Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Wild-type (WT) and Plscr1 -/- mice were exposed to sublethal (300 pfu, B, C, and F–H ) or lethal (900 pfu, D and E ) influenza A virus (IAV) (WSN) infection. ( A ) Scheme of experiment. ( B ) Whole lungs of WT mice were analyzed for Plscr1 RNA by qRT-PCR. ( C, D ) Mean relative weight of mice post-sublethal or lethal infection. ( E ) Survival rate of mice post-lethal IAV infection. ( F ) Viral RNA load in the lungs was assessed by quantifying M gene by qRT-PCR. ( G ) Infectious viral titer in the lungs was assessed by plaque assays. ( H ) Representative staining for H1N1 in lungs. The scale bars represent 1 mm. Quantification was performed using ImageJ. Data are expressed as mean ± SEM of n=30 mice/group for weight loss post-sublethal infection and n=8 mice/group for weight loss and survival rate post-lethal infection. For the rest analysis, n=5–10 mice/group. All data were pooled from three independent experiments and described biological replicates. Log-rank (Mantel-Cox) test was used to compare survival rates. Ordinary two-way ANOVA tests were used to compare weight losses. * p <0.05, ** p <0.01, *** p <0.001, **** P <0.0001. dpi, days post-infection. CTCF, Corrected Total Cell Fluorescence.

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Virus, Infection, Quantitative RT-PCR, Staining, Fluorescence

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Wild-type (WT) and Plscr1 -/- mice were exposed to sublethal (300 pfu) influenza A virus (IAV) (WSN) infection. ( A ) Total Bronchoalveolar lavage (BAL) leukocyte numbers. ( B ) Differential cell counts in BAL. ( C ) Representative lung sections stained with Hematoxylin and Eosin (H&E). Scale bars represent 3 mm (main) and 200 μm (inlays). ( D ) Whole lungs were analyzed for Ifna , Ifnb , Ifng, and Ifnl RNA by qRT-PCR. ( E ) Tnf-α and Ifn-λ concentrations in BAL by ELISA. Data are expressed as mean ± SEM of n=3–14 mice/group. All data were pooled from three independent experiments and described biological replicates. * p <0.05, ** p <0.01. dpi, days post-infection.

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Virus, Infection, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: ( A–E ) Wild-type (WT) and Plscr1 -/- mice were exposed to sublethal (300 pfu) IAV (WSN) infection. ( A ) Heatmap of interferons and their receptors in whole lungs by RNA-seq. ( B ) Whole lungs were analyzed for Ifnlr1 by qRT-PCR. ( C ) Heatmap of differential expressions of all interferon-stimulated genes (ISGs) in whole lungs by RNA-seq. Gene expressions were compared between groups within each row and color-labeled from row minimum (blue) to row maximum (red). ( D ) Localization of Ifn-λr1+ cells in the lungs of IAV-infected WT mice at 7 dpi. Sections stained for Ifn-λr1 (red), Foxj1, uteroglobin, or Sftpc (green), and DAPI (blue) are shown. Scale bars represent 50 μm (main) and 20 μm (inlays). ( E ) Representative staining for Ifn-λr1 in airways or alveoli of IAV-infected WT and Plscr1 -/- mice at 7dpi. Scale bars represent 50 μm. Quantifications were performed using ImageJ. ( F, G ) Calu-3 cells were analyzed for PLSCR1 ( F ) and IFNLR1 ( G ) RNA by qRT-PCR after recombinant IFN-λ and/or α-IFN-λR1 antibody treatment. Data are presented as fold change compared to non-treated group. ( H, I ) Chromatin-Immunoprecipitation of PLSCR1 and IFNLR1 promoter in Calu-3 cells followed by standard PCR ( H ) and real-time quantitative PCR ( I ). Data are expressed as mean ± SEM of n=4–12 mice or wells/group. For transcriptomic analysis, 9 mice from each PBS-treated group and 4 mice from each IAV-infected group were pooled together. All data were pooled from three independent experiments and described biological replicates. * p <0.05, ** p <0.01, *** p <0.001. dpi, days post-infection. CTCF, Corrected Total Cell Fluorescence. Scale bars represent 50 μm. Figure 3—source data 1. PDF file containing original gel for , indicating the relevant treatments. Figure 3—source data 2. Original gel corresponding to .

