Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: HSV1 recombinant viruses expressing cGAS and/or STING exhibit restricted replication in human cancer cells (A) Schematic diagram of rHSV1 constructs. (B–N) 2 × 10 5 293T, hTERT, HT29, and SW48 cells infected with HSV1-Δγ34.5, HSV1-STING, HSV1-cGAS, and HSV1-STING-P2A-cGAS (HSV1-2A) at the MOI indicated. (B) Immunoblot analysis of cGAS, STING, phospho-STING, phospho-TBK1, phospho-IRF3, and β-actin 6 h post infection, (C) percentage of viable cells ( n = 4 biological replicates), and (D) virus titers ( n = 2 biological replicates). (E) Measurement by ELISA of the quantity of 2′3′ cGAMP in 5 × 10 5 293T cells 24 h post infection ( n = 3 biological replicates). (F) IFN-β-luciferase activity in 293T cells 24 h after plasmid transfection followed by 6 h of infection ( n = 3 technical replicates). (G, I, and K) Percentage of viable cells ( n = 3 biological replicates) and (H, J, and L) virus titers ( n = 3 biological replicates) on infected hTERT, HT29, and SW48 cells at MOI 1. (M) qPCR of Cxcl10 ( n = 2 biological replicates) and (N) ELISA analysis of human IFNβ production in hTERT, HT29, and SW48 cells 24 h after infection ( n = 6 biological replicates). Error bars indicate mean ± SEM; Student’s t test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Recombinant, Expressing, Construct, Infection, Western Blot, Virus, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Plasmid Preparation, Transfection

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Recombinant HSV1 exhibits diminutive oncolytic activity yet retains in vivo anti-tumor properties dependent on extrinsic STING signaling (A) Immunoblot analysis of cGAS, STING, and β-actin in B16-OVA, B16-OVA cGAS KO (CKO), B16-OVA STING KO (SKO), and B16-OVA STING/cGAS KO (S/CKO) cells. (B and C) 2 × 10 5 B16 cells were infected with HSV1-Δγ34.5, HSV1-STING, HSV1-cGAS, and HSV1-STING-P2A-cGAS (HSV1-2A) at MOI 5 for 24 h. (B) Virus titers ( n = 2 biological replicates) and (C) percentage of viable cells were measured ( n = 2 biological replicates). (D–F) 2 × 10 5 293T and B16-OVA cells were infected at MOI 0.1 or 5 with HSV1- Δγ34.5-GFP. (D) The virus titer was determined by plaque assay ( n = 3 biological replicates), (E) the percentage of GFP + cells were measured by cytometry ( n = 3 biological replicates), and (F) the quantification of HSV1- Δγ34.5 genome was done by qPCR at 3 or 6 h, 24 h, and 48 h post infection ( n = 3 biological replicates). (G) B16 cells were infected with HSV1- Δγ34.5, HSV1-STING, HSV1-cGAS, or HSV1-2A at MOI 5 for 6 h. B16-OVA cells were treated with 3 μg/mL dsDNA90 as a control, and Cxcl10 was analyzed by qPCR ( n = 4 technical replicates). (H–Q) Wild-type (H–J: n = 6–8 mice per group; L–N: n = 4 mice per group), STING KO C57BL/6J ( n = 11–12 mice per group on 2 independent experiments), and BALB/c nude mice ( n = 7 mice by groups) were subcutaneously injected as indicated with B16-OVA, B16-OVA CKO, or B16-OVA S/CKO cells on the flank (5 × 10 5 cells/mouse). 5 × 10 6 PFU of replicating HSV1- Δγ34.5, HSV1-STING, HSV1-cGAS, or HSV1-2A was injected intratumorally (black arrows) three times. (H–L and O) The tumor volume was measured on the indicated days and calculated with the formula V = (length × width 2 )/2. At 16 or 17 days, the spleen and the tumors were extracted. (M and P) Digital photograph of tumors and (N and Q) ELISpot to measure IFNg release from CD8 + T cells. (R and S) Phagocytosis of B16-OVA and B16-OVA S/CKO cells by murine WT and STING KO macrophages. 1 × 10 6 cells were infected with HSV1-Δγ34.5 for 40 h at MOI 20 then irradiated by UV (120 mJ/cm) and incubated for 24 h. The irradiated cells were fed to macrophages (MØ) (2 × 10 5 cells). (R) Schematic representation and (S) ELISA analysis of IFN-β at 24 h in macrophages following engulfment of B16 ( n = 5 [WT macrophages] and 3 [STING macrophages] technical replicates). Error bars indicate mean ± SEM; Student’s t test and (H–L and O) ordinary one-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Recombinant, Activity Assay, In Vivo, Western Blot, Infection, Virus, Plaque Assay, Cytometry, Control, Injection, Enzyme-linked Immunospot, Irradiation, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Nano-STAVs are readily opsonized by macrophages to stimulate STING signaling (A) Transmission electron microscopy image of nano-empty and nano-STAVs. (B and C) Western blot analysis of phosphorylated and total cGAS, STING, TBK1, and IRF3 proteins in WT or SKO murine macrophages (bone marrow-derived macrophages, BMDMs) and B16-OVA (2 × 10 