Journal: Microbiology Spectrum

Article Title: Condition-dependent effects of Elexacaftor/Tezacaftor/Ivacaftor (Trikafta) on Aspergillus fumigatus growth

doi: 10.1128/spectrum.02275-24

Figure Lengend Snippet: High concentration of CFTR modulators (Elexacaftor [VX445], Tezacaftor [VX661], and Ivacaftor [VX770]) impairs macrophage control of A. fumigatus growth. ( A ) Measurement of A. fumigatus fungal length (Strain: DAL-DSred) infecting bone marrow-derived macrophages (BMDM) at a multiplicity of infection (MOI) of 5, using the IncuCyte live-cell analysis system in the presence of 3VX (VX445/VX661/VX770) treatment at [3VX] S , [3VX] E , or [3VX] 10μM , as well as with [Veh]. * P < 0.05, ** P < 0.01 (two-way ANOVA with Dunnett’s multiple comparisons test); data are presented as mean ± SEM, n = 3. ( B ) Release of TNF⍺ in BMDMs was assessed after 20 h with A. fumigatus (MOI of 15). ** P < 0.01 (one-way ANOVA with Dunnett’s multiple comparisons test); data are presented as mean ± SEM, n = 4. ( C ) Normalized phagocytosis to vehicle control ([Veh]) at 20 h time point. **P < 0.01 , ****P < 0.005 (one sample t test, theoretical mean of 1); data are presented as mean ± SEM, n = 4. ( D ) Representative measurement of zymosan phagocytosis by primed BMDMs in the presence of 3VX treatment at concentrations [3VX] S , [3VX] E or [3VX] 10μM , as well as in the presence of cytochalasine D or [Veh]. ( E ) Quantification of viable conidia following 4 h of BMDM infection in the presence of 3VX treatment at [3VX] S , [3VX] E , or [3VX] 10μM , as well as with [Veh]. ( F ) Immunoblot analysis of pro-caspase-1 ( P45 ), the caspase-1 subunit p20 ( P20 ), and β-actin of primed BMDMs left untreated (medium alone [Med]) or in the presence of 3VX treatment at [3VX] S , [3VX] E , or [3VX] 10μM , as well as with [Veh] during 20 h after infection with A. fumigatus (MOI of 15). ( G ) Release of IL-1β in BMDMs assessed after 20 h infection with A. fumigatus (MOI of 15) left untreated (medium alone [Med]) or in the presence of 3VX treatment at [3VX] S , [3VX] E , or [3VX] 10μM , as well as with [Veh]. *P < 0.05 , **P < 0.01 (one-way ANOVA with Dunnett’s multiple comparisons test); data are presented as mean ± SEM, n = 3.

Article Snippet: Cytokine levels were determined by ELISA using kits according to the manufacturer’s instructions: TNF⍺ DuoSet ELISA (DY410, R&D Systems) and IL-1β (DY401, R&D Systems).

Techniques: Concentration Assay, Control, Derivative Assay, Infection, Cell Analysis, Western Blot

Journal: BMB Reports

Article Title: Effects of α-lipoic acid on LPS-induced neuroinflammation and NLRP3 inflammasome activation through the regulation of BV-2 microglial cells activation

doi: 10.5483/BMBRep.2019.52.10.026

Figure Lengend Snippet: α-LA inhibits the expression of pro-inflammatory cytokines in LPS-treated BV-2 microglial cells. (A) Effects of α-LA on cell viability. BV-2 microglial cells were incubated with LPS (1 μg/ml) for 30 min followed by treatment with the indicated concentrations of α-LA for 24 h. Thereafter, cell viability was assessed through the MTT assay. (B and C) BV-2 microglial cells were treated with LPS (1 μg/ml) for 30 min followed by treatment with the indicated concentrations of α-LA at the suggested times. The cell-free conditioned culture medium was collected and analyzed with ELISA for TNF-α, IL-6. Data from three independent experiments are presented as means ± S.D. *≤ 0.05, **< 0.01, ***< 0.001 and are related to both LPS-induced cells and α-LA treated cells.