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Infection, RNA Sequencing, Quantitative RT-PCR, Labeling, Staining, Recombinant, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Fluorescence

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Wild-type (WT) and Plscr1-/- mice were intranasally given 2.5 μg/g of body weight of poly(I:C) (HMW) constitutively for 6 days and sacrificed on day 7. ( A ) Scheme of experiment. ( B ) Total Bronchoalveolar lavage (BAL) leukocyte numbers. ( C ) Differential cell counts in BAL. ( D, F, G ) Whole lungs were analyzed for Ifna, Ifnb, Ifng, Ifnl ( D ); Plscr1 ( F ); and Ifnlr1 ( G ) RNA by qRT-PCR. ( E ) Representative lung sections stained with Hematoxylin and Eosin (H&E). Scale bars represent 3 mm (main) and 200 μm (inlays). Data are expressed as mean ± SEM of n=5–12 mice/group. All data were pooled from three independent experiments and described biological replicates. ns, not significant, *p<0.05, ***p<0.001.

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Quantitative RT-PCR, Staining

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: ( A ) Co-Immunoprecipitation of Plscr1 and Ifn-λr1 in whole mouse lungs followed by western blot. ( B ) Proximity ligation assay of Ifn-λr1 and Plscr1 in the lungs of wild-type (WT) mice infected or uninfected with IAV. Scale bars represent 50 μm. Quantifications were performed using ImageJ. ( C ) Colocalization of IFN-λR1 (green) and PLSCR1 (red) on Calu-3 cell membranes infected or uninfected with IAV in a non-permeabilized staining. Scale bars represent 10 μm. Data are expressed as mean ± SEM of n=6–7 lungs/group. All data were pooled from three independent experiments and described biological replicates. * p <0.05, ** p <0.01. PLA, Proximity Ligation Assay. CTCF, Corrected Total Cell Fluorescence. Figure 4—source data 1. PDF file containing original membrane for , indicating the relevant bands and treatments. The exposure time was adjusted to visualize Plscr1 (top) or Ifn-λr1 (bottom). Figure 4—source data 2. Original membrane corresponding to .

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Immunoprecipitation, Western Blot, Proximity Ligation Assay, Infection, Staining, Fluorescence, Membrane

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: PLSCR1 plasmids on PLV-EF1a-IRES-Hygro backbone were packaged into GFP-expressing lentivirus. PLSCR1 -/- A549 cells were transduced using lentivirus. After a 10 day hygromycin selection, cells were analyzed using flow cytometry. ( A ) Gating strategy for live, GFP+ and PLSCR1+A549 cells. ( B ) Surface, cytoplasm, and nuclear expression of PLSCR1. Data are presented as fold change compared to WT -PLSCR1 -/- A549 cells. ( C ) Lentiviral transduction efficiency.

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Expressing, Selection, Flow Cytometry, Transduction

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: PLSCR1 -/- A549 cells were transduced with mutated PLSCR1 plasmids using lentivirus and infected with influenza A virus (IA)V (PR8) for 24 hr at 1 MOI ( A–E ) or 10 MOI ( F ). ( A ) IFNLR1 RNA by qRT-PCR. ( B ) IFN-λR1 proteins by western blotting. ( C ) Proximity ligation assay of IFN-λR1 and PLSCR1. Scale bars represent 20 μm. Quantifications were performed using ImageJ. ( D ) Viral RNA load was assessed by quantifying M gene by qRT-PCR. ( E ) Infectious viral titer was assessed by plaque assays. ( F ) Cells were stained with crystal violet. Cell viability was quantified using ImageJ. Data are expressed as mean ± SEM of n=4–13 wells/group. All data were pooled from three independent experiments and described biological replicates. ns, not significant, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, ***** p <0.00001. CTCF, Corrected Total Cell Fluorescence. Figure 5—source data 1. PDF file containing original membrane for , indicating the relevant bands and treatments. The membrane was cut just under 50 kDa marker after transfer. The top part was incubated with α-Ifn-λr1 antibody and the bottom part was incubated with α-β-actin antibody. They were then incubated with corresponding secondary antibodies separately. The exposure time was adjusted to visualize Ifn-λr1 (top) or β-actin (middle). A colorimetric photo was taken to visualize the molecular weight markers (bottom). Lanes 2–5 were from an unrelated experiment. Figure 5—source data 2. Original membrane corresponding to .

