Journal: Cell reports

Article Title: Pyroptosis of pulmonary fibroblasts and macrophages through NLRC4 inflammasome leads to acute respiratory failure.

doi: 10.1016/j.celrep.2025.115479

Figure Lengend Snippet: Figure 1. Flagellin-induced acute lung injury requires NAIP/NLRC4 inflammasome (A and B) Kaplan-Meier survival plots (A) and monitoring of arterial oxygen saturation (B) were conducted in WT or Casp1/ mice intravenously injected with 5 mg LFn-flagellin/PA. (C) Lung capillary filtration coefficient (Kf,c) was measured before or 1 h after flagellin administration. (D and E) Kaplan-Meier survival plots for WT mice, TLR5/, or NAIP/-deficient mice challenged with 5 mg LFn-flagellin/PA (D), and monitoring of arterial oxygen saturation using infrared pulse oximetry (E). (F and G) WT mice intravenously injected with 5 mg LFn-FlicEC/PA or LFn-FlicPA/PA. Arterial oxygen saturation (F) and lung Kf,c were determined (G). (H and I) WT mice injected intravenously with 5 mg LFn-EprJ/PA, with determination of arterial oxygen saturation (H) and lung Kf,c (I). Error bars represent the mean ± SEM. *p < 0.05; **p < 0.01;****p < 0.0001; ns, not significant, by log rank (Mantel-Cox) test (A and D), two-tailed unpaired t test (I), one-way ANOVA (G and H), or two-way ANOVA with Holm-Sidak multiple comparisons test (B, C, E, and F).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Thromboplastin-D Pacific Hemostasis Cat#100357 TAT ELISA kit Abcam Cat#ab137994 CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega Cat#G1780 Plastic cuvette CHRONO-LOG Cat#367 IL-6 ELISA kit ThermoFisher Scientific Cat#88-7064-88 IL-1b ELISA kit ThermoFisher Scientific Cat#88-7013A-88 TNF-a ELISA kit ThermoFisher Scientific Cat#88-7324-88 Deposited data Raw data for single-cell RNA-seq This paper GEO:GSE269196 Experimental models: Cell lines Mouse Primary Bone Marrow Derived Macrophages This paper N/A Mouse Lung Epithelial Cells This paper N/A Mouse Lung Endothelial Cells This paper N/A Mouse Lung Fibroblast Cells This paper N/A Mouse Primary Alveolar Epithelial Cells Cell Biologics Cat#C57-6053 Mouse Primary Lung Microvascular Endothelial Cells Cell Biologics Cat#C57-6011 Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory JAX:000664; RRID:IMSR_JAX:000664 Casp1 / Genentech Inc; Nobuhiko Kayagaki; Kayagaki et al.47 N/A Gsdmd / National Institute of Genetics, Japan; Toshihiko Shiroishi; Wu et al.21 N/A Gsdmdfl/fl National Institute of Genetics, Japan; Toshihiko Shiroishi; Wu et al.21 N/A NAIP / The Jackson Laboratory JAX:032660; RRID:IMSR_JAX:032660 Gsdme / The Jackson Laboratory JAX:032411; RRID:IMSR_JAX:032411 Tlr5 / The Jackson Laboratory JAX:008377; RRID:IMSR_JAX:008377 B6.Cg-Tg(Col1a2-cre/ERT,-ALPP) 7Cpd/2J The Jackson Laboratory JAX:029567; RRID:IMSR_JAX:029567 Casp8 / /Ripk3 / Genentech Inc; Nobuhiko Kayagaki; Lee et al.45 N/A Nlrc4 / Genentech Inc; Lee et al.45 N/A Software and algorithms FlowJo v10.07 FLOWJO https://www.flowjo.com/learn/flowjo- university/flowjo/getting-started-in-flowjo/ 131 GraphPad Prism 6 GraphPad https://www.graphpad.com/scientific- software/prism/ R Studio v. 4.3.6 RStudio https://cran.r-project.org/ (Continued on next page) 16 Cell Reports 44, 115479, April 22, 2025

Techniques: Injection, Two Tailed Test

Journal: Cell reports

Article Title: Pyroptosis of pulmonary fibroblasts and macrophages through NLRC4 inflammasome leads to acute respiratory failure.

