Journal: Nature Communications

Article Title: Cleavage of DNA and RNA by PLD3 and PLD4 limits autoinflammatory triggering by multiple sensors

doi: 10.1038/s41467-021-26150-w

Figure Lengend Snippet: Sequence length distributions of RNA isolated from the livers of a 2-week-old C57BL/6 (B6), Pld3 −/− , Pld4 −/− or Pld3 −/− Pld4 −/− mice, b Two-month-old B6, Tlr9 CpG11/CpG11 , Tlr9 CpG11/CpG11 Pld3 −/− Pld4 −/− or Unc93b1 3d/3d Pld3 −/− Pld4 −/− mice. Shown are means and SEM of three mice/groups. In a, p values show a significant difference in Pld3 −/− Pld4 −/− over C57BL/6 by unpaired two-tailed T -test. In ( b ), p values are shown for Tlr9 CpG11/CpG11 Pld3 −/− Pld4 −/− or Unc93b1 3d/3d Pld3 −/− Pld4 −/− sequences, compared to C57BL/6 controls. Raw data for a , b are provided in Supplementary Tables and . c Biochemical analysis of lysosome-associated nucleic acids. Lysosomal fractions were enriched from spleen or liver tissue of Unc93b1 3d/3d Pld3 −/− Pld4 −/− or Unc93b1 3d/3d mice, or from HEK Blue-hTLR9 cells that were PLD3-sufficient or -deficient, nucleic acids were 5′ labeled with [ 32 P] phosphate using T4 polynucleotide kinase and electrophoresed on histidine-buffered 20% polyacrylamide gel. Radioactivity was revealed by phosphorimaging. Marker lanes show oligo dT lengths in nt. < symbol depicts the region of gel with differing abundance of nucleic acid signal. Sequence analysis shown in ( a ) and ( b ) was performed once ( n = 3 independent mice/group). Lysosomal nucleic acid analysis was performed twice with similar results.

Article Snippet: Lysosome containing fractions from liver, spleen, and HEK293 Blue-hTLR9 cells were enriched using a kit (Thermo Scientific, Catalog # 89839).

Techniques: Sequencing, Isolation, Two Tailed Test, Labeling, Radioactivity, Marker

Journal: International Journal of Molecular Sciences

Article Title: Role Played by Receptors for Advanced Glycosylation End Products in Corneal Endothelial Cells after HSV-1 Infection

doi: 10.3390/ijms22115833

Figure Lengend Snippet: Cellular localization of TLR9, RAGE, and HSV-1. HCEn cells were infected with GFP-HSV (at MOI 50, arrow (green)) and stained for cell surface RAGE (arrow (red): PE-labeled) and TLR9 (Alexa647-labeled, arrow (yellow)) without permeabilization. HSV-1 is colocalized with cell surface RAGE, which partly overlapped with the accumulated TLR9 expression (upper panel). Lower panel: GFP-HSV (arrow (green)) was associated with TLR9 (arrow (yellow)) near cell surface. TLR9 expression was transitioned to endoplasmic reticulum (ER, blue) surrounding nucleus (lower panel). Nuclear transition of GFP-HSV is also observed at 3 h PI. Bar indicates 10 µm.

Article Snippet: The cells were fixed in 1% paraformaldehyde and were stained with a mouse anti-RAGE antibody (ab89911, Abcam, Cambridge, UK) or rabbit anti-human TLR9 (bs-2717R, Bioss, Woburn, MA, USA) and made visible by Alexa 647 (Thermo Fisher Scientific, Waltham, MA, USA) or PE-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA), respectively.

Techniques: Infection, Staining, Labeling, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Role Played by Receptors for Advanced Glycosylation End Products in Corneal Endothelial Cells after HSV-1 Infection

doi: 10.3390/ijms22115833

Figure Lengend Snippet: Association of TLR9, RAGE, and HSV-1. ( A ) Association of HSV-1 and RAGE by a GFP-pull-down assay. HCEn cells were infected with HSV-1 at MOI of 50, and the cell lysates as input were pulled down by anti-GFP antibody. The proteins associated with GFP-HSV-1 were detected by SDS PAGE. SDS PAGE, gel stained by Coomassie Blue, is shown as the input, pull down, and flow through fraction (upper panel). To observe RAGE and TLR9, Western blot for each fraction was conducted for RAGE and TLR9 (lower panel). ( B ) Tubulin and GFP in each fraction are shown by Western blot.

Article Snippet: The cells were fixed in 1% paraformaldehyde and were stained with a mouse anti-RAGE antibody (ab89911, Abcam, Cambridge, UK) or rabbit anti-human TLR9 (bs-2717R, Bioss, Woburn, MA, USA) and made visible by Alexa 647 (Thermo Fisher Scientific, Waltham, MA, USA) or PE-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA), respectively.

Techniques: Pull Down Assay, Infection, SDS Page, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Role Played by Receptors for Advanced Glycosylation End Products in Corneal Endothelial Cells after HSV-1 Infection

doi: 10.3390/ijms22115833

Figure Lengend Snippet: Network analysis of RAGE ( AGER ) and HSV-1 infection-induced transcriptional responses of HCEn cells. Functional analysis was conducted to determine the associations of the HSV-1 infection-induced genes with biological functions. A set of 430 infection-induced genes (fold induction > 4) were analyzed for network generation of biological functions. The 3 highest significant networks ( p < 1 × 10 −32 ) are summarized as merged networks. RAGE ( AGER ) and TLR9 were significantly associated with type I interferon responses.

Article Snippet: The cells were fixed in 1% paraformaldehyde and were stained with a mouse anti-RAGE antibody (ab89911, Abcam, Cambridge, UK) or rabbit anti-human TLR9 (bs-2717R, Bioss, Woburn, MA, USA) and made visible by Alexa 647 (Thermo Fisher Scientific, Waltham, MA, USA) or PE-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA), respectively.

Techniques: Infection, Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Role Played by Receptors for Advanced Glycosylation End Products in Corneal Endothelial Cells after HSV-1 Infection

doi: 10.3390/ijms22115833

Figure Lengend Snippet: Role of RAGE and TLR9 in the interferon-β production by HSV-1 infection of corneal endothelial cells. ( A ) HCEn cells were transfected by siRNA of RAGE or control siRNA, and they were assessed for cell surface expression of RAGE with or without HSV-1 infection (multiplicity of infection (MOI) 1. Plated cells were stained for the surface expression of RAGE, and 1300 cells in high powered field per group were assessed for total fluorescence intensity. Si RNA transfection abolished HSV-1-induced-RAGE expression. HCEn cells were infected by HSV-1 infection at MOI 1 and were assessed for the expression of the mRNA of interferon-β at 12 h by real-time RT-PCR. The HSV-1 infection induced-interferon-β mRNA were significantly impaired by the RAGE blockade using siRAGE. ( B ) HCEn cells were infected by HSV-1 and were assessed for interferon-β production at 24 h by ELISA. HCEn cells stimulated with HSV-1 infection at MOI 1 induced interferon-β which was significantly reduced by anti-RAGE antibody and abolished by TLR9 inhibitory oligonucleotide (2 µM). *: p < 0.01; **: p <0.001; n = 6.

Article Snippet: The cells were fixed in 1% paraformaldehyde and were stained with a mouse anti-RAGE antibody (ab89911, Abcam, Cambridge, UK) or rabbit anti-human TLR9 (bs-2717R, Bioss, Woburn, MA, USA) and made visible by Alexa 647 (Thermo Fisher Scientific, Waltham, MA, USA) or PE-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA), respectively.

Techniques: Infection, Transfection, Expressing, Staining, Fluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay