Journal: International Journal of Biological Sciences

Article Title: Antrodia cinnamomea Galactomannan Elicits Immuno-stimulatory Activity Through Toll-like Receptor 4

doi: 10.7150/ijbs.24564

Figure Lengend Snippet: ACP induces cytokine expression through TLR4. ( A, B ) J774A.1 macrophages stably transfected with a control shRNA plasmid (sh-SC), a TLR4 shRNA plasmid (sh-TLR4), and a TLR2 shRNA plasmid (sh-TLR2) were incubated for 24 h with or without ACP (100 µg/mL), PGN (10 µg/mL) or LPS (1 µg/mL). The levels of TNF-α ( A ) and IL-6 ( B ) in the culture medium were measured by ELISA. ( C, D ) Mouse peritoneal macrophages isolated from wild-type (WT) and TLR4 knockout (TLR4-/-) mice were incubated for 24 h with or without ACP (100 µg/mL), PGN (10 µg/mL) or LPS (1 µg/mL). The levels of TNF-α ( C ) and IL-6 ( D ) in the culture medium were measured by ELISA. ( E ) J774A.1 macrophages were incubated for 30 min with or without ACP (100 µg/mL) and then for 24 h with or without LPS (1 µg/mL). The levels of TNF-α in the culture medium were measured by ELISA. The data are expressed as the means ± SD of three separate experiments. *** indicates a significant difference at the level of p < 0.001.

Article Snippet: All plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): ERK1 shRNA plasmid (sh-ERK1, sc-29308-SH), JNK1 shRNA plasmid (sh-JNK1, sc-29381-SH), p38α shRNA plasmid (sh-p38α, sc-29434-SH), p38β shRNA plasmid (sh-p38β, sc-39117-SH), TLR2 shRNA plasmid (sh-TLR2, sc-40257-SH) and TLR4 shRNA plasmid (sh-TLR4, sc-40261-SH).

Techniques: Expressing, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Incubation, Enzyme-linked Immunosorbent Assay, Isolation, Knock-Out

Journal: International Journal of Biological Sciences

Article Title: Antrodia cinnamomea Galactomannan Elicits Immuno-stimulatory Activity Through Toll-like Receptor 4

doi: 10.7150/ijbs.24564

Figure Lengend Snippet: ACP induces cytokine expression through MAPK. ( A ) J774A.1 macrophages were incubated for 0-60 min with or without ACP (100 µg/mL). The phosphorylation levels of ERK1/2, JNK1/2 and p38 were assayed by Western blot. ( B, C ) J774A.1 macrophages stably transfected with a control shRNA plasmid (sh-SC), a ERK1 shRNA plasmid (sh-ERK1), a JNK1 shRNA plasmid (sh-TLR2), a p38α shRNA plasmid (sh-p38α), and a p38β shRNA plasmid (sh-p38β) were incubated for 24 h with or without ACP (100 μg/mL). The levels of TNF-α ( B ) and IL-6 ( C ) in the culture medium were measured by ELISA. The Western blot results are representative of those obtained in three different experiments. The ELISA data are expressed as the means ± SD of three separate experiments. *** indicates a significant difference at the level of p < 0.001.

Article Snippet: All plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): ERK1 shRNA plasmid (sh-ERK1, sc-29308-SH), JNK1 shRNA plasmid (sh-JNK1, sc-29381-SH), p38α shRNA plasmid (sh-p38α, sc-29434-SH), p38β shRNA plasmid (sh-p38β, sc-39117-SH), TLR2 shRNA plasmid (sh-TLR2, sc-40257-SH) and TLR4 shRNA plasmid (sh-TLR4, sc-40261-SH).

