aR1 surface expression (right panel) in peritoneal B-1 cells. Values shown in (A–C) are the mean ± SD. Statistical differences between non-adherent and adherent PerC cells with or without stimulation were determined using Kruskal–Wallis test with Dunn’s post hoc test (A) . Statistical differences between MFI expression in B-1a and B-1b cells were determined using unpaired t -test (B) ; statistical differences in B-1 cell numbers between control mice and B cell-specific IL-10 knockout mice were determined using Mann–Whitney test (C) ; statistical differences between different stimulation conditions were determined using a paired t -test (D,E) . * p
Figure Legend Snippet: Impact of the C5/C5a/C5aR axes on IL-10 production from peritoneal B-1 cells under steady-state and inflammatory conditions. (A) IL-10 production from non-adherent and adherent PerC cells in response to Pam3CSK4. (B) Determination of IL-10 production in peritoneal B-1a, B-1b, and B-2 cells using GFP-IL-10 knockin mice (Vert-X) (grey histogram = wildtype control, blue line = unstimulated Vert-X mouse). The right panel shows the quantification of IL-10 production (shown as MFI of the GFP signal) from B-1a and B-1b cells. (C) Impact of IL-10 deletion in CD19 + B cells on the number of peritoneal B-1 (left), B-1a (middle), and B-1b (right) cells under steady-state conditions. (D) Impact of C5, C5aR1, or C5aR2 deficiency on IL-10 production from non-adherent PerC cells in response to Pam3CSK4 stimulation. (E) Impact of C5aR1/2 blockade (using the C5aRA A8 Δ71–73 ) on Pam3CSK4-induced IL-10 production. (F) GFP-C5aR1 expression (left panel) or C5aR1 surface expression (right panel) in peritoneal B-1 cells. Values shown in (A–C) are the mean ± SD. Statistical differences between non-adherent and adherent PerC cells with or without stimulation were determined using Kruskal–Wallis test with Dunn’s post hoc test (A) . Statistical differences between MFI expression in B-1a and B-1b cells were determined using unpaired t -test (B) ; statistical differences in B-1 cell numbers between control mice and B cell-specific IL-10 knockout mice were determined using Mann–Whitney test (C) ; statistical differences between different stimulation conditions were determined using a paired t -test (D,E) . * p

Techniques Used: Knock-In, Mouse Assay, Expressing, Knock-Out, MANN-WHITNEY

C5 production and non-canonical C5a generation in peritoneal macrophages following IL-10 and Pam3CSK4 stimulation. (A) Immunohistochemical analysis of C5 production from wild-type (wt) peritoneal macrophages (C57BL/6J background) in response to 24 h PBS, Pam3CSK4 or combined Pam3CSK4 + IL-10 stimulation (three pictures on the left). Quantification of C5 production based on the immunohistochemical analysis of C5 production (right panel). (B) Real-time RT-PCR for C5 using wt peritoneal macrophages (C57BL/6J background) 24 h after in vitro Pam3CSK4 + IL-10 stimulation compared to unstimulated controls. (C) Western Blot to determine C5 cleavage and C5a generation from wt peritoneal macrophages (C57BL/6J background) or their supernatant (SN) 24 h after in vitro Pam3CSK4 + IL-10 stimulation. Lane 1 = marker; lane 2 = Pam3CSK4 + IL-10-stimulated peritoneal macrophages in the presence of exogenous hC5; lane 3 = as in lane 2 but without cells; lane 4 = SN of Pam3CSK4 + IL-10-activated peritoneal macrophages incubated 4 h with exogenous hC5; lane 5 = as in lane 4 but without SN; lane 6 = hC5a purified from serum. The arrow depicts the monomeric C5a band. (D) Impact of MyD88-dependent cell signaling on Pam3CKS4 + IL-10-induced C5 production from peritoneal macrophages. The immunohistochemical analysis of C5 production from wt and MyD88 −/− peritoneal macrophages (both C57BL/6J background) is shown on the left. On the right, the quantification of the C5 production based on the evaluation of the immunohistochemical staining is shown. (E) CXCL13 production from unstimulated MyD88 −/− adherent PerC cells as well as after stimulation with IL-10, Pam3CSK4, or both compared to wt cells (both C57BL/6J background) and (F) from unstimulated (left panel) and Pam3CSK4 + IL-10-stimulated (right panel) wt adherent PerC cells in the presence or absence of the Stat-3 inhibitor Stattic (50 µM). Values shown in (A,B,D,E) are the mean ± SD. Statistical differences between unstimulated and stimulated samples or between wt and knockout cells were determined by Kruskal–Wallis test with Dunn’s post hoc test (A,E) , paired t -test (B,F) , or Mann–Whitney test (D) . * p
Figure Legend Snippet: C5 production and non-canonical C5a generation in peritoneal macrophages following IL-10 and Pam3CSK4 stimulation. (A) Immunohistochemical analysis of C5 production from wild-type (wt) peritoneal macrophages (C57BL/6J background) in response to 24 h PBS, Pam3CSK4 or combined Pam3CSK4 + IL-10 stimulation (three pictures on the left). Quantification of C5 production based on the immunohistochemical analysis of C5 production (right panel). (B) Real-time RT-PCR for C5 using wt peritoneal macrophages (C57BL/6J background) 24 h after in vitro Pam3CSK4 + IL-10 stimulation compared to unstimulated controls. (C) Western Blot to determine C5 cleavage and C5a generation from wt peritoneal macrophages (C57BL/6J background) or their supernatant (SN) 24 h after in vitro Pam3CSK4 + IL-10 stimulation. Lane 1 = marker; lane 2 = Pam3CSK4 + IL-10-stimulated peritoneal macrophages in the presence of exogenous hC5; lane 3 = as in lane 2 but without cells; lane 4 = SN of Pam3CSK4 + IL-10-activated peritoneal macrophages incubated 4 h with exogenous hC5; lane 5 = as in lane 4 but without SN; lane 6 = hC5a purified from serum. The arrow depicts the monomeric C5a band. (D) Impact of MyD88-dependent cell signaling on Pam3CKS4 + IL-10-induced C5 production from peritoneal macrophages. The immunohistochemical analysis of C5 production from wt and MyD88 −/− peritoneal macrophages (both C57BL/6J background) is shown on the left. On the right, the quantification of the C5 production based on the evaluation of the immunohistochemical staining is shown. (E) CXCL13 production from unstimulated MyD88 −/− adherent PerC cells as well as after stimulation with IL-10, Pam3CSK4, or both compared to wt cells (both C57BL/6J background) and (F) from unstimulated (left panel) and Pam3CSK4 + IL-10-stimulated (right panel) wt adherent PerC cells in the presence or absence of the Stat-3 inhibitor Stattic (50 µM). Values shown in (A,B,D,E) are the mean ± SD. Statistical differences between unstimulated and stimulated samples or between wt and knockout cells were determined by Kruskal–Wallis test with Dunn’s post hoc test (A,E) , paired t -test (B,F) , or Mann–Whitney test (D) . * p

Techniques Used: Immunohistochemistry, Quantitative RT-PCR, In Vitro, Western Blot, Marker, Incubation, Purification, Staining, Knock-Out, MANN-WHITNEY

28) Product Images from "Proinflammatory Role of Monocyte-Derived CX3CR1int Macrophages in Helicobacter hepaticus-Induced Colitis"

Article Title: Proinflammatory Role of Monocyte-Derived CX3CR1int Macrophages in Helicobacter hepaticus-Induced Colitis

Journal: Infection and Immunity

doi: 10.1128/IAI.00579-17

Large intestinal CD11b + myeloid cells from H. hepaticus -infected Il10 −/− mice secrete elevated amounts of proinflammatory cytokines following TLR stimulation. Il10 −/− mice were inoculated with H. hepaticus , and LP cells were isolated from pooled cecum and colon 2 weeks later. Uninfected mice were included as controls. (A and B) Large intestinal LP cells were cultured in medium alone or stimulated with 10 μg/ml Pam3CSK4 for 4 h with 10 μg/ml brefeldin A during the last 3 h and then stained for CD45, CD11b, and TNF-α or appropriate isotype control. Dot plots in panel A are gated on CD45 + CD11b + LP cells from uninfected (Uninf.; upper panels) and 2-week H. hepaticus -infected Il10 −/− mice ( Hh -inf.; lower panels), and numbers (+ standard deviations) of TNF-α + CD11b + cells per mouse in panel B were calculated from the percentages in panel A and the total number of cells isolated from each mouse. Data in panels A and B are representative of two independent experiments performed with 3 individual mice per group. Significance was determined by one-way ANOVA followed by Tukey's multiple-comparison test. **, P
Figure Legend Snippet: Large intestinal CD11b + myeloid cells from H. hepaticus -infected Il10 −/− mice secrete elevated amounts of proinflammatory cytokines following TLR stimulation. Il10 −/− mice were inoculated with H. hepaticus , and LP cells were isolated from pooled cecum and colon 2 weeks later. Uninfected mice were included as controls. (A and B) Large intestinal LP cells were cultured in medium alone or stimulated with 10 μg/ml Pam3CSK4 for 4 h with 10 μg/ml brefeldin A during the last 3 h and then stained for CD45, CD11b, and TNF-α or appropriate isotype control. Dot plots in panel A are gated on CD45 + CD11b + LP cells from uninfected (Uninf.; upper panels) and 2-week H. hepaticus -infected Il10 −/− mice ( Hh -inf.; lower panels), and numbers (+ standard deviations) of TNF-α + CD11b + cells per mouse in panel B were calculated from the percentages in panel A and the total number of cells isolated from each mouse. Data in panels A and B are representative of two independent experiments performed with 3 individual mice per group. Significance was determined by one-way ANOVA followed by Tukey's multiple-comparison test. **, P

Techniques Used: Infection, Mouse Assay, Isolation, Cell Culture, Staining

29) Product Images from "Magnoliae Cortex and maize modulate Porphyromonas gingivalis-induced inflammatory reactions"

Article Title: Magnoliae Cortex and maize modulate Porphyromonas gingivalis-induced inflammatory reactions

Journal: Journal of Periodontal & Implant Science

doi: 10.5051/jpis.2018.48.2.70

Effects of M and Z on Pam3CSK4-induced IL-6 formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , IL-6: interleukin-6, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on Pam3CSK4-induced IL-6 formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , IL-6: interleukin-6, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Standard Deviation

Effects of M and Z on Pam3CSK4-induced NF-κB transactivation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes prior to treatment with 10 μg/mL Pam3CSK4 for 2 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , NF-κB: nuclear transcription factor κB, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide, p65: transcription factor p65. a) P
Figure Legend Snippet: Effects of M and Z on Pam3CSK4-induced NF-κB transactivation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes prior to treatment with 10 μg/mL Pam3CSK4 for 2 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , NF-κB: nuclear transcription factor κB, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide, p65: transcription factor p65. a) P