doi: 10.1016/j.celrep.2025.115479

Figure Lengend Snippet: Figure 3. Flagellin-induced lung injury extends beyond macrophage pyroptosis (A and B) WT or TLR5/ mice were intravenously injected with 5 mg LFn-flagellin/PA. After 1 h, the total number of macrophages (A) and cytokine levels (B) in BAL fluid were measured. (C) WT mice received PBS, control liposomes (Lipo), or clodronate-containing liposomes (Cldn) 24 h prior to injection of 5 mg LFn-flagellin/PA. Arterial oxygen saturation was monitored. (D and E) Lung cells were incubated with PA (Ctrl) or 1 mg/mL LFn-flagellin/PA for 6 h. LDH concentration (D) and levels of p20 caspase-1 and p17 IL-1b by immunoblotting (E) were detected. (F) Lung cells were pre-incubated with PBS, 100 mg/mL Lipo, or Cldn, then transfected with PA (Ctrl) or 1 mg/mL LFn-flagellin/PA for 6 h. LDH concentrations were measured. (G and H) Epithelial (G) and endothelial (H) cells were incubated with PA (Ctrl) or 1 mg/mL LFn-flagellin/PA for 6 h. As a positive control, cells were primed with 1 mg/mL LPS for 4 h, followed by stimulation with 20 mM nigericin for 4 h, with or without caspase-1 inhibitor Ac-YVAD-cmk (5 mM). LDH concentrations in the supernatant were measured to determine cytotoxicity. Error bars represent the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant, by two-tailed unpaired t test (A and D), one-way ANOVA (G and H), or two-way ANOVA with Holm-Sidak multiple comparisons test (B, C, and F).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Thromboplastin-D Pacific Hemostasis Cat#100357 TAT ELISA kit Abcam Cat#ab137994 CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega Cat#G1780 Plastic cuvette CHRONO-LOG Cat#367 IL-6 ELISA kit ThermoFisher Scientific Cat#88-7064-88 IL-1b ELISA kit ThermoFisher Scientific Cat#88-7013A-88 TNF-a ELISA kit ThermoFisher Scientific Cat#88-7324-88 Deposited data Raw data for single-cell RNA-seq This paper GEO:GSE269196 Experimental models: Cell lines Mouse Primary Bone Marrow Derived Macrophages This paper N/A Mouse Lung Epithelial Cells This paper N/A Mouse Lung Endothelial Cells This paper N/A Mouse Lung Fibroblast Cells This paper N/A Mouse Primary Alveolar Epithelial Cells Cell Biologics Cat#C57-6053 Mouse Primary Lung Microvascular Endothelial Cells Cell Biologics Cat#C57-6011 Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory JAX:000664; RRID:IMSR_JAX:000664 Casp1 / Genentech Inc; Nobuhiko Kayagaki; Kayagaki et al.47 N/A Gsdmd / National Institute of Genetics, Japan; Toshihiko Shiroishi; Wu et al.21 N/A Gsdmdfl/fl National Institute of Genetics, Japan; Toshihiko Shiroishi; Wu et al.21 N/A NAIP / The Jackson Laboratory JAX:032660; RRID:IMSR_JAX:032660 Gsdme / The Jackson Laboratory JAX:032411; RRID:IMSR_JAX:032411 Tlr5 / The Jackson Laboratory JAX:008377; RRID:IMSR_JAX:008377 B6.Cg-Tg(Col1a2-cre/ERT,-ALPP) 7Cpd/2J The Jackson Laboratory JAX:029567; RRID:IMSR_JAX:029567 Casp8 / /Ripk3 / Genentech Inc; Nobuhiko Kayagaki; Lee et al.45 N/A Nlrc4 / Genentech Inc; Lee et al.45 N/A Software and algorithms FlowJo v10.07 FLOWJO https://www.flowjo.com/learn/flowjo- university/flowjo/getting-started-in-flowjo/ 131 GraphPad Prism 6 GraphPad https://www.graphpad.com/scientific- software/prism/ R Studio v. 4.3.6 RStudio https://cran.r-project.org/ (Continued on next page) 16 Cell Reports 44, 115479, April 22, 2025

Techniques: Injection, Control, Liposomes, Incubation, Concentration Assay, Western Blot, Transfection, Positive Control, Two Tailed Test

