Journal: Nature Communications

Article Title: Microglia clear neuron-released α-synuclein via selective autophagy and prevent neurodegeneration

doi: 10.1038/s41467-020-15119-w

Figure Lengend Snippet: a WT microglia were treated with 250 nM h α-Syn protein for the indicated time and assayed for W.B using antibodies as indicated. The levels of proteins were quantified in the right panel ( n = 6). p -values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(4,24) = 9.196, p = 0.0001 for p-NF-κB, F(4,24) = 4.530, p = 0.0072 for IkB, F(4,24) = 0.8644, p = 0.4994 for p-ERK1/2, F(4,24) = 1.369, p = 0.2741 for p-JNK, F(4,24) = 5.372, p = 0.0031 for p-p38. b , c Cells were pretreated with ML-120B, an IKK-2 inhibitor, or SB203580 and SB202190, two different p38 inhibitors, for indicated concentrations before 250 nM h α-Syn protein treatment, and assayed for W.B ( b , n = 3–5, F(3,12) = 3.750, p = 0.0413) or immunostaining ( c , n = 33, F(4,160) = 56.21, p = 1.53E-29). The levels of proteins were quantified in the right panel. The number of h α-Syn/ubiquitin-positive puncta was counted and quantified ( c ). p -values were calculated by one-way ANOVA with Newman–Keuls post hoc test. d – f Microglia obtained from WT mice and Tlr4 -KO mice were treated with h α-Syn protein for 3 h with indicated concentration without ( d , n = 6) or with ( e ( n = 6), f ( n = 4)) TLR2-blocking antibody (T2.5), and assayed for RT-qPCR using primers for Il-1b (left panel, F(3,40) = 20.70, p = 2.95E-08 for hα-Syn-genotype interaction; F(3,40) = 69.43, p = 6.55E-16 for hα-Syn; F(1,40) = 69.57, p = 2.74E-10 for genotype) and Tnf (right panel, F(3,40) = 10.12, p = 4.32E-05 for hα-Syn-genotype interaction; F(3,40) = 21.73, p = 1.64E-08 for hα-Syn; F(1,40) = 34.36, p = 7.39E-07 for genotype). p -values were calculated by two-way ANOVA with Bonferroni post hoc test (wt vs ko in d ), one-way ANOVA with Newman–Keuls post hoc test (non-treated vs. treated in d , e ; F(3,20) = 22.11, p = 1.46E-06 for Il-1b , F(3,20) = 11.88, p = 0.0001 for Tnf ), and unpaired two-tailed Student’s t test ( f ). g – i HEK293T cells were transfected with a luciferase vector that expresses luciferase under the promoter containing NF-κB-binding elements, and various TLRs. Then, cells were treated h α-Syn protein with indicated concentration ( g ( n = 9), h ( n = 9–12), i ( n = 9)), 50 ng/ml LPS ( i ), or Pam 3 CSK 4 ( i ) for 24 h without ( g , h ) or with ( i ) 10 µg/ml TLR2-blocking antibody (T2.5) and control IgG. p -values were calculated by two-way ANOVA with Bonferroni post hoc test. In g , F(4,80) = 19.63, p = 2.74E-11 for interaction; F(4,80) = 27.31, p = 2.66E-14 for hα-Syn; F(1,80) = 47.95, p = 9.90E-10 for protein overexpression. In h , F(5,126) = 6.070, p = 4.54E-05 for interaction; F(5,126) =6.070, p = 4.54E-05 for hα-Syn; F(1,126) = 73.83, p = 2.77E-14 for protein overexpression. In ( i ), F(3,64) = 16.98, p = 3.19E-08 for interaction; F(3,64) = 177.4, p = 5.90E-31 for stimuli; F(1,64) = 17.47, p = 9.03E-05 for antibody. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.

Article Snippet: Cells were transfected using Lipofectamine ® 2000 (Invitrogen) with pcDNA3 vectors encoding either TLR1 (Addgene #13014), TLR2 (Addgene #13015), TLR4 (Addgene #13018), MD2 (Addgene #13028), CD14 (Addgene #13645), TLR5 (Addgene #13019), or TLR6 (Addgene #13020) with pGL4.32[luc2P/NF-κB-RE/Hygro] vector containing five copies of an NF-κB response element (NF-κB-RE) that drives transcription of the luciferase reporter gene luc2P ( Photinus pyralis ) (#N111A, Promega, Madison, WI).

Techniques: Immunostaining, Ubiquitin Proteomics, Concentration Assay, Blocking Assay, Quantitative RT-PCR, Two Tailed Test, Transfection, Luciferase, Plasmid Preparation, Binding Assay, Control, Over Expression

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: 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