Techniques Used: Standard Deviation

Effects of M and Z on nuclear iNOS and COX-2 expression levels in Pam3CSK4-induced RAW 264.7 cells. The levels of iNOS, COX-2, and β-actin expression were detected by western blotting using specific antibodies. Cells (1.9×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before being treated with 10 μg/mL Pam3CSK4. To detect iNOS and COX-2 expression, Pam3CSK4 was added for 24 hours at 37°C. The blots shown here are representative of 3 independent experiments. Each sample contained 10 μg of total protein. (A) The levels of iNOS, COX-2, and β-actin expression; (B) ratio of iNOS to β-actin expressed as a percentage. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on nuclear iNOS and COX-2 expression levels in Pam3CSK4-induced RAW 264.7 cells. The levels of iNOS, COX-2, and β-actin expression were detected by western blotting using specific antibodies. Cells (1.9×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before being treated with 10 μg/mL Pam3CSK4. To detect iNOS and COX-2 expression, Pam3CSK4 was added for 24 hours at 37°C. The blots shown here are representative of 3 independent experiments. Each sample contained 10 μg of total protein. (A) The levels of iNOS, COX-2, and β-actin expression; (B) ratio of iNOS to β-actin expressed as a percentage. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Expressing, Western Blot

Schematic model for the anti-inflammatory mechanism of M and Z in Pam3CSK4-induced RAW 264.7 cells. ML soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, JNK 1/2: c-Jun N-terminal kinase 1/2, p38: p38 mitogen-activated protein kinase, ERK 1/2: extracellular signal-regulated kinase 1/2, NF-κB/IκBα: nuclear transcription factor κB pathway, JAK/STAT3: Janus kinase/signal transduction and activator of transcription 3 signaling pathway, iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, IL: interleukin; PGE 2 : prostaglandin E 2 , NO: nitric oxide, DMSO: dimethyl sulfoxide.
Figure Legend Snippet: Schematic model for the anti-inflammatory mechanism of M and Z in Pam3CSK4-induced RAW 264.7 cells. ML soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, JNK 1/2: c-Jun N-terminal kinase 1/2, p38: p38 mitogen-activated protein kinase, ERK 1/2: extracellular signal-regulated kinase 1/2, NF-κB/IκBα: nuclear transcription factor κB pathway, JAK/STAT3: Janus kinase/signal transduction and activator of transcription 3 signaling pathway, iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, IL: interleukin; PGE 2 : prostaglandin E 2 , NO: nitric oxide, DMSO: dimethyl sulfoxide.

Techniques Used: Transduction

Effects of M and Z on Pam3CSK4-induced PGE 2 formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , PGE 2 : prostaglandin E 2 , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on Pam3CSK4-induced PGE 2 formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , PGE 2 : prostaglandin E 2 , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Standard Deviation

Effects of M and Z on Pam3CSK4-induced IL-1β formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , IL-1β: interleukin-1β, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on Pam3CSK4-induced IL-1β formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , IL-1β: interleukin-1β, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Standard Deviation

Effects of M and Z on nuclear p44/42 MAPK expression levels in Pam3CSK4-induced RAW 264.7 cells. The levels of p44/42 MAPK and β-actin expression were detected by western blotting using specific antibodies. Cells (1.9×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before being treated with 10 μg/mL Pam3CSK4. To detect p44/42 MAPK expression, Pam3CSK4 was added for 30 minutes at 37°C. The blots shown here are representative of 3 independent experiments. Each sample contained 10 μg of total protein. (A) The levels of p44/42 MAPK, P-p44/42 MAPK and β-actin expression; (B) ratio of P-p44/42 MAPK to p44/42 MAPK expressed as a percentage. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), MAPK: mitogen-activated protein kinase, Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on nuclear p44/42 MAPK expression levels in Pam3CSK4-induced RAW 264.7 cells. The levels of p44/42 MAPK and β-actin expression were detected by western blotting using specific antibodies. Cells (1.9×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before being treated with 10 μg/mL Pam3CSK4. To detect p44/42 MAPK expression, Pam3CSK4 was added for 30 minutes at 37°C. The blots shown here are representative of 3 independent experiments. Each sample contained 10 μg of total protein. (A) The levels of p44/42 MAPK, P-p44/42 MAPK and β-actin expression; (B) ratio of P-p44/42 MAPK to p44/42 MAPK expressed as a percentage. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), MAPK: mitogen-activated protein kinase, Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Expressing, Western Blot

Effects of M and Z on Pam3CSK4-induced NO production in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU and 10 μg/mL Pam3CSK4 for 24 hours at 37°C. In experiments without Pam3CSK4, the medium alone was used as a negative control, and IBU was used as a positive control. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , NO: nitric oxide, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on Pam3CSK4-induced NO production in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU and 10 μg/mL Pam3CSK4 for 24 hours at 37°C. In experiments without Pam3CSK4, the medium alone was used as a negative control, and IBU was used as a positive control. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , NO: nitric oxide, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Negative Control, Positive Control, Standard Deviation

30) Product Images from "Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model"

Article Title: Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0120278

THP-1 Cytokine Response to Test Antigens. THP-1 monocytes were extremely sensitive to activation by LPS, Lipid A, and Pam3CSK4. Production of IL-8 (A) and TNF-a (B) was observed at antigen concentrations as low as 1 ng/mL and cytokine production increased with increasing antigen concentration in a dose-dependent manner.
Figure Legend Snippet: THP-1 Cytokine Response to Test Antigens. THP-1 monocytes were extremely sensitive to activation by LPS, Lipid A, and Pam3CSK4. Production of IL-8 (A) and TNF-a (B) was observed at antigen concentrations as low as 1 ng/mL and cytokine production increased with increasing antigen concentration in a dose-dependent manner.

Techniques Used: Activation Assay, Concentration Assay

Binding Affinities of Immunoglobulins in SBI for Test Antigens. The binding affinities of bovine IgG, IgM, and IgA for LPS (Panel A), Lipid A (Panel B), and Pam3CSK4 (Panel C) were demonstrated using a modified ELISA technique. ELISA control conditions (D-F) confirmed IgG had a greater affinity for the antigens of interest (Complete ELISA) when compared with non-specific binding to blocking proteins (-antigen, +blocking) or the surface of the ELISA plate (-antigen,-blocking).
Figure Legend Snippet: Binding Affinities of Immunoglobulins in SBI for Test Antigens. The binding affinities of bovine IgG, IgM, and IgA for LPS (Panel A), Lipid A (Panel B), and Pam3CSK4 (Panel C) were demonstrated using a modified ELISA technique. ELISA control conditions (D-F) confirmed IgG had a greater affinity for the antigens of interest (Complete ELISA) when compared with non-specific binding to blocking proteins (-antigen, +blocking) or the surface of the ELISA plate (-antigen,-blocking).

Techniques Used: Binding Assay, Modification, Enzyme-linked Immunosorbent Assay, Blocking Assay

Translocation of Test Antigens Across Damaged Co-Culture Monolayers. Each test antigen was added to the apical compartment of undamaged and PPS damaged C2BBe1 co-cultures. LPS, Lipid A, and Pam3CSK4 were added at 10 ng/mL, 200 ng/mL, and 10 ng/mL, respectively. Pam3CSK4 was the only antigen tested capable of translocating a PPS-damaged barrier and stimulating IL-8 production by basal THP-1 monocytes. The small increases in IL-8 production observed for both LPS and Lipid A added to the apical compartment were not statistically different from PPS alone indicating these antigens were unable to translocate the PPS damaged barrier. * indicates P
Figure Legend Snippet: Translocation of Test Antigens Across Damaged Co-Culture Monolayers. Each test antigen was added to the apical compartment of undamaged and PPS damaged C2BBe1 co-cultures. LPS, Lipid A, and Pam3CSK4 were added at 10 ng/mL, 200 ng/mL, and 10 ng/mL, respectively. Pam3CSK4 was the only antigen tested capable of translocating a PPS-damaged barrier and stimulating IL-8 production by basal THP-1 monocytes. The small increases in IL-8 production observed for both LPS and Lipid A added to the apical compartment were not statistically different from PPS alone indicating these antigens were unable to translocate the PPS damaged barrier. * indicates P

Techniques Used: Translocation Assay, Co-Culture Assay

Immune Exclusion of Test Antigens. Immune exclusion of LPS (A) and Lipid A (B) induced IL-8 pro-inflammatory cytokine production was observed by the addition of SBI. Cytokine production by LPS and Lipid A was increasingly inhibited as the SBI concentration was increased from 1 mg/mL to 50 mg/mL. Immune exclusion of Pam3CSK4 (C) was not observed as the addition of SBI did not affect cytokine production. The control protein collagen did not affect cytokine production induced by any test antigen. * indicates P
Figure Legend Snippet: Immune Exclusion of Test Antigens. Immune exclusion of LPS (A) and Lipid A (B) induced IL-8 pro-inflammatory cytokine production was observed by the addition of SBI. Cytokine production by LPS and Lipid A was increasingly inhibited as the SBI concentration was increased from 1 mg/mL to 50 mg/mL. Immune exclusion of Pam3CSK4 (C) was not observed as the addition of SBI did not affect cytokine production. The control protein collagen did not affect cytokine production induced by any test antigen. * indicates P

Techniques Used: Concentration Assay

Steric Exclusion of Pam3CSK4 by Immunoglobulin Binding. Pam3CSK4 translocation across a PPS damaged epithelial barrier was inhibited by steric exclusion when 10 ng/mL of Pam3CSK4 was bound by the immunoglobulin in 50 mg/mL of SBI. All co-cultures were incubated with 0.01% PPS for 4 hours prior to experimentation to ensure epithelial barriers were damaged. Pam3CSK4 translocates from the apical to basal compartment across a PPS damaged epithelial barrier. Binding of Pam3CSK4 by the immunoglobulins in SBI completely inhibits antigen translocation as shown by the complete elimination of IL-8 production by basal THP-1 monocytes. * indicates P
Figure Legend Snippet: Steric Exclusion of Pam3CSK4 by Immunoglobulin Binding. Pam3CSK4 translocation across a PPS damaged epithelial barrier was inhibited by steric exclusion when 10 ng/mL of Pam3CSK4 was bound by the immunoglobulin in 50 mg/mL of SBI. All co-cultures were incubated with 0.01% PPS for 4 hours prior to experimentation to ensure epithelial barriers were damaged. Pam3CSK4 translocates from the apical to basal compartment across a PPS damaged epithelial barrier. Binding of Pam3CSK4 by the immunoglobulins in SBI completely inhibits antigen translocation as shown by the complete elimination of IL-8 production by basal THP-1 monocytes. * indicates P

Techniques Used: Binding Assay, Translocation Assay, Incubation

31) Product Images from "Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model"

Article Title: Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0120278

THP-1 Cytokine Response to Test Antigens. THP-1 monocytes were extremely sensitive to activation by LPS, Lipid A, and Pam3CSK4. Production of IL-8 (A) and TNF-a (B) was observed at antigen concentrations as low as 1 ng/mL and cytokine production increased with increasing antigen concentration in a dose-dependent manner.
Figure Legend Snippet: THP-1 Cytokine Response to Test Antigens. THP-1 monocytes were extremely sensitive to activation by LPS, Lipid A, and Pam3CSK4. Production of IL-8 (A) and TNF-a (B) was observed at antigen concentrations as low as 1 ng/mL and cytokine production increased with increasing antigen concentration in a dose-dependent manner.