Journal: Cell reports

Article Title: Pyroptosis of pulmonary fibroblasts and macrophages through NLRC4 inflammasome leads to acute respiratory failure.

doi: 10.1016/j.celrep.2025.115479

Figure Lengend Snippet: Figure 5. Caspase-8/GSDME pathway in macrophage contributed to acute lung injury induced by high-dose flagellin (A and B) WT mice, Casp1/, NAIP/, or Nlrc4/ mice were injected with 10 mg LFn-flagellin/PA. Kaplan-Meier survival plots (A) and arterial oxygen saturation were monitored (B). (C) Casp1/ mice received 10 mg LFn-flagellin/PA. The lung Kf,c was determined. (D) WT mice or TLR5/-deficient mice received 10 mg LFn-flagellin/PA. Arterial oxygen saturation was monitored. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Thromboplastin-D Pacific Hemostasis Cat#100357 TAT ELISA kit Abcam Cat#ab137994 CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega Cat#G1780 Plastic cuvette CHRONO-LOG Cat#367 IL-6 ELISA kit ThermoFisher Scientific Cat#88-7064-88 IL-1b ELISA kit ThermoFisher Scientific Cat#88-7013A-88 TNF-a ELISA kit ThermoFisher Scientific Cat#88-7324-88 Deposited data Raw data for single-cell RNA-seq This paper GEO:GSE269196 Experimental models: Cell lines Mouse Primary Bone Marrow Derived Macrophages This paper N/A Mouse Lung Epithelial Cells This paper N/A Mouse Lung Endothelial Cells This paper N/A Mouse Lung Fibroblast Cells This paper N/A Mouse Primary Alveolar Epithelial Cells Cell Biologics Cat#C57-6053 Mouse Primary Lung Microvascular Endothelial Cells Cell Biologics Cat#C57-6011 Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory JAX:000664; RRID:IMSR_JAX:000664 Casp1 / Genentech Inc; Nobuhiko Kayagaki; Kayagaki et al.47 N/A Gsdmd / National Institute of Genetics, Japan; Toshihiko Shiroishi; Wu et al.21 N/A Gsdmdfl/fl National Institute of Genetics, Japan; Toshihiko Shiroishi; Wu et al.21 N/A NAIP / The Jackson Laboratory JAX:032660; RRID:IMSR_JAX:032660 Gsdme / The Jackson Laboratory JAX:032411; RRID:IMSR_JAX:032411 Tlr5 / The Jackson Laboratory JAX:008377; RRID:IMSR_JAX:008377 B6.Cg-Tg(Col1a2-cre/ERT,-ALPP) 7Cpd/2J The Jackson Laboratory JAX:029567; RRID:IMSR_JAX:029567 Casp8 / /Ripk3 / Genentech Inc; Nobuhiko Kayagaki; Lee et al.45 N/A Nlrc4 / Genentech Inc; Lee et al.45 N/A Software and algorithms FlowJo v10.07 FLOWJO https://www.flowjo.com/learn/flowjo- university/flowjo/getting-started-in-flowjo/ 131 GraphPad Prism 6 GraphPad https://www.graphpad.com/scientific- software/prism/ R Studio v. 4.3.6 RStudio https://cran.r-project.org/ (Continued on next page) 16 Cell Reports 44, 115479, April 22, 2025

Techniques: Injection

Journal: Biochimica et biophysica acta

Article Title: Toll-like receptor 5 deficiency attenuates interstitial cardiac fibrosis and dysfunction induced by pressure overload by inhibiting inflammation and the endothelial-mesenchymal transition.

doi: 10.1016/j.bbadis.2015.08.013

Figure Lengend Snippet: Figure 1 TLR5 expression in the myocardium after AB-induced pressure overload. Western

Article Snippet: Before TGF-β1 treatment, the HUVEC-12 cells were transfected with a plasmid over-expressing TLR5 (Ad-TLR5, Addgene, 13019) using FuGENE®HD Transfection Reagent (Promega, E2311).