Techniques: Expressing, Incubation, Western Blot, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet: ( A, B ) Bone marrow-derived macrophages (BMDMs) from WT and Myd88 −/ − mice were stimulated with S2 protein (500 ng/ml). ( A ) The activation of the NF-κB pathway was measured by Western blot analysis of P-P65 and P-IκBα. ( B ) The induction of Il6 , Il1b, and Tnfa was measured by real-time RT-PCR. ( C ) BMDMs from WT, Tlr2 −/ − , and Tlr4 −/ − mice were treated with S2 protein (500 ng/ml). Cell lysates collected at different times were analyzed for the activation of the NF-κB pathway by Western blotting of P-P65 and P-IκBα. ( D ) BMDMs from WT and Tlr2 −/ − mice were treated with S1 protein (500 ng/ml), and the activation of P65 and IκBα was measured by Western blotting. ( E, F ) WT, Tlr2 −/ − , and Tlr4 −/ − macrophages were treated with S2 protein (500 ng/ml) or S-tri (500 ng/ml). The expression of cytokines was measured by real-time RT-PCR at 4 hr post-stimulation. Data represent mean ± SD (n=3); ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments were repeated two times and data of representative experiments are presented. ( G ) THP1 cells were stimulated with S2 protein (500 ng/ml), Pam3CSK4 (500 ng/ml), or LPS (100 ng/ml) in the presence or absence of Tlr2 inhibitor C29 (150 mM) for 4 hr. The expression of IL6, IL1B , and TNFA was measured by real-time RT-PCR. ( H ) WT and Tlr2 −/ − mice were administered with S1 and S2 protein (1 μg each/mouse). Blood collected before and 16 hr post S protein administration was measured for IL-6, IL-1β, and TNFα by ELISA. Data represent mean ± SEM (n=5); ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments were repeated two times and data of representative experiments are presented. Figure 5—source data 1. Raw source data for B, E, F, G, H.

Article Snippet: Mouse macrophage cells RAW 264.7 (TIB-71, ATCC) were transfected with GFP-Flag or TLR1 CRISPR/Cas9 (sc-423418, Santa Cruz) or TLR2 CRISPR/Cas9 (sc-423981, Santa Cruz), or TLR6 CRISPR/Cas9 (sc-423420, Santa Cruz) constructs (1.5 μg/ml) using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Activation Assay, Western Blot, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet: BMDMs from WT and Tlr2 −/ − mice were stimulated with LPS (1 μg/l) or Pam3CSK4 (1 μg/ml) for 4 hr. The expression of inflammatory cytokines was measured by real-time RT-PCR. Data represent mean ± SD (n=3). Figure 5—figure supplement 1—source data 1. Raw source data.

Article Snippet: Mouse macrophage cells RAW 264.7 (TIB-71, ATCC) were transfected with GFP-Flag or TLR1 CRISPR/Cas9 (sc-423418, Santa Cruz) or TLR2 CRISPR/Cas9 (sc-423981, Santa Cruz), or TLR6 CRISPR/Cas9 (sc-423420, Santa Cruz) constructs (1.5 μg/ml) using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet: Calu3 cells were stimulated with S2 (500 ng/ml) in the presence or absence of TLR2-inhibitor C29 (150 mM) for 24 hr. The expression of inflammatory cytokines was measured by real-time RT-PCR. Data represent mean ± SD (n=3); ***p<0.0001 by unpaired Student’s t-test. Experiments were repeated two times and data of representative experiments are presented. Figure 5—figure supplement 2—source data 1. Raw source data.

Article Snippet: Mouse macrophage cells RAW 264.7 (TIB-71, ATCC) were transfected with GFP-Flag or TLR1 CRISPR/Cas9 (sc-423418, Santa Cruz) or TLR2 CRISPR/Cas9 (sc-423981, Santa Cruz), or TLR6 CRISPR/Cas9 (sc-423420, Santa Cruz) constructs (1.5 μg/ml) using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet: ( A, B ) HEK-Blue-Null, HEK-Blue-TLR2, HEK-Blue-TLR1/2, HEK-Blue-TLR2/6, and HEK-Blue-TLR4 were stimulated with S1, S2, or S-tri for 6 hr. FSL1, Pam3CSK4, and LPS were used as ligands for TLR2/1, TLR2/6, and TLR4, respectively. The activation of NF-κB was monitored by the blue color development ( A ), which was measured at 620 nm ( B ). ( C ) HEK-Blue-Null, HEK-Blue-TLR2, HEK-Blue-TLR1/2, HEK-Blue-TLR2/6, and HEK-Blue-TLR4 cells were stimulated with S2 (500 ng/ml) at indicated times. The activation of P-P65 and P-IκBα was measured by Western blot analysis. ( D ) HEK-Blue-Null, HEK2-Blue-TLR2, HEK-Blue-TLR1/2, HEK-Blue-TLR2/6, and HEK-Blue-TLR4 cells were stimulated with S2 (500 ng/ml) for 6 hr. The induction of IL6 and IL1B was measured by real-time RT-PCR. ( E, F ) HEK-Blue-TLR2, HEK-Blue-TLR2/1, and HEK-Blue-TLR2/6 cells were stimulated with S1 or S2 in the presence or absence of TLR2 inhibitor C29 (150 mM) for 6 hr. The NF-κB activity was monitored colorimetrically at 620 nm. ( G, H ) TLR1, TLR2, TLR6, or TLR1/6 were knocked out in Raw264.7 cells with CRISPR/Cas9. Cells were then stimulated with S2 protein (500 ng/ml) for 4 hr. ( G ) The expression of cytokines was measured by real-time RT-PCR. Data represent mean ± SD (n=5); ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. All experiments were repeated three times and data of representative experiments are presented. Figure 6—source data 1. Raw source data for B, D, F, G.