Techniques Used: Activation Assay, Concentration Assay

Binding Affinities of Immunoglobulins in SBI for Test Antigens. The binding affinities of bovine IgG, IgM, and IgA for LPS (Panel A), Lipid A (Panel B), and Pam3CSK4 (Panel C) were demonstrated using a modified ELISA technique. ELISA control conditions (D-F) confirmed IgG had a greater affinity for the antigens of interest (Complete ELISA) when compared with non-specific binding to blocking proteins (-antigen, +blocking) or the surface of the ELISA plate (-antigen,-blocking).
Figure Legend Snippet: Binding Affinities of Immunoglobulins in SBI for Test Antigens. The binding affinities of bovine IgG, IgM, and IgA for LPS (Panel A), Lipid A (Panel B), and Pam3CSK4 (Panel C) were demonstrated using a modified ELISA technique. ELISA control conditions (D-F) confirmed IgG had a greater affinity for the antigens of interest (Complete ELISA) when compared with non-specific binding to blocking proteins (-antigen, +blocking) or the surface of the ELISA plate (-antigen,-blocking).

Techniques Used: Binding Assay, Modification, Enzyme-linked Immunosorbent Assay, Blocking Assay

Translocation of Test Antigens Across Damaged Co-Culture Monolayers. Each test antigen was added to the apical compartment of undamaged and PPS damaged C2BBe1 co-cultures. LPS, Lipid A, and Pam3CSK4 were added at 10 ng/mL, 200 ng/mL, and 10 ng/mL, respectively. Pam3CSK4 was the only antigen tested capable of translocating a PPS-damaged barrier and stimulating IL-8 production by basal THP-1 monocytes. The small increases in IL-8 production observed for both LPS and Lipid A added to the apical compartment were not statistically different from PPS alone indicating these antigens were unable to translocate the PPS damaged barrier. * indicates P
Figure Legend Snippet: Translocation of Test Antigens Across Damaged Co-Culture Monolayers. Each test antigen was added to the apical compartment of undamaged and PPS damaged C2BBe1 co-cultures. LPS, Lipid A, and Pam3CSK4 were added at 10 ng/mL, 200 ng/mL, and 10 ng/mL, respectively. Pam3CSK4 was the only antigen tested capable of translocating a PPS-damaged barrier and stimulating IL-8 production by basal THP-1 monocytes. The small increases in IL-8 production observed for both LPS and Lipid A added to the apical compartment were not statistically different from PPS alone indicating these antigens were unable to translocate the PPS damaged barrier. * indicates P

Techniques Used: Translocation Assay, Co-Culture Assay

Immune Exclusion of Test Antigens. Immune exclusion of LPS (A) and Lipid A (B) induced IL-8 pro-inflammatory cytokine production was observed by the addition of SBI. Cytokine production by LPS and Lipid A was increasingly inhibited as the SBI concentration was increased from 1 mg/mL to 50 mg/mL. Immune exclusion of Pam3CSK4 (C) was not observed as the addition of SBI did not affect cytokine production. The control protein collagen did not affect cytokine production induced by any test antigen. * indicates P
Figure Legend Snippet: Immune Exclusion of Test Antigens. Immune exclusion of LPS (A) and Lipid A (B) induced IL-8 pro-inflammatory cytokine production was observed by the addition of SBI. Cytokine production by LPS and Lipid A was increasingly inhibited as the SBI concentration was increased from 1 mg/mL to 50 mg/mL. Immune exclusion of Pam3CSK4 (C) was not observed as the addition of SBI did not affect cytokine production. The control protein collagen did not affect cytokine production induced by any test antigen. * indicates P

Techniques Used: Concentration Assay

Steric Exclusion of Pam3CSK4 by Immunoglobulin Binding. Pam3CSK4 translocation across a PPS damaged epithelial barrier was inhibited by steric exclusion when 10 ng/mL of Pam3CSK4 was bound by the immunoglobulin in 50 mg/mL of SBI. All co-cultures were incubated with 0.01% PPS for 4 hours prior to experimentation to ensure epithelial barriers were damaged. Pam3CSK4 translocates from the apical to basal compartment across a PPS damaged epithelial barrier. Binding of Pam3CSK4 by the immunoglobulins in SBI completely inhibits antigen translocation as shown by the complete elimination of IL-8 production by basal THP-1 monocytes. * indicates P
Figure Legend Snippet: Steric Exclusion of Pam3CSK4 by Immunoglobulin Binding. Pam3CSK4 translocation across a PPS damaged epithelial barrier was inhibited by steric exclusion when 10 ng/mL of Pam3CSK4 was bound by the immunoglobulin in 50 mg/mL of SBI. All co-cultures were incubated with 0.01% PPS for 4 hours prior to experimentation to ensure epithelial barriers were damaged. Pam3CSK4 translocates from the apical to basal compartment across a PPS damaged epithelial barrier. Binding of Pam3CSK4 by the immunoglobulins in SBI completely inhibits antigen translocation as shown by the complete elimination of IL-8 production by basal THP-1 monocytes. * indicates P

Techniques Used: Binding Assay, Translocation Assay, Incubation

32) Product Images from "Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model"

Article Title: Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0120278

THP-1 Cytokine Response to Test Antigens. THP-1 monocytes were extremely sensitive to activation by LPS, Lipid A, and Pam3CSK4. Production of IL-8 (A) and TNF-a (B) was observed at antigen concentrations as low as 1 ng/mL and cytokine production increased with increasing antigen concentration in a dose-dependent manner.
Figure Legend Snippet: THP-1 Cytokine Response to Test Antigens. THP-1 monocytes were extremely sensitive to activation by LPS, Lipid A, and Pam3CSK4. Production of IL-8 (A) and TNF-a (B) was observed at antigen concentrations as low as 1 ng/mL and cytokine production increased with increasing antigen concentration in a dose-dependent manner.

Techniques Used: Activation Assay, Concentration Assay

Binding Affinities of Immunoglobulins in SBI for Test Antigens. The binding affinities of bovine IgG, IgM, and IgA for LPS (Panel A), Lipid A (Panel B), and Pam3CSK4 (Panel C) were demonstrated using a modified ELISA technique. ELISA control conditions (D-F) confirmed IgG had a greater affinity for the antigens of interest (Complete ELISA) when compared with non-specific binding to blocking proteins (-antigen, +blocking) or the surface of the ELISA plate (-antigen,-blocking).
Figure Legend Snippet: Binding Affinities of Immunoglobulins in SBI for Test Antigens. The binding affinities of bovine IgG, IgM, and IgA for LPS (Panel A), Lipid A (Panel B), and Pam3CSK4 (Panel C) were demonstrated using a modified ELISA technique. ELISA control conditions (D-F) confirmed IgG had a greater affinity for the antigens of interest (Complete ELISA) when compared with non-specific binding to blocking proteins (-antigen, +blocking) or the surface of the ELISA plate (-antigen,-blocking).

Techniques Used: Binding Assay, Modification, Enzyme-linked Immunosorbent Assay, Blocking Assay

Translocation of Test Antigens Across Damaged Co-Culture Monolayers. Each test antigen was added to the apical compartment of undamaged and PPS damaged C2BBe1 co-cultures. LPS, Lipid A, and Pam3CSK4 were added at 10 ng/mL, 200 ng/mL, and 10 ng/mL, respectively. Pam3CSK4 was the only antigen tested capable of translocating a PPS-damaged barrier and stimulating IL-8 production by basal THP-1 monocytes. The small increases in IL-8 production observed for both LPS and Lipid A added to the apical compartment were not statistically different from PPS alone indicating these antigens were unable to translocate the PPS damaged barrier. * indicates P
Figure Legend Snippet: Translocation of Test Antigens Across Damaged Co-Culture Monolayers. Each test antigen was added to the apical compartment of undamaged and PPS damaged C2BBe1 co-cultures. LPS, Lipid A, and Pam3CSK4 were added at 10 ng/mL, 200 ng/mL, and 10 ng/mL, respectively. Pam3CSK4 was the only antigen tested capable of translocating a PPS-damaged barrier and stimulating IL-8 production by basal THP-1 monocytes. The small increases in IL-8 production observed for both LPS and Lipid A added to the apical compartment were not statistically different from PPS alone indicating these antigens were unable to translocate the PPS damaged barrier. * indicates P

Techniques Used: Translocation Assay, Co-Culture Assay

Immune Exclusion of Test Antigens. Immune exclusion of LPS (A) and Lipid A (B) induced IL-8 pro-inflammatory cytokine production was observed by the addition of SBI. Cytokine production by LPS and Lipid A was increasingly inhibited as the SBI concentration was increased from 1 mg/mL to 50 mg/mL. Immune exclusion of Pam3CSK4 (C) was not observed as the addition of SBI did not affect cytokine production. The control protein collagen did not affect cytokine production induced by any test antigen. * indicates P
Figure Legend Snippet: Immune Exclusion of Test Antigens. Immune exclusion of LPS (A) and Lipid A (B) induced IL-8 pro-inflammatory cytokine production was observed by the addition of SBI. Cytokine production by LPS and Lipid A was increasingly inhibited as the SBI concentration was increased from 1 mg/mL to 50 mg/mL. Immune exclusion of Pam3CSK4 (C) was not observed as the addition of SBI did not affect cytokine production. The control protein collagen did not affect cytokine production induced by any test antigen. * indicates P

Techniques Used: Concentration Assay

Steric Exclusion of Pam3CSK4 by Immunoglobulin Binding. Pam3CSK4 translocation across a PPS damaged epithelial barrier was inhibited by steric exclusion when 10 ng/mL of Pam3CSK4 was bound by the immunoglobulin in 50 mg/mL of SBI. All co-cultures were incubated with 0.01% PPS for 4 hours prior to experimentation to ensure epithelial barriers were damaged. Pam3CSK4 translocates from the apical to basal compartment across a PPS damaged epithelial barrier. Binding of Pam3CSK4 by the immunoglobulins in SBI completely inhibits antigen translocation as shown by the complete elimination of IL-8 production by basal THP-1 monocytes. * indicates P
Figure Legend Snippet: Steric Exclusion of Pam3CSK4 by Immunoglobulin Binding. Pam3CSK4 translocation across a PPS damaged epithelial barrier was inhibited by steric exclusion when 10 ng/mL of Pam3CSK4 was bound by the immunoglobulin in 50 mg/mL of SBI. All co-cultures were incubated with 0.01% PPS for 4 hours prior to experimentation to ensure epithelial barriers were damaged. Pam3CSK4 translocates from the apical to basal compartment across a PPS damaged epithelial barrier. Binding of Pam3CSK4 by the immunoglobulins in SBI completely inhibits antigen translocation as shown by the complete elimination of IL-8 production by basal THP-1 monocytes. * indicates P

Techniques Used: Binding Assay, Translocation Assay, Incubation

33) Product Images from "TLR2 Expression Is Increased in Rosacea and Stimulates Enhanced Serine Protease Production by Keratinocytes"

Article Title: TLR2 Expression Is Increased in Rosacea and Stimulates Enhanced Serine Protease Production by Keratinocytes