Article Snippet: Both TNF-α and IL-6 were quantitatively measured through an enzyme-linked immunosorbent assay (ELISA) using the mouse TNF-α and IL-6 DuoSet ELISA kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

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

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: Hypoxia increased the production of S100 calcium-binding protein A8 (S100A8) in neuron and microglia and induced the release of S100A8 in SH-SY5Y cells. ( A , B ) S100A8 expression (red) were detected by immunocytochemical analysis in primary cultured neurons (NeuN, neuron marker) and cultured mixed glia (Iba1, microglial marker and GFAP, astrocyte marker) exposed to hypoxic conditions for 48 h. Scheme 25 μm. S100A8 expression was detected by western blot analysis in ( C , D ) SH-SY5Y cells and ( E , F ) BV-2 cells exposed to hypoxic conditions for 48 h. ( G , H ) S100A8 protein expression in BV-2 cells were confirmed by immunocytochemistry and ( I ) S100A8 release in SH-SY5Y was measured by enzyme-linked immunosorbent assay (ELISA) at 48 h after hypoxia. Values of * p < 0.05, ** p < 0.01, *** p < 0.001 versus control were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Cell Culture, Marker, Western Blot, Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 induces pro-inflammatory cytokines and inflammation in BV-2 cells. BV-2 cells were stimulated with S100A8 (10 μg/mL) for 24 h. ( A ) The supernatant was collected and TNF-α and interleukin-6 (IL-6) analyzed by ELISA. ( B ) The protein and mRNA were extracted, and the expression levels of IL-1β were assessed by ELISA and RT-qPCR. ( C – E ) The protein was extracted, separated on 10% SDS-acrylamide gels (15 μg/lane) and transferred to nitrocellulose membrane. The protein expression level was detected by western blotting with anti-ERK1/2, anti-phospho-ERK1/2 (p-ERK1/2), anti-JNK and anti-p-JNK. ( F ) Cells were pre-treated with ERK inhibitor (PD98059, 20 μM), JNK inhibitor (SP600125, 10 μM) or the equivalent volume of DMSO for 1 h, then stimulated for 24 h with LPS or S100A8 for ELISA of TNF-α, IL-6. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, *** p < 0.001 versus control; ### p < 0.001 versus S100A8-treated sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Membrane, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 induces inflammasome priming by toll-like receptor (TLR)-4 receptors associated with ERK and JNK pathway in BV-2 cells. BV-2 cells were incubated for 24 h with LPS (1 μg/mL) or S100A8 (10 μg/mL) followed by Adenosine 5′-triphosphate disodium salt hydrate (ATP) (1 mM) for 1 h. ( A , B ) The NLRP3, ASC, and ( C , D ) cleaved caspase-1 were detected by western blotting. β-actin was used as an internal control. ( E , F ) BV-2 cells were lysed to whole lysates and IκB-α phosphorylation was analyzed by western blotting. ( G , H ) The translocation of nuclear factor- κB (NF-κB) was also detected by western blotting. BV-2 cells were lysed to cytosolic extracts and nucleic extracts. Lamin-B1 was used as internal controls. ( I , J ) BV-2 microglial cells were pre-treated with PD98059 (ERK inhibitor, 20 μM), SP600125 (JNK inhibitor, 10 μM), TAK-202 (TLR4 inhibitor, 10 μg/mL) or an equivalent volume of DMSO and stimulated for 24 h with LPS or S100A8. Cells harvested and lysed in RIPA buffer for western blotting of NLRP3. Results are from one experiment that is representative of at least three others. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, ** p < 0.01 versus control; # p < 0.05, ## p < 0.01 versus S100A8-treated sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Incubation, Western Blot, Control, Phospho-proteomics, Translocation Assay