Techniques: Expressing, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Toll-like receptor 5 deficiency attenuates interstitial cardiac fibrosis and dysfunction induced by pressure overload by inhibiting inflammation and the endothelial-mesenchymal transition.

doi: 10.1016/j.bbadis.2015.08.013

Figure Lengend Snippet: Figure 2 the expression of TLR5 in cultured neonatal rat cardiomyocytes, H9c2

Article Snippet: Before TGF-β1 treatment, the HUVEC-12 cells were transfected with a plasmid over-expressing TLR5 (Ad-TLR5, Addgene, 13019) using FuGENE®HD Transfection Reagent (Promega, E2311).

Techniques: Expressing, Cell Culture

Journal: Biochimica et biophysica acta

Article Title: Toll-like receptor 5 deficiency attenuates interstitial cardiac fibrosis and dysfunction induced by pressure overload by inhibiting inflammation and the endothelial-mesenchymal transition.

doi: 10.1016/j.bbadis.2015.08.013

Figure Lengend Snippet: Figure 3 The effects of TLR5 on cardiac dysfunction. Statistical analysis of the HW/BW ratio,

Article Snippet: Before TGF-β1 treatment, the HUVEC-12 cells were transfected with a plasmid over-expressing TLR5 (Ad-TLR5, Addgene, 13019) using FuGENE®HD Transfection Reagent (Promega, E2311).

Techniques:

Journal: Biochimica et biophysica acta

Article Title: Toll-like receptor 5 deficiency attenuates interstitial cardiac fibrosis and dysfunction induced by pressure overload by inhibiting inflammation and the endothelial-mesenchymal transition.

doi: 10.1016/j.bbadis.2015.08.013

Figure Lengend Snippet: Figure 4 TLR5 deficiency attenuates the fibrotic response induced by pressure overload.

Article Snippet: Before TGF-β1 treatment, the HUVEC-12 cells were transfected with a plasmid over-expressing TLR5 (Ad-TLR5, Addgene, 13019) using FuGENE®HD Transfection Reagent (Promega, E2311).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Cross-Talk of Toll-Like Receptor 5 and Mu-Opioid Receptor Attenuates Chronic Constriction Injury-Induced Mechanical Hyperalgesia through a Protein Kinase C Alpha-Dependent Signaling

doi: 10.3390/ijms22041891

Figure Lengend Snippet: Influence of nerve decompression in the CCI-induced changes of Toll-like receptor 5 (TLR5) expression in dorsal horn. ( A , C , E ) Standard pattern of TLR5-immunoreactive (ir) expression was exposed on the contralateral sides of CCI and Decompression groups at post-operative week (POW)2 and POW 8. While on contralateral sides, TLR5-ir expression which showed a dotted appearance, mainly expressed in the inner part of laminae II. Related laminae of dorsal horn were demonstrated in ( A ), which involved lamina I (I), the outer part of lamina II (IIo) and the inner part of lamina II (IIi). ( B , D , F ) On ipsilateral sides, TLR5-ir expressions were exhibited in both groups. ( B ) At POW 2, CCI-induced reduction of TLR5-ir expression was revealed mainly in the medial portion of dorsal horn in both groups. ( D , F ) In Decompression group, more significant increase of TLR5-ir expression was observed at POW 8 (arrowheads in ( F )), as compared with those in CCI group (arrow in ( D )), especially in the corresponding lamina. Scale bar = 100 μm.

Article Snippet: In brief, these sections are blocked with 5% normal goat serum with 0.5% Triton X-100 in 0.5 M Tris for 1 h at room temperature and incubated with one of a mixture of primary antiserum at 4 °C overnight: (1) rabbit polyclonal TLR5 (1:100, 19810-1-AP, Proteintech Group, Inc., Chicago, IL, USA); (2) rabbit polyclonal pPKCα (1:200, 1195-1,Epitomics, Burlingame, CA, USA); (3) rabbit polyclonal pPKA RII (1:200, 1151-1, Epitomics, Burlingame, CA, USA); and (4) rabbit polyclonal MOR (1:200, RA10104, Neuromics, Edina, MN, USA) were mixed with mouse monoclonal SP (1:100, sc-58591, Santa Cruz Biotechnology; Santa Cruz, CA, USA), mouse monoclonal CGRP (1:100, sc-57053,Santa Cruz Biotechnology; Santa Cruz, CA, USA) or biotinylated IB4 (1:1000, L 2140, Sigma, UK) respectively.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Cross-Talk of Toll-Like Receptor 5 and Mu-Opioid Receptor Attenuates Chronic Constriction Injury-Induced Mechanical Hyperalgesia through a Protein Kinase C Alpha-Dependent Signaling