Journal: The Journal of investigative dermatology

doi: 10.1038/jid.2010.351

Toll-like receptor 2 (TLR2) ligands induce a post-transcriptional increase in kallikrein 5 (KLK5) release in a calcium-dependent manner ( a , b ) Normal human epidermal keratinocytes (NHEKs) were cultured in low calcium media and treated with cycloheximide (10 μgml −1 ) or brefeldin A (5 μgml −1 ) for 30 minutes, and stimulated with TLR2/1L (Pam3CSK4: 1 μgml −1 ) for a further 6 hours. Then, ( b ) KLK5 mRNA and ( a ) KLK5 protein in the culture media were measured. * P
Figure Legend Snippet: Toll-like receptor 2 (TLR2) ligands induce a post-transcriptional increase in kallikrein 5 (KLK5) release in a calcium-dependent manner ( a , b ) Normal human epidermal keratinocytes (NHEKs) were cultured in low calcium media and treated with cycloheximide (10 μgml −1 ) or brefeldin A (5 μgml −1 ) for 30 minutes, and stimulated with TLR2/1L (Pam3CSK4: 1 μgml −1 ) for a further 6 hours. Then, ( b ) KLK5 mRNA and ( a ) KLK5 protein in the culture media were measured. * P

Techniques Used: Cell Culture

34) Product Images from "TLR2 Deficiency Exacerbates Imiquimod-Induced Psoriasis-Like Skin Inflammation through Decrease in Regulatory T Cells and Impaired IL-10 Production"

Article Title: TLR2 Deficiency Exacerbates Imiquimod-Induced Psoriasis-Like Skin Inflammation through Decrease in Regulatory T Cells and Impaired IL-10 Production

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21228560

Systemic treatment with Pam3CSK4 and Pam2CSK4 attenuates imiquimod-induced skin inflammation. Shaved back skin of wild-type (WT) mice were topically treated with imiquimod for 5 consecutive days. Wild-type mice were injected with 50 g Pam3CSK4 and 50 g Pam2CSK4 (PAM) or PBS intravenously two days before, on the same day, and two days after imiquimod application. ( A ) The phenotypical manifestation of back skin induced by imiquimod application at day 5. ( B ) Disease severity during imiquimod treatment. Clinical scores for disease severity were calculated daily using a scoring system based on the clinical psoriasis area and severity index. Data are presented as mean ± SEM of three independent experiments ( n = 9 for each group). * p
Figure Legend Snippet: Systemic treatment with Pam3CSK4 and Pam2CSK4 attenuates imiquimod-induced skin inflammation. Shaved back skin of wild-type (WT) mice were topically treated with imiquimod for 5 consecutive days. Wild-type mice were injected with 50 g Pam3CSK4 and 50 g Pam2CSK4 (PAM) or PBS intravenously two days before, on the same day, and two days after imiquimod application. ( A ) The phenotypical manifestation of back skin induced by imiquimod application at day 5. ( B ) Disease severity during imiquimod treatment. Clinical scores for disease severity were calculated daily using a scoring system based on the clinical psoriasis area and severity index. Data are presented as mean ± SEM of three independent experiments ( n = 9 for each group). * p

Techniques Used: Mouse Assay, Injection

35) Product Images from "First Insight into the Modulation of Noncanonical NF-κB Signaling Components by Poxviruses in Established Immune-Derived Cell Lines: An In Vitro Model of Ectromelia Virus Infection"

Article Title: First Insight into the Modulation of Noncanonical NF-κB Signaling Components by Poxviruses in Established Immune-Derived Cell Lines: An In Vitro Model of Ectromelia Virus Infection

Journal: Pathogens

doi: 10.3390/pathogens9100814

ECTV inhibits phosphorylation of p100 and RelB and affects p100 processing in JAWS II and RAW 264.7 cells. ( A ) Immunoblot analysis of phosphorylation of RelB in unstimulated or PMA + Io-stimulated mock-, uvi-ECTV-, and ECTV-infected JAWS II cells. ( B ) Immunoblot analysis of the content of p-RelB and p-p100 in unstimulated or rmIFN-γ + Escherichia coli O111:B4 LPS-stimulated mock- and ECTV-infected JAWS II cells. ( C ) Immunoblot analysis of p-RelB and p-p100 in unstimulated and Pam3CSK4- or rmIFN-γ + LPS O111:B4-stimulated mock- and ECTV-infected RAW 264.7 cells. ( D ) Immunoblot analysis of p100/p52 content in mock- and ECTV-infected JAWS II cells treated with poly(I:C) or rmIFN-γ + LPS O111:B4. ( E ) Densitometric analysis of the p100/p52 ratio and p52 content in JAWS II cells. Data obtained from two independent experiments are shown on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control. ( F ) Immunoblot analysis of p100/p52 proteins in mock- and ECTV-infected RAW 264.7 cells at 4 hpi. The cells were left untreated or treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( G ) Densitometric analysis of the p100/p52 ratio and p52 content in RAW 264.7 cells based on the data obtained from two independent experiments. Data are presented on histograms with a logarithmic scale. GAPDH—loading control. ( H ) Immunoblot analysis of p100/p52 proteins in mock- and ECTV-infected RAW 264.7 cells at 18 hpi. The cells were left untreated or treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( I ) Densitometric analysis of the p100/p52 ratio and p52 content in RAW 264.7 cells based on the data obtained from two independent experiments. Data are presented on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control.
Figure Legend Snippet: ECTV inhibits phosphorylation of p100 and RelB and affects p100 processing in JAWS II and RAW 264.7 cells. ( A ) Immunoblot analysis of phosphorylation of RelB in unstimulated or PMA + Io-stimulated mock-, uvi-ECTV-, and ECTV-infected JAWS II cells. ( B ) Immunoblot analysis of the content of p-RelB and p-p100 in unstimulated or rmIFN-γ + Escherichia coli O111:B4 LPS-stimulated mock- and ECTV-infected JAWS II cells. ( C ) Immunoblot analysis of p-RelB and p-p100 in unstimulated and Pam3CSK4- or rmIFN-γ + LPS O111:B4-stimulated mock- and ECTV-infected RAW 264.7 cells. ( D ) Immunoblot analysis of p100/p52 content in mock- and ECTV-infected JAWS II cells treated with poly(I:C) or rmIFN-γ + LPS O111:B4. ( E ) Densitometric analysis of the p100/p52 ratio and p52 content in JAWS II cells. Data obtained from two independent experiments are shown on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control. ( F ) Immunoblot analysis of p100/p52 proteins in mock- and ECTV-infected RAW 264.7 cells at 4 hpi. The cells were left untreated or treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( G ) Densitometric analysis of the p100/p52 ratio and p52 content in RAW 264.7 cells based on the data obtained from two independent experiments. Data are presented on histograms with a logarithmic scale. GAPDH—loading control. ( H ) Immunoblot analysis of p100/p52 proteins in mock- and ECTV-infected RAW 264.7 cells at 18 hpi. The cells were left untreated or treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( I ) Densitometric analysis of the p100/p52 ratio and p52 content in RAW 264.7 cells based on the data obtained from two independent experiments. Data are presented on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control.

Techniques Used: Infection

ECTV influences the expression of components of the noncanonical NF-κB signaling pathway in JAWS II and RAW 264.7 cells. ( A ) Immunoblots of mock- and ECTV-infected JAWS II cells untreated or treated with poly(I:C) or rmIFN-γ + Escherichia coli LPS O111:B4. ( B ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in JAWS II cells. The analysis was based on the data of two independent biological experiments. The data are shown on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control. ( C ) Immunoblots of mock- and ECTV-infected RAW 264.7 cells at 4 hpi. Cells were left untreated or were treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( D ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in RAW 264.7 cells. The analysis was based on the data of two independent biological experiments and is plotted on histograms with a logarithmic scale. GAPDH—loading control. ( E ) Immunoblots of mock- and ECTV-infected RAW 264.7 cells at 18 hpi. Cells were left untreated or were treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( F ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in RAW 264.7 cells. The analysis was based on the data of two independent biological experiments and is plotted on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control.
Figure Legend Snippet: ECTV influences the expression of components of the noncanonical NF-κB signaling pathway in JAWS II and RAW 264.7 cells. ( A ) Immunoblots of mock- and ECTV-infected JAWS II cells untreated or treated with poly(I:C) or rmIFN-γ + Escherichia coli LPS O111:B4. ( B ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in JAWS II cells. The analysis was based on the data of two independent biological experiments. The data are shown on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control. ( C ) Immunoblots of mock- and ECTV-infected RAW 264.7 cells at 4 hpi. Cells were left untreated or were treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( D ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in RAW 264.7 cells. The analysis was based on the data of two independent biological experiments and is plotted on histograms with a logarithmic scale. GAPDH—loading control. ( E ) Immunoblots of mock- and ECTV-infected RAW 264.7 cells at 18 hpi. Cells were left untreated or were treated with poly(I:C), Pam3CSK4, rmIFN-γ + E. coli LPS O55:B5, or rmIFN-γ + LPS O111:B4. ( F ) Densitometric evaluation of RelB, cIAP1, and TRAF2 expression in RAW 264.7 cells. The analysis was based on the data of two independent biological experiments and is plotted on histograms with a logarithmic scale. * p ≤ 0.05, ** p ≤ 0.01. GAPDH—loading control.

Techniques Used: Expressing, Western Blot, Infection

36) Product Images from "Immunotherapy targeting toll-like receptor 2 alleviates neurodegeneration in models of synucleinopathy by modulating α-synuclein transmission and neuroinflammation"

Article Title: Immunotherapy targeting toll-like receptor 2 alleviates neurodegeneration in models of synucleinopathy by modulating α-synuclein transmission and neuroinflammation