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 derived from neuronal cells induces NLRP3 inflammasome priming in microglia under hypoxic conditions. BV-2 cells were pre-treated with TAK-202 (TLR4 inhibitor, 10 μg/mL) for 1 h, then stimulated for 48 h in hypoxic condition with SH-SY5Y cells indirectly co-cultured in 0.4 μm pore transwell. ( A ) The protein expression level was detected by western blotting with NLRP3. β-actin was used as an internal control. ( B ) Quantitative analysis of NLRP3 levels. Data from three independent experiments are presented as the means ± S.D. Values of ** p < 0.01 versus control; # p < 0.05 versus co-cultured sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Derivative Assay, Cell Culture, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: The expression of S100A8 in microglial cell induces apoptosis of neuronal cells in hypoxic condition. ( A , B ) SH-SY5Y cells incubated without or with S100A8 KD BV-2 cells for 48 h in hypoxic condition. Cleaved caspase-3 immunofluorescence images and were detected and quantitative analysis of the number of cleaved-caspase3-positive cells are shown in lower panel. ( C , D ) Representative Annexin-V/PI images were detected by flow cytometry. Quantitative analysis of the apoptotic rate of SH-SY5Y cells are shown in lower panel. ( E , F ) Primary neuron-glial mixed cells were transfected with S100A8 shRNA vector for 24 h followed by 48 h in hypoxic condition. Cells were harvested, and the expression protein levels of S100A8 and cleaved caspase-3 were analyzed by Western blotting. Data from three independent experiments are presented as the means ± S.D. Values of *** p < 0.001 versus control; # p < 0.05, ### p < 0.001 versus hypoxia-exposed sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, Immunofluorescence, Flow Cytometry, Transfection, shRNA, Plasmid Preparation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: The expression of S100A8 in microglial cell induces the Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE 2) pathway. BV-2 cells were transfected with S100A8 shRNA or Scramble vector. After 24 h, cells were incubated in hypoxic condition for 48 h. ( A ) The mRNA and ( B ) the protein levels of S100A8 and COX-2 were detected by real-time PCR and western blotting. ( C ) Secretion of PGE 2 level analyzed by ELISA. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, ** p < 0.01 versus control; # p < 0.05, ### p < 0.001 versus hypoxia-exposed sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: IRAK4 autophosphorylation controls inflammatory signaling by activating IRAK oligomerization

doi: 10.1101/2023.12.21.572799

Figure Lengend Snippet: a) IL-2 release in WT and EL4-MyD88-GFP/IRAK4-mScarlet cells treated with DMSO or IRAK4 kinase inhibitor. IL-2 release was measured by ELISA 24 h after IL-1β stimulation. Values shown are the fold change in IL-2 release. Average values calculated from three independent experiments. Bars represent mean ± SEM. b) Treatment with the IRAK4 kinase inhibitor blocks pIRAK4 production after IL-1 stimulation. WT and EL4-MyD88-GFP/IRAK4-mScarlet cell lysates analyzed for pIRAK4 production by Western blot analysis. Cells treated with 20 µM of IRAK4 kinase inhibitor (or DMSO) for 4 h before being stimulated with 1 ng/ml of IL-1β for 30 mins in the presence of the inhibitor. c) TIRF images and kymograph analysis of DMSO control treated EL4-MyD88-GFP/IRAK4-mScarlet stimulated on IL-1 functionalized SLBs. Kymographs derived from red line overlaid TIRF images (left panel). Scale bar, 5 µm.

Article Snippet: To measure IL-2 release, we used the Mouse IL-2 DuoSet ELISA kit (R&D Systems, DY402-05) using the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control, Derivative Assay

Journal: bioRxiv

Article Title: IRAK4 autophosphorylation controls inflammatory signaling by activating IRAK oligomerization

doi: 10.1101/2023.12.21.572799

Figure Lengend Snippet: a) IL-2 release in EL4-MyD88-GFP/IRAK4-KO cells reconstituted with IRAK4 WT , IRAK4 K213/14A and IRAK4 DD . IL-2 release was measured by ELISA 24 h after IL-1β stimulation. Values shown are the fold change in IL-2 release. Average values calculated from three independent experiments. Bars represent mean ± SEM. One way Anova was used to compare the replicate means. b) TIRF images and kymograph of EL4-MyD88-GFP/IRAK4-KO cells reconstituted with IRAK4 WT -mScarlet stimulated on IL-1 functionalized SLBs. Kymographs derived from red line overlaid TIRF images (left panel). Scale bar, 5 µm. c) Time-series TIRF images and fluorescence-intensity time series showing the formation of a single MyD88-GFP:IRAK4 WT assembly in EL4-MyD88-GFP/IRAK4-KO cells reconstituted with IRAK4 WT -mScarlet. Scale bar, 1 µm.

Article Snippet: To measure IL-2 release, we used the Mouse IL-2 DuoSet ELISA kit (R&D Systems, DY402-05) using the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Fluorescence