doi: 10.3390/ijms22041891

Figure Lengend Snippet: Quantitation of CCI-induced changes of TLR5 expression in dorsal horn after nerve decompression. In ( A , B ) CCI group (dark blue bars) andDecompression group (dark green bars), panels revealed the temporal changes of TLR5-ir expression on ipsilateral sides, which were quantified as the dorsal horn area (μm 2 ) in the ( A ) medial and ( B ) lateral portions of dorsal horn respectively (quantitative areas were exhibited in panel B, ). The values on contralateral sides in CCI group (light blue bars) and Decompression group (light green bars) were also shown in panels for comparison. All the values were expressed as mean ± SD ( n = 5 per time points). Student’s t test was applied to examine contralateral side vs. ipsilateral side at each time points. * p < 0.05, ** p < 0.01 and *** p < 0.001, indicated a significant difference and ns mean no significant difference.

Article Snippet: In brief, these sections are blocked with 5% normal goat serum with 0.5% Triton X-100 in 0.5 M Tris for 1 h at room temperature and incubated with one of a mixture of primary antiserum at 4 °C overnight: (1) rabbit polyclonal TLR5 (1:100, 19810-1-AP, Proteintech Group, Inc., Chicago, IL, USA); (2) rabbit polyclonal pPKCα (1:200, 1195-1,Epitomics, Burlingame, CA, USA); (3) rabbit polyclonal pPKA RII (1:200, 1151-1, Epitomics, Burlingame, CA, USA); and (4) rabbit polyclonal MOR (1:200, RA10104, Neuromics, Edina, MN, USA) were mixed with mouse monoclonal SP (1:100, sc-58591, Santa Cruz Biotechnology; Santa Cruz, CA, USA), mouse monoclonal CGRP (1:100, sc-57053,Santa Cruz Biotechnology; Santa Cruz, CA, USA) or biotinylated IB4 (1:1000, L 2140, Sigma, UK) respectively.

Techniques: Quantitation Assay, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Cross-Talk of Toll-Like Receptor 5 and Mu-Opioid Receptor Attenuates Chronic Constriction Injury-Induced Mechanical Hyperalgesia through a Protein Kinase C Alpha-Dependent Signaling

doi: 10.3390/ijms22041891

Figure Lengend Snippet: Identification of TLR5 expression in dorsal horn by double immunofluorescence.On ipsilateral sides, ( A , D , G ) CCI and ( B , E , H ) Decompression groups exhibited their related co-expressions in the medial portion of dorsal horn at POW 8. ( A , B ) The merged images illustrated the increase of TLR5-ir (green) expression in Decompression group almost co-expressed with SP-ir (red) expression in lamina I. ( D , E ) In the merged images, TLR5- (green) and CGRP-ir (red) expressions partially co-expressed, especially near the border between the outer and inner part of lamina II. ( E , F ) Importantly, TLR5-ir (green) expression mainly co-expressed with IB4-ir (red) expression in the inner part of lamina II (arrowheads in ( H )). Also, the similar pattern of TLR5- and IB4-ir co-expression was demonstrated in CCI group (arrow in ( G )). ( C , F , I ) Panels showed the quantitation of individual ir dorsal horn areas and their co-expressed areas. Scale bar = 40 μm.

Article Snippet: In brief, these sections are blocked with 5% normal goat serum with 0.5% Triton X-100 in 0.5 M Tris for 1 h at room temperature and incubated with one of a mixture of primary antiserum at 4 °C overnight: (1) rabbit polyclonal TLR5 (1:100, 19810-1-AP, Proteintech Group, Inc., Chicago, IL, USA); (2) rabbit polyclonal pPKCα (1:200, 1195-1,Epitomics, Burlingame, CA, USA); (3) rabbit polyclonal pPKA RII (1:200, 1151-1, Epitomics, Burlingame, CA, USA); and (4) rabbit polyclonal MOR (1:200, RA10104, Neuromics, Edina, MN, USA) were mixed with mouse monoclonal SP (1:100, sc-58591, Santa Cruz Biotechnology; Santa Cruz, CA, USA), mouse monoclonal CGRP (1:100, sc-57053,Santa Cruz Biotechnology; Santa Cruz, CA, USA) or biotinylated IB4 (1:1000, L 2140, Sigma, UK) respectively.