Journal: Molecular Neurodegeneration

doi: 10.1186/s13024-018-0276-2

TLR2 mediates neurotoxic neuron-to-neuron α-synuclein transmission. a Overview diagram. Donor neuronal cells (V1S), expressing α-synuclein-conjugated with N-terminus of venus were plated in trans-well insert and the recipient neuronal cells (SV2), expressing α-synuclein conjugated with C-terminus of venus were seeded onto cover slips in the bottom well. Only SV2 cells were treated with pam3CSK4 (10 μg/ml), lentiviral vectors, or antibodies. Images were taken from SV2 cells after a 3-days co-culture. b – e Representative confocal images for BiFC fluorescence and caspase-3 activity in SV2 cells. Middle panels are enlargements of cropped regions outlined with dashed lines from upper panels. Lower panels are double-immunolabeling assay with active casepase-3. The average numbers of venus fluorescence intensity in each cell, the average size of the venus punctum diameters, and caspase-3 fluorescence intensity were analyzed. b V1S and SV2 cells were co-cultured in the presence or absence of pam3CSK4 (10 μg/ml) (n = 3). c V1S and SV2 cells were co-cultured with either LV-control or LV-TLR2 (n = 3). d V1S and SV2 cells were co-cultured with either LV-sh.control or LV-shTLR2 (n = 3). e V1S and SV2 cells were co-cultured with either IgG (5 μg/ml) or T2.5 (5 μg/ml) (n = 3). f The kinetics of α-synuclein internalization in the presence of antibodies. dSY5Y cells were incubated with αSCM for indicated hours in the presence of either IgG (5 μg/ml) or T2.5 (5 μg/ml). The kinetics was analyzed by immunolabeling assay (n = 3). g Neuronal internalization of α-synuclein in the presence of antibodies. Mouse primary cortical neurons were incubated with αSCM or LZCM for indicated hours in the presence of either IgG (5 μg/ml) or T2.5 (5 μg/ml). Neurons were double immunolabelled with human α-synuclein (Middle panels) and active form of caspase-3 (low panels) (n = 3). Data are mean ± SEM (n = 3 per group). * p
Figure Legend Snippet: TLR2 mediates neurotoxic neuron-to-neuron α-synuclein transmission. a Overview diagram. Donor neuronal cells (V1S), expressing α-synuclein-conjugated with N-terminus of venus were plated in trans-well insert and the recipient neuronal cells (SV2), expressing α-synuclein conjugated with C-terminus of venus were seeded onto cover slips in the bottom well. Only SV2 cells were treated with pam3CSK4 (10 μg/ml), lentiviral vectors, or antibodies. Images were taken from SV2 cells after a 3-days co-culture. b – e Representative confocal images for BiFC fluorescence and caspase-3 activity in SV2 cells. Middle panels are enlargements of cropped regions outlined with dashed lines from upper panels. Lower panels are double-immunolabeling assay with active casepase-3. The average numbers of venus fluorescence intensity in each cell, the average size of the venus punctum diameters, and caspase-3 fluorescence intensity were analyzed. b V1S and SV2 cells were co-cultured in the presence or absence of pam3CSK4 (10 μg/ml) (n = 3). c V1S and SV2 cells were co-cultured with either LV-control or LV-TLR2 (n = 3). d V1S and SV2 cells were co-cultured with either LV-sh.control or LV-shTLR2 (n = 3). e V1S and SV2 cells were co-cultured with either IgG (5 μg/ml) or T2.5 (5 μg/ml) (n = 3). f The kinetics of α-synuclein internalization in the presence of antibodies. dSY5Y cells were incubated with αSCM for indicated hours in the presence of either IgG (5 μg/ml) or T2.5 (5 μg/ml). The kinetics was analyzed by immunolabeling assay (n = 3). g Neuronal internalization of α-synuclein in the presence of antibodies. Mouse primary cortical neurons were incubated with αSCM or LZCM for indicated hours in the presence of either IgG (5 μg/ml) or T2.5 (5 μg/ml). Neurons were double immunolabelled with human α-synuclein (Middle panels) and active form of caspase-3 (low panels) (n = 3). Data are mean ± SEM (n = 3 per group). * p

Techniques Used: Transmission Assay, Expressing, Co-Culture Assay, Bimolecular Fluorescence Complementation Assay, Fluorescence, Activity Assay, Immunolabeling, Cell Culture, Incubation

37) Product Images from "H2-Dd-mediated upregulation of interleukin-4 production by natural killer T-cell and dendritic cell interaction"

Article Title: H2-Dd-mediated upregulation of interleukin-4 production by natural killer T-cell and dendritic cell interaction

Journal: Immunology

doi: 10.1111/j.1365-2567.2007.02744.x

The effect of Pam3CSK4 on DC function for NKT-cell activation. BC1 cells were unstimulated (iDCs) or stimulated with CpG-ODN (CpG-DCs) or Pam3CSK4 (P3C-DCs). (a) α-GalCer-loaded iDCs, CpG-DCs, or P3C-DCs (1·5 × 10 4 cells) were cocultured with nylon non-adherent splenocytes (5 × 10 5 cells) for 48 hr. The amount of IL-4 in the culture supernatants was measured by ELISA. Each column represents the mean ± SE of three or four independent experiments. (b) Expression of H-2D d , CD80 and CD86 on iDCs, CpG-DCs and P3C-DCs. Mean fluorescence intensity (MFI) of each expression is shown. Each column represents the mean ± SE of three (CD86) or four (H2-D d and CD80) independent experiments. Statistical significance was calculated by Student's t -test (* P
Figure Legend Snippet: The effect of Pam3CSK4 on DC function for NKT-cell activation. BC1 cells were unstimulated (iDCs) or stimulated with CpG-ODN (CpG-DCs) or Pam3CSK4 (P3C-DCs). (a) α-GalCer-loaded iDCs, CpG-DCs, or P3C-DCs (1·5 × 10 4 cells) were cocultured with nylon non-adherent splenocytes (5 × 10 5 cells) for 48 hr. The amount of IL-4 in the culture supernatants was measured by ELISA. Each column represents the mean ± SE of three or four independent experiments. (b) Expression of H-2D d , CD80 and CD86 on iDCs, CpG-DCs and P3C-DCs. Mean fluorescence intensity (MFI) of each expression is shown. Each column represents the mean ± SE of three (CD86) or four (H2-D d and CD80) independent experiments. Statistical significance was calculated by Student's t -test (* P

Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence

38) Product Images from "Use of Therapeutic Pathogen Recognition Receptor Ligands for Osteo-Immunomodulation"

Article Title: Use of Therapeutic Pathogen Recognition Receptor Ligands for Osteo-Immunomodulation

Journal: Materials

doi: 10.3390/ma14051119

Cytokine production by hMSCs after stimulation with PRR ligands for 24 h. Cytokines measured were ( a ) TNF-α, ( b ) IL-10, ( c ) IL-6, and ( d ) IL-8. Concentrations used are the following: Pam3CSK4 (0.1 µg/mL), Curdlan (1 µg/mL), MPLA (10 µg/mL), Resiquimod (0.1 µg/mL), Murabutide (10 µg/mL), CpG ODN C (1 µg/mL), and Poly(I:C) (1 µg/mL). All results shown here are represented as mean ± standard deviation ( n = 3 donors) and normalized to the control. Absolute values of the controls can be found in supplementary Table S1 . Significance was tested using repeated-measures ANOVA with Sidak’s post hoc test for multiple comparisons. *** p
Figure Legend Snippet: Cytokine production by hMSCs after stimulation with PRR ligands for 24 h. Cytokines measured were ( a ) TNF-α, ( b ) IL-10, ( c ) IL-6, and ( d ) IL-8. Concentrations used are the following: Pam3CSK4 (0.1 µg/mL), Curdlan (1 µg/mL), MPLA (10 µg/mL), Resiquimod (0.1 µg/mL), Murabutide (10 µg/mL), CpG ODN C (1 µg/mL), and Poly(I:C) (1 µg/mL). All results shown here are represented as mean ± standard deviation ( n = 3 donors) and normalized to the control. Absolute values of the controls can be found in supplementary Table S1 . Significance was tested using repeated-measures ANOVA with Sidak’s post hoc test for multiple comparisons. *** p

Techniques Used: Standard Deviation

Effect of PRR ligands on osteoclast formation. ( A ) Tartrate-resistant acid phosphate (TRAP) staining performed on human monocytes cultured for 6 days +/- receptor activator of NF-κB ligand (RANKL). Osteoclasts are shown as TRAP-positive (red/pink) cells with ≥ 3 nuclei. ( a ) and ( b ) show variations in the shape and size of osteoclasts. ( B ) Representative images of monocytes stimulated with PRR ligands. Concentrations used are the following: Pam3CSK4 (0.1 µg/mL), Curdlan (1 µg/mL), Monophosphoryl Lipid A (MPLA) (10 µg/mL), Resiquimod (0.1 µg/mL), Murabutide (10 µg/mL), CpG ODN C (1 µg/mL), and Poly(I:C) (1 µg/mL). Scale bars correspond to 50 µm. ( C ) Osteoclast counts are represented as mean ± standard deviation ( n = 3–4 donors) and normalized to the control. Significance was tested using a repeated-measures mixed model approach with Sidak’s post hoc test for multiple comparisons. * p
Figure Legend Snippet: Effect of PRR ligands on osteoclast formation. ( A ) Tartrate-resistant acid phosphate (TRAP) staining performed on human monocytes cultured for 6 days +/- receptor activator of NF-κB ligand (RANKL). Osteoclasts are shown as TRAP-positive (red/pink) cells with ≥ 3 nuclei. ( a ) and ( b ) show variations in the shape and size of osteoclasts. ( B ) Representative images of monocytes stimulated with PRR ligands. Concentrations used are the following: Pam3CSK4 (0.1 µg/mL), Curdlan (1 µg/mL), Monophosphoryl Lipid A (MPLA) (10 µg/mL), Resiquimod (0.1 µg/mL), Murabutide (10 µg/mL), CpG ODN C (1 µg/mL), and Poly(I:C) (1 µg/mL). Scale bars correspond to 50 µm. ( C ) Osteoclast counts are represented as mean ± standard deviation ( n = 3–4 donors) and normalized to the control. Significance was tested using a repeated-measures mixed model approach with Sidak’s post hoc test for multiple comparisons. * p

Techniques Used: Staining, Cell Culture, Standard Deviation

39) Product Images from "Magnoliae Cortex and maize modulate Porphyromonas gingivalis-induced inflammatory reactions"

Article Title: Magnoliae Cortex and maize modulate Porphyromonas gingivalis-induced inflammatory reactions

Journal: Journal of Periodontal & Implant Science

doi: 10.5051/jpis.2018.48.2.70

Effects of M and Z on Pam3CSK4-induced IL-6 formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , IL-6: interleukin-6, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on Pam3CSK4-induced IL-6 formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , IL-6: interleukin-6, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Standard Deviation

Effects of M and Z on Pam3CSK4-induced NF-κB transactivation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes prior to treatment with 10 μg/mL Pam3CSK4 for 2 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , NF-κB: nuclear transcription factor κB, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide, p65: transcription factor p65. a) P
Figure Legend Snippet: Effects of M and Z on Pam3CSK4-induced NF-κB transactivation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes prior to treatment with 10 μg/mL Pam3CSK4 for 2 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , NF-κB: nuclear transcription factor κB, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide, p65: transcription factor p65. a) P

Techniques Used: Standard Deviation

Effects of M and Z on nuclear iNOS and COX-2 expression levels in Pam3CSK4-induced RAW 264.7 cells. The levels of iNOS, COX-2, and β-actin expression were detected by western blotting using specific antibodies. Cells (1.9×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before being treated with 10 μg/mL Pam3CSK4. To detect iNOS and COX-2 expression, Pam3CSK4 was added for 24 hours at 37°C. The blots shown here are representative of 3 independent experiments. Each sample contained 10 μg of total protein. (A) The levels of iNOS, COX-2, and β-actin expression; (B) ratio of iNOS to β-actin expressed as a percentage. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on nuclear iNOS and COX-2 expression levels in Pam3CSK4-induced RAW 264.7 cells. The levels of iNOS, COX-2, and β-actin expression were detected by western blotting using specific antibodies. Cells (1.9×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before being treated with 10 μg/mL Pam3CSK4. To detect iNOS and COX-2 expression, Pam3CSK4 was added for 24 hours at 37°C. The blots shown here are representative of 3 independent experiments. Each sample contained 10 μg of total protein. (A) The levels of iNOS, COX-2, and β-actin expression; (B) ratio of iNOS to β-actin expressed as a percentage. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Expressing, Western Blot

Schematic model for the anti-inflammatory mechanism of M and Z in Pam3CSK4-induced RAW 264.7 cells. ML soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, JNK 1/2: c-Jun N-terminal kinase 1/2, p38: p38 mitogen-activated protein kinase, ERK 1/2: extracellular signal-regulated kinase 1/2, NF-κB/IκBα: nuclear transcription factor κB pathway, JAK/STAT3: Janus kinase/signal transduction and activator of transcription 3 signaling pathway, iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, IL: interleukin; PGE 2 : prostaglandin E 2 , NO: nitric oxide, DMSO: dimethyl sulfoxide.
Figure Legend Snippet: Schematic model for the anti-inflammatory mechanism of M and Z in Pam3CSK4-induced RAW 264.7 cells. ML soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, JNK 1/2: c-Jun N-terminal kinase 1/2, p38: p38 mitogen-activated protein kinase, ERK 1/2: extracellular signal-regulated kinase 1/2, NF-κB/IκBα: nuclear transcription factor κB pathway, JAK/STAT3: Janus kinase/signal transduction and activator of transcription 3 signaling pathway, iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, IL: interleukin; PGE 2 : prostaglandin E 2 , NO: nitric oxide, DMSO: dimethyl sulfoxide.