Article Snippet: Mouse macrophage cells RAW 264.7 (TIB-71, ATCC) were transfected with GFP-Flag or TLR1 CRISPR/Cas9 (sc-423418, Santa Cruz) or TLR2 CRISPR/Cas9 (sc-423981, Santa Cruz), or TLR6 CRISPR/Cas9 (sc-423420, Santa Cruz) constructs (1.5 μg/ml) using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions.

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Activity Assay, CRISPR, Expressing

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet: HEK-Blue-TLR2 and HEK-Blue-TLR4 cells were stimulated with S1, S2, M, N, or E proteins (500 ng/ml of each protein). Six hours following stimulation, blue substrate activation was measured by optical density taken at 620 nm. Data represent mean ± SD (n=3). Experiments were repeated three times and data of representative experiments are presented. Figure 6—figure supplement 1—source data 1. Raw source data.

Article Snippet: Mouse macrophage cells RAW 264.7 (TIB-71, ATCC) were transfected with GFP-Flag or TLR1 CRISPR/Cas9 (sc-423418, Santa Cruz) or TLR2 CRISPR/Cas9 (sc-423981, Santa Cruz), or TLR6 CRISPR/Cas9 (sc-423420, Santa Cruz) constructs (1.5 μg/ml) using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions.

Techniques: Activation Assay

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet: Tlr1 , Tlr2 , Tlr6 , or Tlr1 / 6 were knocked out in Raw264.7 cells with CRISPR/Cas9. ( A ) The expression of Tlr1 , Tlr2 , Tlr6 , and Tlr4 was measured by real-time RT-PCR. ( B ) Knockout cells were stimulated with Pam3CSK4 (500 ng/ml), FSL1 (100 ng/ml), or LPS (100 ng/ml) for 4 hr. The expression of Il6 was measured by real-time RT-PCR. Data represent mean ± SD (n=3). Experiments were repeated two times and data of representative experiments are presented. Figure 6—figure supplement 2—source data 1. Raw source data for A-B.

Article Snippet: Mouse macrophage cells RAW 264.7 (TIB-71, ATCC) were transfected with GFP-Flag or TLR1 CRISPR/Cas9 (sc-423418, Santa Cruz) or TLR2 CRISPR/Cas9 (sc-423981, Santa Cruz), or TLR6 CRISPR/Cas9 (sc-423420, Santa Cruz) constructs (1.5 μg/ml) using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions.

Techniques: CRISPR, Expressing, Quantitative RT-PCR, Knock-Out

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet:

Article Snippet: Mouse macrophage cells RAW 264.7 (TIB-71, ATCC) were transfected with GFP-Flag or TLR1 CRISPR/Cas9 (sc-423418, Santa Cruz) or TLR2 CRISPR/Cas9 (sc-423981, Santa Cruz), or TLR6 CRISPR/Cas9 (sc-423420, Santa Cruz) constructs (1.5 μg/ml) using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Recombinant, Plasmid Preparation, CRISPR, Sequencing, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: PLOS ONE

Article Title: ECDD-S16 targets vacuolar ATPase: A potential inhibitor compound for pyroptosis-induced inflammation

doi: 10.1371/journal.pone.0292340

Figure Lengend Snippet: Raw264.7 macrophages were transfected with TLR2-YFP (green) for 48 h. After that, the transfected cells were stimulated with Pam2CSK4 (1 μg/ml) for 1 h. Acidification of endosomes/lysosomes was then determined by adding the Lysotracker Red (LTR) dye to these cells in the presence or absence of ECDD-S16 and incubation was continued for 2 h. The treated cells were then fixed and processed for CLSM image analysis. (A) Confocal microscopy showing the colocalization of TLR2-YFP and LTR. Scale bar is 10 μm. (B) The co-localization of TLR2-YFP and LTR was quantified. All data were determined by one-way ANOVA followed by Tukey’s multiple comparison test. **** p < 0.0001.

Article Snippet: Raw264.7 cells were transiently transfected with TLR2 plasmid (5 μg) using Nucleofector solution kit V (Amaxa, London, UK) according to the manufacturer’s instructions.

Techniques: Transfection, Incubation, Confocal Microscopy, Comparison