Journal: International Journal of Inflammation

Article Title: NPT1220-312, a TLR2/TLR9 Small Molecule Antagonist, Inhibits Pro-Inflammatory Signaling, Cytokine Release, and NLRP3 Inflammasome Activation

doi: 10.1155/2022/2337363

Figure Lengend Snippet: NPT1220-312 selectively inhibits TLR2 and TLR9. NPT1220-312 inhibition of (a) TLR1/2 as activated by Pam 3 CSK 4 and TLR2/6 as activated by Pam 2 CSK 4 in HEK293 cells overexpressing hTLR2; (b) TLR3 as activated by Poly I:C in HEK293 cells overexpressing hTLR3; (c) TLR4 as activated by LPS in HEK293 cells overexpressing hTLR4; (d) TLR7 as activated by R848 and as activated by CL-264 in HEK293 cells overexpressing hTLR7; (e) TLR8 as activated by R848 in HEK293 cells overexpressing hTLR8; and (f) TLR9 as activated by ODN2006 in HEK293 cells overexpressing hTLR9. TNF α was incorporated as a control for non-specific inhibition of signaling in each assay.

Article Snippet: The growth medium was supplemented with antibiotics, 1x HEK-Blue Selection for hTLR2 or hTLR4 cells, 30 μ g/ml blasiticidin and 100 μ g/ml zeocin for hTLR3, and hTLR8 cells, 10 μ g/ml blasticidin and 100 μ g/ml zeocin for hTLR7 and hTLR9 cells, and 10 μ g/ml blasticidin for murine RAW-Blue cells (InvivoGen).

Techniques: Inhibition, Control

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor and DNA sensor cGAS dictate ionizing radiation-mediated proinflammatory macrophage activation

doi: 10.1101/2024.12.19.629560

Figure Lengend Snippet: Ionizing radiation induces cytoplasmic accumulation of DNA breaks and cGAS expression during proinflammatory activation of macrophages. A, B Confocal microscopy images (A) and percentages (B) of PMA-differentiated human THP1 macrophages showing cytoplasmic double strand (ds) DNA + foci 30 minutes after treatment with control or 2 Gy single- dose ionizing radiation (IR) are shown. Nuclei are stained with Hoechst 33342 (scale bar, 3 μm). C, D Confocal microscopy images (C) and percentages (D) of PMA-differentiated human THP1 macrophages showing cytoplasmic single strand (ss) DNA + foci 30 minutes after treatment with control or 2 Gy single-dose IR are shown. Nuclei are stained with Hoechst 33342 (scale bar, 3 μm). E cGAS expression level detected 3 hours after the treatment of murine RAW264.7 macrophages with control or 2 Gy single-dose IR. Representative western blots are shown. β-actin is used as loading control. F cGAS expression level detected 6 hours after the treatment of PMA- differentiated human THP1 macrophages with control or 2 Gy single-dose IR. Representative western blot is shown. GAPDH is used as loading control. G, H Confocal microscopy images (G) and percentages (H) of PMA-differentiated human THP1 macrophages expressing iNOS (iNOS + ) 24 hours after treatment with control or 2 Gy single-dose IR. Nuclei are stained with Hoechst 33342 (scale bar, 5 μm). I IRF5 expression level detected after 96 hours of treatment of PMA- differentiated human THP1 macrophages with control or 2 Gy single-dose IR. Representative western blots are shown. GAPDH is used as loading control. J IRF5 expression level detected after 6 hours of treatment of murine RAW264.7 macrophages with control or 2 Gy single-dose IR. Representative western blots are shown. β-actin is used as loading control. Images are representative from 3 independent experiments. Data are means ± S.E.M from three independent experiments. P-values (**P< 0.01) determined with two-tailed unpaired t-test (B, D, H).

Article Snippet: As described above, human THP1 cells were differentiated into macrophages with 320 nM of PMA (Invivogen, #tlrl-PMA) in 12-well plates (10 6 cells/well) or in 24-well plates (5x10 5 cells/well).