Techniques Used: Transduction

Effects of M and Z on Pam3CSK4-induced PGE 2 formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , PGE 2 : prostaglandin E 2 , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on Pam3CSK4-induced PGE 2 formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , PGE 2 : prostaglandin E 2 , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Standard Deviation

Effects of M and Z on Pam3CSK4-induced IL-1β formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , IL-1β: interleukin-1β, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on Pam3CSK4-induced IL-1β formation in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , IL-1β: interleukin-1β, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Standard Deviation

Effects of M and Z on nuclear p44/42 MAPK expression levels in Pam3CSK4-induced RAW 264.7 cells. The levels of p44/42 MAPK and β-actin expression were detected by western blotting using specific antibodies. Cells (1.9×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before being treated with 10 μg/mL Pam3CSK4. To detect p44/42 MAPK expression, Pam3CSK4 was added for 30 minutes at 37°C. The blots shown here are representative of 3 independent experiments. Each sample contained 10 μg of total protein. (A) The levels of p44/42 MAPK, P-p44/42 MAPK and β-actin expression; (B) ratio of P-p44/42 MAPK to p44/42 MAPK expressed as a percentage. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), MAPK: mitogen-activated protein kinase, Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on nuclear p44/42 MAPK expression levels in Pam3CSK4-induced RAW 264.7 cells. The levels of p44/42 MAPK and β-actin expression were detected by western blotting using specific antibodies. Cells (1.9×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU for 30 minutes before being treated with 10 μg/mL Pam3CSK4. To detect p44/42 MAPK expression, Pam3CSK4 was added for 30 minutes at 37°C. The blots shown here are representative of 3 independent experiments. Each sample contained 10 μg of total protein. (A) The levels of p44/42 MAPK, P-p44/42 MAPK and β-actin expression; (B) ratio of P-p44/42 MAPK to p44/42 MAPK expressed as a percentage. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), MAPK: mitogen-activated protein kinase, Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Expressing, Western Blot

Effects of M and Z on Pam3CSK4-induced NO production in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU and 10 μg/mL Pam3CSK4 for 24 hours at 37°C. In experiments without Pam3CSK4, the medium alone was used as a negative control, and IBU was used as a positive control. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , NO: nitric oxide, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P
Figure Legend Snippet: Effects of M and Z on Pam3CSK4-induced NO production in RAW 264.7 cells. Cells (5×10 4 /cm 2 ) were treated with M, Z, MZ, or IBU and 10 μg/mL Pam3CSK4 for 24 hours at 37°C. In experiments without Pam3CSK4, the medium alone was used as a negative control, and IBU was used as a positive control. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments. M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis , NO: nitric oxide, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide. a) P

Techniques Used: Negative Control, Positive Control, Standard Deviation

40) Product Images from "Mitochondrial assembly of the NLRP3 inflammasome complex is initiated at priming"

Article Title: Mitochondrial assembly of the NLRP3 inflammasome complex is initiated at priming

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1701723

NLRP3 and caspase-1 independently associate with the mitochondria at priming. (A, B) J774A.1 cells were unstimulated, LPS-primed, or LPS-primed followed by stimulation with nigericin (A) or primed with Pam3CSK4 or HMW poly(I:C) (B) . Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (C) J774A.1 cells were LPS-primed for the indicated time in the presence of 10 μM cycloheximide. Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (D) J774A.1 cells were unstimulated or LPS-primed followed by stimulation with nigericin as indicated. Mitochondrial and cytosolic fractions were isolated and mitochondria treated with proteinase K in the presence or absence of Triton X-100. Cytosolic and mitochondrial fractions were then immunoblotted as shown. (E-G) WT, Nlrp3 −/− , Casp1 −/− , and Asc −/− BMM were unstimulated or LPS-primed and stimulated with nigericin as indicated. Mitochondrial and cytosolic fractions were subjected to immunoblot. Data shown are representative of three (A, E, G) or two (B-D, F) independent experiments.
Figure Legend Snippet: NLRP3 and caspase-1 independently associate with the mitochondria at priming. (A, B) J774A.1 cells were unstimulated, LPS-primed, or LPS-primed followed by stimulation with nigericin (A) or primed with Pam3CSK4 or HMW poly(I:C) (B) . Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (C) J774A.1 cells were LPS-primed for the indicated time in the presence of 10 μM cycloheximide. Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (D) J774A.1 cells were unstimulated or LPS-primed followed by stimulation with nigericin as indicated. Mitochondrial and cytosolic fractions were isolated and mitochondria treated with proteinase K in the presence or absence of Triton X-100. Cytosolic and mitochondrial fractions were then immunoblotted as shown. (E-G) WT, Nlrp3 −/− , Casp1 −/− , and Asc −/− BMM were unstimulated or LPS-primed and stimulated with nigericin as indicated. Mitochondrial and cytosolic fractions were subjected to immunoblot. Data shown are representative of three (A, E, G) or two (B-D, F) independent experiments.

Techniques Used: Isolation

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    InvivoGen pam3csk4
    Ezh2 regulates TRAF6 degradation and its downstream signaling. (A and B) Analysis of TRAF6 Lys48-linked ubiquitination in WT and Ezh2 -deficient macrophages (A) or in Ezh2- KO BV2 cell that infected with EV or lentiviral vector encoding WT Ezh2 (B) that was stimulated with LPS at the indicated time points. (C–E) IB analysis of TRAF6 protein levels in WT and Ezh2 -deficient macrophages (C), in microglia (D), or in Ezh2- KO BV2 cells that were infected with EV or lentiviral vector encoding WT Ezh2 ( E ) that was stimulated without or with LPS at the indicated time points. The relative expression of Traf6 compared with loading controls (Actin or Hsp60) was quantified and presented below the Traf6 IB panels. (F and G) qRT-PCR analysis of Traf6 mRNA expression in WT and Ezh2 -deficient macrophages (F) or microglia (G) that was stimulated with LPS at the indicated time points. (H–K) Immunoblot analysis of phosphorylated (P-) and total NF-κB and MAPKs signaling proteins, H3K27me3, H3, Ezh2, or Hsp60 (loading control) in whole-cell lysates of WT and Ezh2 -deficient macrophages that were left unstimulated or stimulated with LPS (100 ng/ml; H) or <t>Pam3CSK4</t> (100 ng/ml; I) or poly I:C (pI:C, 20 µg/ml; J), and IB of the indicated signal protein in the whole-cell lysates of WT and Ezh2 -deficient primary microglia that were left unstimulated or stimulated with LPS (100 ng/ml; K) at the indicated time points. The qRT-PCR data were normalized to a reference gene, Actb (β-actin), and other data are shown as mean ± SD based on three independent experiments.
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    Ezh2 regulates TRAF6 degradation and its downstream signaling. (A and B) Analysis of TRAF6 Lys48-linked ubiquitination in WT and Ezh2 -deficient macrophages (A) or in Ezh2- KO BV2 cell that infected with EV or lentiviral vector encoding WT Ezh2 (B) that was stimulated with LPS at the indicated time points. (C–E) IB analysis of TRAF6 protein levels in WT and Ezh2 -deficient macrophages (C), in microglia (D), or in Ezh2- KO BV2 cells that were infected with EV or lentiviral vector encoding WT Ezh2 ( E ) that was stimulated without or with LPS at the indicated time points. The relative expression of Traf6 compared with loading controls (Actin or Hsp60) was quantified and presented below the Traf6 IB panels. (F and G) qRT-PCR analysis of Traf6 mRNA expression in WT and Ezh2 -deficient macrophages (F) or microglia (G) that was stimulated with LPS at the indicated time points. (H–K) Immunoblot analysis of phosphorylated (P-) and total NF-κB and MAPKs signaling proteins, H3K27me3, H3, Ezh2, or Hsp60 (loading control) in whole-cell lysates of WT and Ezh2 -deficient macrophages that were left unstimulated or stimulated with LPS (100 ng/ml; H) or Pam3CSK4 (100 ng/ml; I) or poly I:C (pI:C, 20 µg/ml; J), and IB of the indicated signal protein in the whole-cell lysates of WT and Ezh2 -deficient primary microglia that were left unstimulated or stimulated with LPS (100 ng/ml; K) at the indicated time points. The qRT-PCR data were normalized to a reference gene, Actb (β-actin), and other data are shown as mean ± SD based on three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Macrophage/microglial Ezh2 facilitates autoimmune inflammation through inhibition of Socs3

    doi: 10.1084/jem.20171417

    Figure Lengend Snippet: Ezh2 regulates TRAF6 degradation and its downstream signaling. (A and B) Analysis of TRAF6 Lys48-linked ubiquitination in WT and Ezh2 -deficient macrophages (A) or in Ezh2- KO BV2 cell that infected with EV or lentiviral vector encoding WT Ezh2 (B) that was stimulated with LPS at the indicated time points. (C–E) IB analysis of TRAF6 protein levels in WT and Ezh2 -deficient macrophages (C), in microglia (D), or in Ezh2- KO BV2 cells that were infected with EV or lentiviral vector encoding WT Ezh2 ( E ) that was stimulated without or with LPS at the indicated time points. The relative expression of Traf6 compared with loading controls (Actin or Hsp60) was quantified and presented below the Traf6 IB panels. (F and G) qRT-PCR analysis of Traf6 mRNA expression in WT and Ezh2 -deficient macrophages (F) or microglia (G) that was stimulated with LPS at the indicated time points. (H–K) Immunoblot analysis of phosphorylated (P-) and total NF-κB and MAPKs signaling proteins, H3K27me3, H3, Ezh2, or Hsp60 (loading control) in whole-cell lysates of WT and Ezh2 -deficient macrophages that were left unstimulated or stimulated with LPS (100 ng/ml; H) or Pam3CSK4 (100 ng/ml; I) or poly I:C (pI:C, 20 µg/ml; J), and IB of the indicated signal protein in the whole-cell lysates of WT and Ezh2 -deficient primary microglia that were left unstimulated or stimulated with LPS (100 ng/ml; K) at the indicated time points. The qRT-PCR data were normalized to a reference gene, Actb (β-actin), and other data are shown as mean ± SD based on three independent experiments.