Techniques: Expressing, Immunofluorescence, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: Cross-Talk of Toll-Like Receptor 5 and Mu-Opioid Receptor Attenuates Chronic Constriction Injury-Induced Mechanical Hyperalgesia through a Protein Kinase C Alpha-Dependent Signaling

doi: 10.3390/ijms22041891

Figure Lengend Snippet: Role of FLA-ST Ultrapure (flagellin), a TLR5 agonist, in CCI-induced pain hypersensitivity by an intrathecal administration. ( A , C ) Mechanical hyperalgesia and ( B , D ) mechanical allodynia were evaluated in CCI rats at POW 8 and recorded as baseline values at post-injection hour 0. Temporal changes of pain hypersensitivity on ( A , B ) ipsilateral and ( C , D ) contralateral sides were evaluated in Vehicle and various FLA-ST Ultrapure groups. Temporal patterns of ( E ) difference in withdrawal threshold and ( F ) difference in mechanical threshold demonstrated that FLA-ST Ultrapure exerted an analgesic effect on CCI-induced mechanical hyperalgesia but not mechanical allodynia, in a dose-responsive manner. The withdrawal threshold and mechanical threshold were represented as mean ± SD. Vehicle group (white squares, n = 3) was used to demonstrate the effect of intrathecal administration on behavioral assessments. The properties of FLA-ST Ultrapure (flagellin) were tested at the concentration of 0.1 μg (light blue squares), 0.3 μg (blue squares) and 0.9 μg (deep blue squares) ( n = 3 per group). Statistical comparisons in Vehicle and FLA-ST Ultrapure groups were made by the two-way ANOVA, followed by Bonferroni post hoc test, with the concentrations of FLA-ST Ultrapure as between-subjects factors and time as the within-subjects factor. *** p < 0.001, indicated a significant difference.

Article Snippet: In brief, these sections are blocked with 5% normal goat serum with 0.5% Triton X-100 in 0.5 M Tris for 1 h at room temperature and incubated with one of a mixture of primary antiserum at 4 °C overnight: (1) rabbit polyclonal TLR5 (1:100, 19810-1-AP, Proteintech Group, Inc., Chicago, IL, USA); (2) rabbit polyclonal pPKCα (1:200, 1195-1,Epitomics, Burlingame, CA, USA); (3) rabbit polyclonal pPKA RII (1:200, 1151-1, Epitomics, Burlingame, CA, USA); and (4) rabbit polyclonal MOR (1:200, RA10104, Neuromics, Edina, MN, USA) were mixed with mouse monoclonal SP (1:100, sc-58591, Santa Cruz Biotechnology; Santa Cruz, CA, USA), mouse monoclonal CGRP (1:100, sc-57053,Santa Cruz Biotechnology; Santa Cruz, CA, USA) or biotinylated IB4 (1:1000, L 2140, Sigma, UK) respectively.

Techniques: Injection, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Cross-Talk of Toll-Like Receptor 5 and Mu-Opioid Receptor Attenuates Chronic Constriction Injury-Induced Mechanical Hyperalgesia through a Protein Kinase C Alpha-Dependent Signaling

doi: 10.3390/ijms22041891

Figure Lengend Snippet: Role of Go 6976, a protein kinase C (PKC)inhibitor, in TLR5- and MOR-mediated attenuation of pain hypersensitivity by an intrathecal administration. ( A , B ) Mechanical hyperalgesia and ( C , D ) mechanical allodynia were evaluated in CCI rats at POW 8 and recorded as baseline values at post-injection hour 0. Temporal changes of ( A , C ) FLA-ST Ultrapure- and ( B , D ) DAMGO-mediated attenuation of pain hypersensitivity were affected by an intrathecal Go 6976 administration. ( A , B ) Time-based modes of difference in withdrawal threshold demonstrated that Go 6976 exerted a positive effect on the re-induction of FLA-ST Ultrapure- and DAMGO-mediated attenuation of mechanical hyperalgesia, in a dose-responsive manner. The difference in withdrawal threshold and difference in mechanical threshold were represented as mean ± SD. FLA-ST Ultrapure + Go 6976 0 μM group or DAMGO + Go 6976 0 μM group (white squares, n = 3) was used to demonstrate the effect of intrathecal administration on behavioral assessments. The properties of Go 6976 were tested at the concentration of 5 μM (light purple squares in ( A , C ), light green squares in ( B , D )), 20 μM (purple squares in ( A , C ), green squares in ( B , D )) and 50 μM (deep purple squares in ( A , C ), deep green squares in ( B , D )) ( n = 3 per group). Statistical comparisons were made by the two-way ANOVA, followed by Bonferroni post hoc test, with the concentrations of Go 6976 as between-subjects factors and time as the within-subjects factor. * p < 0.05, ** p < 0.01 and *** p < 0.001, indicated a significant difference.