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Transduction, Infection, Virus, Quantitative RT-PCR, Western Blot, Proximity Ligation Assay, Staining, Fluorescence, Membrane, Marker, Incubation, Molecular Weight

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: WIld-type (WT), Plscr1 -/- , Ifnlr1 -/- and Plscr1 -/- ;Ifnlr1 -/- mice were exposed to sublethal (300 pfu) influenza A virus (IAV) (WSN) infection and sacrificed at 3 dpi. ( A ) Representative immunofluorescent staining for DAPI and Ifn-λr1 in lungs. Scale bars represent 100 μm. ( B ) Mean relative weight of mice. ( C ) Total Bronchoalveolar lavage (BAL) leukocyte numbers. ( D ) Neutrophil percentages in BAL. ( E ) Infectious viral titer in the lungs was assessed by plaque assays. Data are expressed as mean ± SEM of n=3–12 mice/group. All data were pooled from three independent experiments and described biological replicates. ns, not significant, * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Virus, Infection, Staining

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Wild-type (WT) mice were exposed to 2500 EID50 IAV (PR8) infection. Lungs were used for single-cell RNA sequencing analysis at 0, 1, 3, 6, and 21 dpi. ( A ) Two-dimensional UMAP representation of individual cells obtained from different timepoints. ( B ) Violin plot of aggregated Plscr1 expressions in all epithelial cell clusters. Red dots represent mean expression levels. ( C ) Violin plot of time-dependent Plscr1 expressions in both ciliated epithelial cell clusters. ( D ) Gene Ontology (GO) analysis for upregulated pathways in Ciliated Epithelial Cells-1. ( E ) Heatmap of the most differentially expressed genes of Ciliated Epithelial Cells-1 at different timepoints. ( F ) Violin plot of aggregated Plscr1 expressions in all immune cell clusters. Red dots represent mean expression levels. ( G ) Violin plot of time-dependent Plscr1 expressions in alveolar macrophage cluster. ( H ) Violin plot of time-dependent Plscr1 expressions in neutrophil cluster.

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Infection, RNA Sequencing, Expressing

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Plscr1 floxStop and Plscr1 floxStop ;Foxj1-Cre + mice were exposed to sublethal (300 pfu) influenza A virus (IAV) (WSN) infection and sacrificed at 3 dpi. ( A ) Schematic representation of the experimental design of ciliated epithelial cell conditional Plscr1 KI mice. ( B ) Validation of Plscr1 overexpression in lungs of Plscr1 floxStop ;Foxj1-Cre + mice by qRT-PCR. ( C ) Representative immunofluorescent staining for Plscr1, Ifn-λr1, and Foxj1 in lungs. Scale bars represent 50 μm (main) and 10 μm (inlays).( D ) Mean relative weight of mice. ( E ) Viral RNA load in the lungs was assessed by quantifying M gene by qRT-PCR. ( F ) Infectious viral titer in the lungs was assessed by plaque assays. ( G ) Total Bronchoalveolar lavage (BAL) leukocyte numbers. ( H ) Neutrophil percentages in BAL. ( I ) Whole lungs were analyzed for Ifnlr1 RNA by qRT-PCR and Ifn-λr1 protein by western blot. ( J ) Whole lungs were analyzed for Ifna , Ifnb , Ifng , and Ifnl RNA by qRT-PCR. ( K ) Model depicting proposed mechanism of PLSCR1-regulated IFN-λ signaling. Data are expressed as mean ± SEM of n=3–10 mice/group. All data were pooled from three independent experiments and described biological replicates. ns, not significant, * p <0.05, ** p <0.01, *** p <0.001. dpi, days post-infection. Figure 8—source data 1. PDF file containing original membrane for , indicating the relevant bands and treatments. The membrane was cut just under 50 kDa marker after transfer. The top part was incubated with α-Ifn-λr1 antibody and the bottom part was incubated with α-β-actin antibody. They were then incubated with corresponding secondary antibodies separately. The exposure time was adjusted to visualize Ifn-λr1 (top) or β-actin (middle). A colorimetric photo was taken to visualize the molecular weight markers (bottom). Lanes 2 and 3 were from an unrelated experiment. Figure 8—source data 2. Original membrane corresponding to .

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Virus, Infection, Biomarker Discovery, Over Expression, Quantitative RT-PCR, Staining, Western Blot, Membrane, Marker, Incubation, Molecular Weight

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Plscr1 floxStop and Plscr1 floxStop ;Lyz2-Cre + mice were exposed to sublethal (300 pfu) influenza A virus (IAV) (WSN) infection. ( A ) Validation of Plscr1 overexpression in lungs of Plscr1 floxStop ;Lyz2-Cre + mice by qRT-PCR. ( B ) Mean relative weight of mice. ( C ) Total Bronchoalveolar lavage (BAL) leukocyte numbers. ( D ) Differential cell counts in BAL. ( E ) Viral RNA load in the lungs was assessed by quantifying M gene by qRT-PCR. ( F ) Representative lung sections stained with Hematoxylin and Eosin (H&E). Scale bars represent 3 mm (main) and 200 μm (inlays). ( G–I ) Whole lungs were analyzed for Ifna , Ifnb , Ifng , Ifnl ( G ); Ifnlr1 ( H ); and Plscr1 ( I ) RNA by qRT-PCR. Data are expressed as mean ± SEM of n=15–16 mice/group for weight loss. For the rest analysis, n=3–7 mice/group. All data were pooled from three independent experiments and described biological replicates. ns, not significant, ** p <0.01. dpi, days post-infection.