    Article Snippet: CpG, poly I:C, and Pam3CSK4 were from InvivoGen.

    Techniques: Infection, Plasmid Preparation, Expressing, Quantitative RT-PCR

    FcαRI stimulation promotes cytokine gene translation and induces caspase-1 activation. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3), IgA immune complexes (IgA-IC), or a combination and analysed for mRNA expression of indicated genes using qPCR (normalized to GAPDH expression, fold increase compared to unstimulated control). b mRNA stability of TNF of CD103 + DCs which were unstimulated or treated with Pam3CSK4 or co-stimulation with IgA-IC was determined after addition of 10 µg/mL actinomycin D (Act D) to prevent de novo synthesis of mRNA. TNF expression was analysed using qPCR (normalized to GAPDH ). c Lysates of CD103 + DCs stimulated for 3 h with Pam3CSK4 or Pam3CSK4 with IgA-IC were loaded on sucrose gradients to measure mRNA translation of TNF (normalized to GAPDH , qPCR was performed in duplicate, mean + s.e.m.). d Induction of caspase-1 activation in CD103 + DCs after stimulation with Pam3CSK4, IgA-IC, or a combination was measured using caspase-1 binding compound FAM-YVAD-FMK (FLICA) by flow cytometry. Representative example ( a – d ) of three independent experiments

    Journal: Nature Communications

    Article Title: FcαRI co-stimulation converts human intestinal CD103+ dendritic cells into pro-inflammatory cells through glycolytic reprogramming

    doi: 10.1038/s41467-018-03318-5

    Figure Lengend Snippet: FcαRI stimulation promotes cytokine gene translation and induces caspase-1 activation. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3), IgA immune complexes (IgA-IC), or a combination and analysed for mRNA expression of indicated genes using qPCR (normalized to GAPDH expression, fold increase compared to unstimulated control). b mRNA stability of TNF of CD103 + DCs which were unstimulated or treated with Pam3CSK4 or co-stimulation with IgA-IC was determined after addition of 10 µg/mL actinomycin D (Act D) to prevent de novo synthesis of mRNA. TNF expression was analysed using qPCR (normalized to GAPDH ). c Lysates of CD103 + DCs stimulated for 3 h with Pam3CSK4 or Pam3CSK4 with IgA-IC were loaded on sucrose gradients to measure mRNA translation of TNF (normalized to GAPDH , qPCR was performed in duplicate, mean + s.e.m.). d Induction of caspase-1 activation in CD103 + DCs after stimulation with Pam3CSK4, IgA-IC, or a combination was measured using caspase-1 binding compound FAM-YVAD-FMK (FLICA) by flow cytometry. Representative example ( a – d ) of three independent experiments

    Article Snippet: 30,000 cells were plated per well and assayed in glucose-free medium after addition of glucose at the indicated time cells were left unstimulated or stimulated with 10 µg/mL Pam3CSK4.

    Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Activated Clotting Time Assay, Binding Assay, Flow Cytometry, Cytometry

    FcαRI-induced upregulation of cytokine production is dependent on glycolytic reprogramming through Syk, PI3K, and TBK1-IKKε. a Real-time changes in the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of CD103 + dendritic cells (DC) left unstimulated (open circles), stimulated with Pam3CSK4 (Pam3) alone (grey circles), or Pam3CSK4 with IgA immune complexes (IgA-IC) (black circles). IgA-IC are present from the start of the experiment. The dotted line indicates initiation of further treatment. Experiments were performed in quadruplicate, mean + s.e.m. b Relative lactate production of CD103 + DCs after 24 h of treatment. Pooled data from five experiments. * p

    Journal: Nature Communications

    Article Title: FcαRI co-stimulation converts human intestinal CD103+ dendritic cells into pro-inflammatory cells through glycolytic reprogramming

    doi: 10.1038/s41467-018-03318-5

    Figure Lengend Snippet: FcαRI-induced upregulation of cytokine production is dependent on glycolytic reprogramming through Syk, PI3K, and TBK1-IKKε. a Real-time changes in the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of CD103 + dendritic cells (DC) left unstimulated (open circles), stimulated with Pam3CSK4 (Pam3) alone (grey circles), or Pam3CSK4 with IgA immune complexes (IgA-IC) (black circles). IgA-IC are present from the start of the experiment. The dotted line indicates initiation of further treatment. Experiments were performed in quadruplicate, mean + s.e.m. b Relative lactate production of CD103 + DCs after 24 h of treatment. Pooled data from five experiments. * p

    Article Snippet: 30,000 cells were plated per well and assayed in glucose-free medium after addition of glucose at the indicated time cells were left unstimulated or stimulated with 10 µg/mL Pam3CSK4.

    Techniques:

    IgA-IC promote proinflammatory cytokine production through synergy with various PRRs. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3), IgA immune complexes (IgA-IC), or a combination. b Cytokine production by CD103 + DCs stimulated with Pam3CSK4 or Pam3CSK4 combined with IgA-IC. Each pair of dots represents one donor. * p

    Journal: Nature Communications

    Article Title: FcαRI co-stimulation converts human intestinal CD103+ dendritic cells into pro-inflammatory cells through glycolytic reprogramming

    doi: 10.1038/s41467-018-03318-5

    Figure Lengend Snippet: IgA-IC promote proinflammatory cytokine production through synergy with various PRRs. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3), IgA immune complexes (IgA-IC), or a combination. b Cytokine production by CD103 + DCs stimulated with Pam3CSK4 or Pam3CSK4 combined with IgA-IC. Each pair of dots represents one donor. * p

    Article Snippet: 30,000 cells were plated per well and assayed in glucose-free medium after addition of glucose at the indicated time cells were left unstimulated or stimulated with 10 µg/mL Pam3CSK4.

    Techniques:

    Co-stimulation of CD103 + DCs with IgA-IC promotes Th17 responses and activates intestinal ILC3. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3) alone or Pam3CSK4 together with IgA immune complexes (IgA-IC) and co-cultured with CD4 + T Cells. After T-cell outgrowth, resting cells were re-stimulated and after 24 h supernatant was assayed with ELISA, mean + s.e.m. Experiments were performed in triplicate. b , c Supernatant from CD103 + DCs which were stimulated with Pam3CSK4 or Pam3CSK4 with IgA-IC was transferred onto human intestinal ILC3 and cultured for 7 days and analysed for mRNA expression ( b ) or protein expression ( c ). ND, not detected. Experiments were performed in triplicate. mRNA expression of indicated genes were assayed using qPCR (normalized to GAPDH expression) mean + s.e.m. Supernatant was analysed using ELISA, mean + s.e.m. Representative example of three ( a , b ) or two ( c ) experiments using different donors

    Journal: Nature Communications

    Article Title: FcαRI co-stimulation converts human intestinal CD103+ dendritic cells into pro-inflammatory cells through glycolytic reprogramming

    doi: 10.1038/s41467-018-03318-5

    Figure Lengend Snippet: Co-stimulation of CD103 + DCs with IgA-IC promotes Th17 responses and activates intestinal ILC3. a CD103 + dendritic cells (DC) were stimulated with Pam3CSK4 (Pam3) alone or Pam3CSK4 together with IgA immune complexes (IgA-IC) and co-cultured with CD4 + T Cells. After T-cell outgrowth, resting cells were re-stimulated and after 24 h supernatant was assayed with ELISA, mean + s.e.m. Experiments were performed in triplicate. b , c Supernatant from CD103 + DCs which were stimulated with Pam3CSK4 or Pam3CSK4 with IgA-IC was transferred onto human intestinal ILC3 and cultured for 7 days and analysed for mRNA expression ( b ) or protein expression ( c ). ND, not detected. Experiments were performed in triplicate. mRNA expression of indicated genes were assayed using qPCR (normalized to GAPDH expression) mean + s.e.m. Supernatant was analysed using ELISA, mean + s.e.m. Representative example of three ( a , b ) or two ( c ) experiments using different donors

    Article Snippet: 30,000 cells were plated per well and assayed in glucose-free medium after addition of glucose at the indicated time cells were left unstimulated or stimulated with 10 µg/mL Pam3CSK4.

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

    Complexed IgA1 and IgA2 amplify proinflammatory cytokine production through FcαRI. a FcαRI expression on unstimulated CD103 + dendritic cells (DC) was analysed using flow cytometry. Light grey histogram indicates background staining. b Prior to stimulation, CD103 + DCs were incubated with a blocking antibody against FcαRI and stimulated with Pam3CSK4 (Pam3) alone or Pam3CSK4 together with IgA immune complexes (IgA-IC). c Cytokine production by CD103 + DCs stimulated with Pam3CSK4 alone or in combination with IgA1-IC or IgA2-IC. Experiments b and c were performed in triplicate. After 24 h cytokine levels were analysed using ELISA, mean + s.e.m. Representative example ( a – c ) of three experiments using different donors

    Journal: Nature Communications

    Article Title: FcαRI co-stimulation converts human intestinal CD103+ dendritic cells into pro-inflammatory cells through glycolytic reprogramming

    doi: 10.1038/s41467-018-03318-5

    Figure Lengend Snippet: Complexed IgA1 and IgA2 amplify proinflammatory cytokine production through FcαRI. a FcαRI expression on unstimulated CD103 + dendritic cells (DC) was analysed using flow cytometry. Light grey histogram indicates background staining. b Prior to stimulation, CD103 + DCs were incubated with a blocking antibody against FcαRI and stimulated with Pam3CSK4 (Pam3) alone or Pam3CSK4 together with IgA immune complexes (IgA-IC). c Cytokine production by CD103 + DCs stimulated with Pam3CSK4 alone or in combination with IgA1-IC or IgA2-IC. Experiments b and c were performed in triplicate. After 24 h cytokine levels were analysed using ELISA, mean + s.e.m. Representative example ( a – c ) of three experiments using different donors

    Article Snippet: 30,000 cells were plated per well and assayed in glucose-free medium after addition of glucose at the indicated time cells were left unstimulated or stimulated with 10 µg/mL Pam3CSK4.

    Techniques: Expressing, Flow Cytometry, Cytometry, Staining, Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay

    Effect of PAM3 treatment on the M1:M2 ratio of NZB/W mice. Female NZB/W mice were treated weekly with 100 μg of PAM3 starting either early (7 wk) or late (13 wk). Controls included littermates treated with PBS and normal (BALB/c) mice. The M1:M2 ratio was calculated based on the ratio of CD206 − :CD206 + F480 + macrophages collected from the peritoneal cavity at 6 months of age. N = 5 NZB/W mice/group, N = 2 BALB/c controls. * p

    Journal: Journal of autoimmunity

    Article Title: PAM3 supports the generation of M2-like macrophages from lupus patient monocytes and improves disease outcome in murine lupus

    doi: 10.1016/j.jaut.2019.01.004

    Figure Lengend Snippet: Effect of PAM3 treatment on the M1:M2 ratio of NZB/W mice. Female NZB/W mice were treated weekly with 100 μg of PAM3 starting either early (7 wk) or late (13 wk). Controls included littermates treated with PBS and normal (BALB/c) mice. The M1:M2 ratio was calculated based on the ratio of CD206 − :CD206 + F480 + macrophages collected from the peritoneal cavity at 6 months of age. N = 5 NZB/W mice/group, N = 2 BALB/c controls. * p

    Article Snippet: Initial experiments examined whether administering PAM3 to female NZB/W mice (a widely accepted model of human lupus) elicited the same alteration in macrophage phenotype in vivo as was observed using human cells in vitro.