Article Snippet: In brief, these sections are blocked with 5% normal goat serum with 0.5% Triton X-100 in 0.5 M Tris for 1 h at room temperature and incubated with one of a mixture of primary antiserum at 4 °C overnight: (1) rabbit polyclonal TLR5 (1:100, 19810-1-AP, Proteintech Group, Inc., Chicago, IL, USA); (2) rabbit polyclonal pPKCα (1:200, 1195-1,Epitomics, Burlingame, CA, USA); (3) rabbit polyclonal pPKA RII (1:200, 1151-1, Epitomics, Burlingame, CA, USA); and (4) rabbit polyclonal MOR (1:200, RA10104, Neuromics, Edina, MN, USA) were mixed with mouse monoclonal SP (1:100, sc-58591, Santa Cruz Biotechnology; Santa Cruz, CA, USA), mouse monoclonal CGRP (1:100, sc-57053,Santa Cruz Biotechnology; Santa Cruz, CA, USA) or biotinylated IB4 (1:1000, L 2140, Sigma, UK) respectively.

Techniques: Injection, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: The Salmonella typhimurium Flagellar Basal Body Protein FliE Is Required for Flagellin Production and to Induce a Proinflammatory Response in Epithelial Cells

doi: 10.1074/jbc.m200149200

Figure Lengend Snippet: FIG. 7. TLR5 is required for S. typhimurium- and flagellin- induced IL-8 secretion. A, filter-grown MDCK monolayers were bi- otinylated at either the apical or basolateral surfaces, and biotinylated proteins were isolated by affinity chromatography. Biotinylated pro- teins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-TLR5 antibody. B, filter-grown MDCK monolayers were incubated for 24 h at 37 °C in the presence or absence of purified flagellin (100 ng/ml) added to the basolateral medium. Cells were then washed and incubated for 5 h in the presence or absence of TNF (100 ng/ml) or purified flagellin (100 ng/ml) added basolaterally or apical WT Salmonella (3306). Basolateral supernatants were analyzed for IL-8 using ELISA. Graphical data are presented as the mean S.D. of triplicate samples from three separate experiments.

Article Snippet: The isolated proteins were separated by SDS-PAGE and immunoblotted with anti-TLR5 antibody (Santa Cruz Biotechnology).

Techniques: Isolation, Affinity Chromatography, SDS Page, Incubation, Purification, Enzyme-linked Immunosorbent Assay

Journal: Journal of Veterinary Science

Article Title: Decreased expression of Toll-like receptor 4 and 5 during progression of prostate transformation in transgenic adenocarcinoma of mouse prostate mice

doi: 10.4142/jvs.2015.16.3.281

Figure Lengend Snippet: TLR5 expression in pathologic grades of TRAMP prostate. Sections from were incubated with anti-TLR5 antibody. Images of normal prostate tissue from WT mice (A) and low-grade PIN (B), high-grade PIN (C), poorly differentiated adenocarcinoma (D). The intensity of TLR5 immunostaining in each pathologic grade is shown (E). Intensity of TLR5 immunostaining was measured in an average of three fields and is presented as mean ± SD ( * p < 0.05 and ** p < 0.01). (A-D) 400× magnification, (inset) 100× magnification.

Article Snippet: Paraffin sections were blocked with 3% hydrogen peroxide (H 2 O 2 ) in phosphate-buffered saline (PBS) for 10 min to remove endogenous peroxidase, then blocked with 10% normal goat serum in PBS for 30 min. Next, the sections were incubated overnight in a solution containing diluted rabbit or mouse IgG isotype, anti-cytokeratin 8 (1:200 dilution; Covance, USA), anti-synaptophysin (1:200 dilution; Abcam, UK), anti-TLR4 (1:200 dilution; Imgenex, USA) or anti-TLR5 antibody (1:200 dilution; Imgenex) at room temperature and subsequently exposed to biotinylated goat anti-rabbit IgG or anti-mouse IgG and streptavidin peroxidase complex (Vector Laboratories, USA).

Techniques: Expressing, Incubation, Immunostaining

Journal: Scientific Reports

Article Title: IL-27 amplifies cytokine responses to Gram-negative bacterial products and Salmonella typhimurium infection

doi: 10.1038/s41598-018-32007-y

Figure Lengend Snippet: Stimulation with IL-27 increased TLR4 and TLR5 expression in monocytes and macrophages. THP-1 cells ( A ) and PMA-THP-1 cells ( B ) were stimulated with or without IL-27 (50 ng/ml) for 16 hours. Cells were stained with anti-human TLR4 ( top panels ) or TLR5 ( bottom panels ) antibodies for receptor expression quantification by flow cytometry. Unstained cells were acquired to quantify autofluorescence of each cell type. Data are representative of at least three independent replicate experiments.

Article Snippet: For surface staining, cells were resuspended in FACS buffer (PBS + 0.01% sodium azide + 2% FBS) and incubated with anti-human TLR4 AlexaFluor488 (Thermo Fisher Scientific Invitrogen eBioscience) and anti-human TLR5 AlexaFluor647 (R&D Systems).

Techniques: Expressing, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: IL-27 amplifies cytokine responses to Gram-negative bacterial products and Salmonella typhimurium infection

doi: 10.1038/s41598-018-32007-y

Figure Lengend Snippet: Simultaneous treatment with IL-27 results in elevated cytokine production in response to TLR4/TLR5 agonists. THP-1 cells ( A–C ) and PMA-THP-1 cells ( D–F ) were stimulated with LPS-S (100 ng/ml), flagellin (Flag) (500 ng/ml), IL-27 (50 ng/ml), or combinations of TLR agonists plus IL-27 concomitantly as indicated for 24 hours. IL-12p40 ( top panels ), TNF-α ( middle panels ), and IL-6 ( bottom panels ) levels were measured in cell-free supernatants by ELISA. Data are representative of at least three independent replicate experiments showing mean and standard deviation of ELISA technical replicates from one experiment. Mann Whitney U tests were used for statistical analyses between pairs as indicated. ns = not significant; *p≤0.05; **p≤0.01; ***p≤0.001.

Article Snippet: For surface staining, cells were resuspended in FACS buffer (PBS + 0.01% sodium azide + 2% FBS) and incubated with anti-human TLR4 AlexaFluor488 (Thermo Fisher Scientific Invitrogen eBioscience) and anti-human TLR5 AlexaFluor647 (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, MANN-WHITNEY

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

Article Title: Stimulation by TLR5 modulates osteoclast differentiation through STAT1/IFN-beta.

doi: 10.4049/jimmunol.180.3.1382

Figure Lengend Snippet: FIGURE 1. Flagellin suppresses RANKL-induced osteoclast differenti- ation from precursors. A, TLR5 expression in osteoblasts and BMMs from C57BL/6 mice. Total RNA from the cells was isolated, and the expression levels of TLR5 (29 cycles) and -actin (18 cycles) mRNA were analyzed by RT-PCR (upper panel). Whole lysates of the cells were subjected to Western blotting (WB) with the indicated Abs (lower panel). B and C, BMMs from C57BL/6 mice were cultured in the presence of M-CSF (30 ng/ml) plus RANKL (100 ng/ml) for 4 days. FlaB (5–500 ng/ml) or LPS (1 ng/ml) was added at the start of culture. , p 0.01 vs untreated control. D, FlaB (500 ng/ml) was added to the cultures on days 0, 1, 2, and 3. The cells were stained for TRAP, and TRAP-positive multinucleated cells were counted as osteoclasts. , p 0.01 vs untreated control. All values are the means SD of three independent experiments.

Article Snippet: Anti-TLR5 Ab was from IMGENEX Corporation.

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Control, Staining