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Virus, Infection, Biomarker Discovery, Over Expression, Quantitative RT-PCR, Staining

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Influenza infection is usually detected by intracellular pattern recognition receptors (PRRs) such as TLR 3 and 7, RIG-I, and MDA5. These PRRs activate the expression of IFNL in early infection stage through IRF-3 and NK-κB-controlled transcriptions. IFN-λ secreted by the infected cells interacts with IL-10R2 and IFN-λR1 on neighboring cell surfaces, which results in activation of expression of various IFN-stimulated genes, including PLSCR1 . In ciliated airway epithelial cells, PLSCR1 can further enhance the transcription of IFNLR1 by directly binding to its promoter region as a transcriptional factor, or interact with IFN-λR1 on the cell membrane.

Article Snippet: Tissues were stained overnight at 4 °C with goat anti-mouse Ifn-λr1 polyclonal antibody (1:300 dilution, Invitrogen, cat #PA1-21360, lot #WL3462592C), rabbit anti-mouse uteroglobin polyclonal antibody (1:300 dilution, Santa Cruz Biotechnology, cat #sc-365992, lot #F0220), rabbit anti-mouse Spc polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology, cat #sc-13979, lot #C2007), mouse anti-mouse Foxj1 monoclonal antibody (1:300 dilution, Invitrogen, cat #14-9965-82, lot #2712325), and/or rabbit anti-mouse Plscr1 polyclonal antibody (1:300 dilution, Proteintech, cat #11582–1-AP, no lot/clone #).

Techniques: Infection, Expressing, Activation Assay, Binding Assay, Membrane

Journal: Journal of Nanobiotechnology

Article Title: Multifunctional nanoagonist enhances photodynamic therapy-driven in situ cancer vaccination by inhibiting tumor thrombosis

doi: 10.1186/s12951-025-03843-8

Figure Lengend Snippet: In vitro evaluation of cDVPMA-induced ICD effects. ( A ) Representative CLSM images of CRT exposure on 4T1 cells following different treatments. Scale bar = 20 μm. ( B ) Representative CLSM images of HMGB1 release from 4T1 cells following different treatments. Scale bar = 20 μm. ( C ) Quantitative analysis of CRT exposure on 4T1 cells following different treatments. ( D ) Quantitative analysis of HMGB1 release from 4T1 cells following different treatments. ELISA analysis of ( E ) HMGB1 and ( F ) ATP release from 4T1 cells following different treatments. ( G ) Western blot analysis and quantification of ( H ) p-STING activation, ( I ) p-TBK1 activation, ( J ) p-IRF3 activation after various treatments. ( K ) Scheme illustration of the transwell co-culture system. Concentrations of ( L ) IFN-β and ( M ) TNF-α in the supernatants of different treatment groups. ( N ) Quantitative analysis and ( O ) representative flow cytometry plots of the proportion of mature DCs after co-incubation with 4T1 cell supernatants pre-stimulated with different formulations. FL., fluorescence intensity. c, free 2′3′-cGAMP. All data are represented as mean ± SD ( n = 3). ns, no significance. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Singlet Oxygen Sensor Green (SOSG) fluorescent probe, dichlorodihydrofluorescein diacetate (DCFH-DA), cytokine detection kits (IFN-β, IFN-γ, IL-6, TNF-α, IL-1β, TGF-β1), ATP detection kit, 4′,6-diamidino-2-phenylindole (DAPI), protease inhibitors, Alexa Fluor 555-labeled donkey anti-rabbit IgG (H + L), rabbit anti-mouse β -actin monoclonal antibody, HRP-labeled goat anti-rabbit IgG (H + L), HMGB1 detection kit, GM-CSF, and IL-4 were all purchased from Shanghai Bio-Tech Biotechnology Co., Ltd. Rabbit anti-mouse calreticulin polyclonal antibody and rabbit anti-mouse HMGB1 polyclonal antibody were purchased from Proteintech Group, Inc. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, and trypsin were obtained from Thermo Fisher Scientific Inc. Phospho-STING (Ser365) (D8F4W) rabbit monoclonal antibody (#72971), and Phospho-IRF-3 (Ser396) (4D4G) rabbit monoclonal antibody (#4947) were purchased from Cell Signaling Technology, Inc. Anti-NAK/TBK1 (phospho S172) antibody [EPR2867(2)] was obtained from Abcam.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Co-Culture Assay, Flow Cytometry, Incubation, Fluorescence