    Techniques: Mouse Assay

    Effect of PAM3 on the phenotype of lupus monocytes. Monocytes were MACS purified from the peripheral blood of lupus patients and normal controls and stimulated in vitro for 5 days with optimal concentrations of PAM3, M-CSF, IFNγ or R848. A) Representative histogram showing the expression of 25F9 and CD163 by cultured cells. Note that

    Journal: Journal of autoimmunity

    Article Title: PAM3 supports the generation of M2-like macrophages from lupus patient monocytes and improves disease outcome in murine lupus

    doi: 10.1016/j.jaut.2019.01.004

    Figure Lengend Snippet: Effect of PAM3 on the phenotype of lupus monocytes. Monocytes were MACS purified from the peripheral blood of lupus patients and normal controls and stimulated in vitro for 5 days with optimal concentrations of PAM3, M-CSF, IFNγ or R848. A) Representative histogram showing the expression of 25F9 and CD163 by cultured cells. Note that

    Article Snippet: Initial experiments examined whether administering PAM3 to female NZB/W mice (a widely accepted model of human lupus) elicited the same alteration in macrophage phenotype in vivo as was observed using human cells in vitro.

    Techniques: Magnetic Cell Separation, Purification, In Vitro, Expressing, Cell Culture

    The effect of Pam3Csk4 is differentially regulated between HMVECs and HUVECs. HMVECs (left) or HUVECs (right) were primed with the TLR2 agonist, Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 24 hours (HUVECs) or 32 hours (HMVECs). Afterward, cells were exposed to Pam3Csk4 (10 μ g/ml) for 6 hours (HUVECs) or 16 hours (HMVECs). Culture supernatants were collected at the completion of the experiments and examined for IL-6 ( A ) or IP-10 ( B ) by ELISA. Data are expressed as means ± standard error of replicated, individual experiments (n = 4). * indicates P

    Journal: Translational research : the journal of laboratory and clinical medicine

    Article Title: Potentiation and tolerance of toll-like receptor priming in human endothelial cells

    doi: 10.1016/j.trsl.2016.08.001

    Figure Lengend Snippet: The effect of Pam3Csk4 is differentially regulated between HMVECs and HUVECs. HMVECs (left) or HUVECs (right) were primed with the TLR2 agonist, Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 24 hours (HUVECs) or 32 hours (HMVECs). Afterward, cells were exposed to Pam3Csk4 (10 μ g/ml) for 6 hours (HUVECs) or 16 hours (HMVECs). Culture supernatants were collected at the completion of the experiments and examined for IL-6 ( A ) or IP-10 ( B ) by ELISA. Data are expressed as means ± standard error of replicated, individual experiments (n = 4). * indicates P

    Article Snippet: These doses were based on their effect on LPS-induced IP-10 production (see ) as well as previously reported doses of Nec-1s inhibition of receptor-interacting serine/threonine-protein kinase 1 (RIP1) phosphorylation and TSA inhibition of interferon regulatory factor 7 (IRF7) transcription., In separate experiments to test TLR2 differences between HUVECs and HMVECs, both cell lines underwent incubation with LPS (100 ng/ml), Pam3Csk4 (10 mg/ml), Purified Lipotechoic Acid from S. aureus (LTA, 10 μ g/ml, Invivogen, San Diego, Calif), or media (control) for 16 hours.

    Techniques: Enzyme-linked Immunosorbent Assay

    IRF7 increased while RIP1 decreased in LPS-treated cells primed with Poly I:C. HMVECs were primed with Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 32 hours. Afterward, cells were exposed to LPS (100 ng/ml) for 1 hour. Cell lysates were collected and run for Western blot analysis. Blots were probed for total and phosphorylated proteins. ( A ) Representative images showing phosphorylated (p-RIP1, p-IKK) and total proteins (IRF7, RIP1, IKK, and tubulin). ( B ) Bar graphs showing the relative abundance of phosphorylated or total proteins after normalization to respective total protein or tubulin expression. Data are expressed as means ± standard error of individual experiments (n = 4) after normalization to control quantities. * indicates P

    Journal: Translational research : the journal of laboratory and clinical medicine

    Article Title: Potentiation and tolerance of toll-like receptor priming in human endothelial cells

    doi: 10.1016/j.trsl.2016.08.001

    Figure Lengend Snippet: IRF7 increased while RIP1 decreased in LPS-treated cells primed with Poly I:C. HMVECs were primed with Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 32 hours. Afterward, cells were exposed to LPS (100 ng/ml) for 1 hour. Cell lysates were collected and run for Western blot analysis. Blots were probed for total and phosphorylated proteins. ( A ) Representative images showing phosphorylated (p-RIP1, p-IKK) and total proteins (IRF7, RIP1, IKK, and tubulin). ( B ) Bar graphs showing the relative abundance of phosphorylated or total proteins after normalization to respective total protein or tubulin expression. Data are expressed as means ± standard error of individual experiments (n = 4) after normalization to control quantities. * indicates P

    Article Snippet: These doses were based on their effect on LPS-induced IP-10 production (see ) as well as previously reported doses of Nec-1s inhibition of receptor-interacting serine/threonine-protein kinase 1 (RIP1) phosphorylation and TSA inhibition of interferon regulatory factor 7 (IRF7) transcription., In separate experiments to test TLR2 differences between HUVECs and HMVECs, both cell lines underwent incubation with LPS (100 ng/ml), Pam3Csk4 (10 mg/ml), Purified Lipotechoic Acid from S. aureus (LTA, 10 μ g/ml, Invivogen, San Diego, Calif), or media (control) for 16 hours.

    Techniques: Western Blot, Expressing

    Pam3Csk4 and LPS priming induced tolerance, while Poly I:C potentiated. HMVECs (left) or HUVECs (right) were primed with the Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 24 hours (HUVECs) or 32 hours (HMVECs). Afterward, cells were exposed to LPS (100 ng/ml) for 6 hours (HUVECs) or 16 hours (HMVECs). Culture supernatants were collected at the completion of the experiments and examined for IL-6 ( A ) or IP-10 ( B ) by ELISA. ( C ) HMVECs (left) and HUVECs (right) were exposed to Poly I:C (10 μ g/ml) or medium (control) for 16 hours, washed in fresh media for 24 hours and then exposed to LPS (100 ng/ml) or medium (control). Supernatants were collected at the end of the 16 hours and examined for IP-10 via ELISA. Data are expressed as means ± standard error of replicated, individual experiments (n = 4). * indicates P

    Journal: Translational research : the journal of laboratory and clinical medicine

    Article Title: Potentiation and tolerance of toll-like receptor priming in human endothelial cells

    doi: 10.1016/j.trsl.2016.08.001

    Figure Lengend Snippet: Pam3Csk4 and LPS priming induced tolerance, while Poly I:C potentiated. HMVECs (left) or HUVECs (right) were primed with the Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 24 hours (HUVECs) or 32 hours (HMVECs). Afterward, cells were exposed to LPS (100 ng/ml) for 6 hours (HUVECs) or 16 hours (HMVECs). Culture supernatants were collected at the completion of the experiments and examined for IL-6 ( A ) or IP-10 ( B ) by ELISA. ( C ) HMVECs (left) and HUVECs (right) were exposed to Poly I:C (10 μ g/ml) or medium (control) for 16 hours, washed in fresh media for 24 hours and then exposed to LPS (100 ng/ml) or medium (control). Supernatants were collected at the end of the 16 hours and examined for IP-10 via ELISA. Data are expressed as means ± standard error of replicated, individual experiments (n = 4). * indicates P

    Article Snippet: These doses were based on their effect on LPS-induced IP-10 production (see ) as well as previously reported doses of Nec-1s inhibition of receptor-interacting serine/threonine-protein kinase 1 (RIP1) phosphorylation and TSA inhibition of interferon regulatory factor 7 (IRF7) transcription., In separate experiments to test TLR2 differences between HUVECs and HMVECs, both cell lines underwent incubation with LPS (100 ng/ml), Pam3Csk4 (10 mg/ml), Purified Lipotechoic Acid from S. aureus (LTA, 10 μ g/ml, Invivogen, San Diego, Calif), or media (control) for 16 hours.

    Techniques: Enzyme-linked Immunosorbent Assay

    MAPKs are increased in Poly I:C primed cells challenged with LPS. HMVECs were primed with Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 32 hours. Afterward, cells were exposed to LPS (100 ng/ml) for 1 hour. Cell lysates were collected and run for Western blot analysis. Blots were probed for total and phosphorylated proteins. ( A ) Representative images showing phosphorylated (p-p38, p-JNK, p-ERK 1/2) and total proteins (p38, JNK, ERK 1/2). ( B ) Bar graphs showing the relative abundance of phosphorylated after normalization to respective total protein. Data are expressed as means ± standard error of individual experiments (n = 4) after normalization to control quantities. * indicates P

    Journal: Translational research : the journal of laboratory and clinical medicine

    Article Title: Potentiation and tolerance of toll-like receptor priming in human endothelial cells

    doi: 10.1016/j.trsl.2016.08.001

    Figure Lengend Snippet: MAPKs are increased in Poly I:C primed cells challenged with LPS. HMVECs were primed with Poly I:C (10 μ g/ml), Pam3Csk4 (10 μ g/ml), LPS (100 ng/ml), or medium (control) for 16 hours and then exposed to fresh media for 32 hours. Afterward, cells were exposed to LPS (100 ng/ml) for 1 hour. Cell lysates were collected and run for Western blot analysis. Blots were probed for total and phosphorylated proteins. ( A ) Representative images showing phosphorylated (p-p38, p-JNK, p-ERK 1/2) and total proteins (p38, JNK, ERK 1/2). ( B ) Bar graphs showing the relative abundance of phosphorylated after normalization to respective total protein. Data are expressed as means ± standard error of individual experiments (n = 4) after normalization to control quantities. * indicates P

    Article Snippet: These doses were based on their effect on LPS-induced IP-10 production (see ) as well as previously reported doses of Nec-1s inhibition of receptor-interacting serine/threonine-protein kinase 1 (RIP1) phosphorylation and TSA inhibition of interferon regulatory factor 7 (IRF7) transcription., In separate experiments to test TLR2 differences between HUVECs and HMVECs, both cell lines underwent incubation with LPS (100 ng/ml), Pam3Csk4 (10 mg/ml), Purified Lipotechoic Acid from S. aureus (LTA, 10 μ g/ml, Invivogen, San Diego, Calif), or media (control) for 16 hours.

    Techniques: Western Blot