Techniques: Expressing, Activation Assay, Confocal Microscopy, Control, Staining, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor and DNA sensor cGAS dictate ionizing radiation-mediated proinflammatory macrophage activation

doi: 10.1101/2024.12.19.629560

Figure Lengend Snippet: B 3D reconstructions of confocal microscopy images of the mitochondrial networks ( A ) and percentages ( B ) of PMA-differentiated human THP1 macrophages showing mitochondrial fragmentation 30 minutes after the treatment with control or 2 Gy single- dose IR are shown. Mitochondria networks are revealed by TOM 20 expression (scale bar, 3 μm). Magnifications are shown (scale bar, 1 μm). Images are representative from 3 independent experiments. C, D DRP1S616* expression level detected 6 hours after the treatment of murine RAW264.7 macrophages ( C ) or PMA-differentiated human THP1 macrophages ( D ) with control or 2 Gy single-dose IR. Representative western blots are shown. β-actin is used as loading control. E 3D reconstructions of confocal microscopy images of the mitochondrial networks of PMA- differentiated human THP1 macrophages detected 30 minutes after the treatment with 10 μM Mdivi-1 with control or 2 Gy single-dose IR are shown. Mitochondria networks are detected as shown in ( A ) (scale bar, 3 μm). Images are representative from 3 independent experiments. F Higher magnification details of the mitochondrial networks detected in 2 Gy-irradiated PMA- differentiated human THP1 macrophages or in PMA-differentiated human THP1 macrophages that were treated with Mdivi-1 and irradiated with a single dose of 2 Gy. a and b are magnifications of dashed regions a and b respectively shown in ( E ). Images are representative from 3 independent experiments (scale bar, 1 μm). G Percentages of PMA-differentiated human THP1 macrophages showing fragmented mitochondria 30 minutes after the treatment with control, 2 Gy single-dose IR, 10 μM Mdivi-1, or 2 Gy single-dose IR combined with 10 μM Mdivi-1 are shown. Data are means ± S.E.M from three independent experiments. P-values (*P< 0.05 and **P< 0.01) were determined with two-tailed unpaired t-test ( B ) or two-way ANOVA with Tukey’s multiple comparisons test ( G ).

Article Snippet: As described above, human THP1 cells were differentiated into macrophages with 320 nM of PMA (Invivogen, #tlrl-PMA) in 12-well plates (10 6 cells/well) or in 24-well plates (5x10 5 cells/well).

Techniques: Confocal Microscopy, Control, Expressing, Western Blot, Irradiation, Two Tailed Test

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor and DNA sensor cGAS dictate ionizing radiation-mediated proinflammatory macrophage activation

doi: 10.1101/2024.12.19.629560

Figure Lengend Snippet: A cGAS and DRP1 expression levels after 6 hours of culture of PMA-differentiated human THP1 macrophages depleted or not for DRP1, and irradiated (or not) with a single dose of 2 Gy. Representative westerns are shown. GAPDH is used as loading control. B, C Confocal microscopy images ( B ) and percentages ( C ) of PMA-differentiated human THP1 macrophages showing iNOS ( B,C ) and ψ-H2AX ( C ) expressions 24 hours after treatment with control, 2 Gy single-dose IR, 5 μM G140, or 2 Gy single-dose IR combined with 5 μM G140 are shown. Nuclei are stained with Hoechst 33342 (scale bar, 5 μm). Images are representative from 3 independent experiments. D IFR5 expression levels detected after 96 hours of culture of PMA-differentiated human THP1 macrophages that have been treated with control, 2 Gy single-dose IR, 5 μM G140, or 2 Gy single-dose IR combined with 5 μM G140. Representative western blots are shown. GAPDH is used as loading control. E IFR5 expression levels detected after 6 hours of culture of murine RAW264.7 macrophages that have been treated with control, 2 Gy single-dose IR, 10 μM RU521, or 2 Gy single-dose IR combined with 10 μM RU521. Representative western blots are shown. GAPDH is used as loading control. F cGAS expression after 96 hours of transfection with control and cGAS-specific siRNA (sicGAS). Representative western blots are shown. GAPDH is used as loading control. G IRF5 expression levels detected after 96 hours of culture of PMA-differentiated human THP1 macrophages depleted or not for cGAS and treated with control or 2 Gy single-dose irradiation. Representative western blots are shown. GAPDH is used as loading control. Data are means ± S.E.M from three independent experiments. P-values (**P< 0.01 and ***P< 0.001) were determined with two-way ANOVA with Tukey’s multiple comparisons test ( C ).

Article Snippet: As described above, human THP1 cells were differentiated into macrophages with 320 nM of PMA (Invivogen, #tlrl-PMA) in 12-well plates (10 6 cells/well) or in 24-well plates (5x10 5 cells/well).

Techniques: Expressing, Irradiation, Control, Confocal Microscopy, Staining, Western Blot, Transfection

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor and DNA sensor cGAS dictate ionizing radiation-mediated proinflammatory macrophage activation

doi: 10.1101/2024.12.19.629560

Figure Lengend Snippet: hMDMs ( A, B, F, H, I, K ) or PMA-differentiated human THP1 macrophages ( C, D, E, G, J, L ) treated with Kaempferol (Kaempf.) ( A-C, H-K ), IFNγ ( B ), AR-C118925XX (AR-C) ( C ), transfected with siRNA ( D-F ) or expressing shRNA ( G, L ) specific for P2Y2 were analyzed for gene expression of known human polarization-specific markers ( A ), IRF5 ( B, C, E-G ), P2Y2 ( D, G ), membrane CD163 ( H, I ) and IL-10 ( J-L ) expressions using microarray assay ( A ), western blots ( B, C, E-G ), RT-PCR ( D ), flow cytometry ( H, I ) or ELISA ( J-L ). Results were obtained from at least 3 independent experiments with the exception of ( C ) with AR-C118925XX (n=2). P- values (*P< 0.05 and **P< 0.01) were determined with one sample Wilcoxon test ( D ), two-tailed Wilcoxon matched-pairs signed rank test ( I ), two-tailed unpaired t-test ( J, L ) and two-tailed Mann- Whitney test ( K ).

Article Snippet: As described above, human THP1 cells were differentiated into macrophages with 320 nM of PMA (Invivogen, #tlrl-PMA) in 12-well plates (10 6 cells/well) or in 24-well plates (5x10 5 cells/well).

Techniques: Transfection, Expressing, shRNA, Membrane, Microarray, Western Blot, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: P2Y2 purinergic receptor and DNA sensor cGAS dictate ionizing radiation-mediated proinflammatory macrophage activation

doi: 10.1101/2024.12.19.629560

Figure Lengend Snippet: Confocal microscopy images ( A, C, E ) and percentages ( B, D, F ) of PMA-differentiated, control or P2Y2-depleted human THP1 macrophages showing cytoplasmic dsDNA + foci ( A, B ), ssDNA + foci ( C, D ) or fragmented mitochondria ( E, F ) 30 minutes after treatment with control or 2 Gy single-dose ionizing radiation (IR) are shown. Nuclei are stained with Hoechst 33342 (scale bar, 3 μm). G, H Confocal microscopy images ( G ) and percentages ( H ) of PMA-differentiated, control or P2Y2-depleted human THP1 macrophages expressing (or not) iNOS (iNOS + ) after 24-hour treatment with control or 2 Gy single-dose IR are shown. Nuclei are stained with Hoechst 33342 (scale bar, 5 μm). Data are means ± S.E.M from three independent experiments. P-values (*P< 0.05, **P< 0.01 and ****P< 0.0001) were determined with two-way ANOVA with Tukey’s multiple comparisons test ( B, D, F, H ).

Article Snippet: As described above, human THP1 cells were differentiated into macrophages with 320 nM of PMA (Invivogen, #tlrl-PMA) in 12-well plates (10 6 cells/well) or in 24-well plates (5x10 5 cells/well).

Techniques: Confocal Microscopy, Control, Staining, Expressing