5 cells) treated with nano-empty, nano-STAVs, or lipofectamine +/− STAVs at 1 μg/ml for 6 h. (D) macrophages and B16-OVA (5 × 10 4 cells) treated with nano-STAVs-Cy5 (red) (3 μg/mL) for 16 h, fixed and stained with DAPI (blue), and analyzed by microscopy confocal. (E and F) B16-OVA and macrophages (5 × 10 4 cells) were treated with nano-STAVs-Cy5 (red) (3 μg/mL) for 16 h, fixed and stained with EEA1-FITC, RAB7-FITC, CD63-FITC, LAMP1-FITC (green), and DAPI (blue), and analyzed by confocal microscopy. (G–N) (G–I and K–M) qPCR analysis of IFNb1, CXCL10, and CCL5 and (J and N) IFNb ELISA in B16-OVA, WT, and SKO macrophages (2 × 10 5 cells), treated with nano-empty, nano-STAVs, or lipofectamine +/− STAVs at 1 μg/ml for 6 h (qPCR) or 24 h (ELISA) ( n = 2 biological replicates). Error bars indicate mean ± SEM; Student’s t test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Derivative Assay, Staining, Microscopy, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Murine and human tumor cells exposed to nano-STAVs activate APCs in trans in an STING-dependent manner, augmenting checkpoint therapeutic activity in vivo (A) Schematic representation of the phagocytosis of mouse and human cells by macrophages. 1 × 10 6 cells were treated with 1 μg/mL of nano-empty or nano-STAVs or transfected with lipofectamine + STAVs and irradiated by UV (120 mJ/cm). The irradiated cells were fed to murine or human macrophages (MØ) (2 × 10 5 cells) 24 h after UV irradiation. (B) Confocal microscopy analysis with nano-STAVs-cy5 (red) in CD11b + FITC murine macrophages (green). Cells were treated for 6 h with nanoparticles, and the phagocytosis was evaluated at 6 h. (C–H) (C, E, and G) RT-qPCR analysis of Cxcl10 at 6 h and (D, F, and H) IFN-β ELISA at 24 h in human and murine WT macrophages following engulfment of B16, SK-MEL-31, and SK-MEL-5 cells in presence or absence of nano-STAVs for 24 h ( n = 4 [mouse cell] and 3 [human cells] biological replicates). (I–L) Mice were subcutaneously injected with B16-OVA cells (5 × 10 5 cells/mouse) ( n = 13–17 mice per group on 2 independent experiments) on the right flank. On days 7, 10, and 13, after tumor inoculation, the mice were intratumorally injected with PBS, STAVs, nano-empty, or nano-STAVs (0.1 μg/mouse) and/or intraperitoneally with PD1 (50 μg/mouse) (black arrows). At day 19, the spleen was extracted to measure IFNg release from CD8 + T cells. (I) Schematic representation of experimental design. (J) The tumor volume was measured and calculated with the formula V = (length × width 2 )/2. (K) Digital photographs of tumors. (L) IFNg ELISpot. (C–H) Error bars indicate mean ± SEM; Student’s t test and (J and L) two-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Activity Assay, In Vivo, Transfection, Irradiation, Confocal Microscopy, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Injection, Enzyme-linked Immunospot

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Nano-STAVs activity is augmented by type I IFN (A–D) Mice were subcutaneously injected with B16 OVA cells (5 × 10 5 cells/mouse) ( n = 17–18 mice per group on 2 independent experiments) on the right flank. On days 7, 10, and 13, after tumor inoculation, the mice were intratumorally injected with PBS, STAVs, nano-empty, or nano-STAVs (0.1 μg/mouse) and/or intraperitoneally with PD1 (50 μg/mouse) and/or IFNa (10,000 U/mouse) (black arrows). At day 17, the spleen was extracted to measure IFNg release from CD8 + T cells. (A) Schematic representation of experimental design. (B) The tumor volume was measured and calculated with the formula V = (length × width 2 )/2. (C) IFNg ELISpot. (D) Digital photographs of tumors. (E–J) qPCR analysis of CXCL10 and CCL5 in B16-OVA, B16-OVA-cGAS KO (CKO), and WT and SKO murine macrophages (2 × 10 5 cells), treated with IFNa at 1000 U/ml for 6 h ( n = 4 biological replicates). (K and L) Flow cytometry for H-2Kb and CD86 on macrophages (2 × 10 5 cells) following IFNa treatment at 1000 U/ml for 24 h ( n = 2 biological replicates). (M) Schematic representation of the phagocytosis of B16-OVA cells by macrophages. 1 × 10 6 B16-OVA cells were treated with IFNa at 1000 U/ml for 24 h and irradiated by UV (120 mJ/cm). The irradiated cells were fed to murine macrophages (MØ) (2 × 10 5 cells) previously treated or not with IFNa at 1000 U/ml for 24 h. (N) RT-qPCR analysis of Cxcl10 at 6 h ( n = 2 biological replicates). (O) IFN-β ELISA at 24 h of murine WT macrophages following engulfment of B16-OVA treated with IFNa ( n = 3 biological replicates). (P) Schematic representation of the phagocytosis of untreated B16-OVA cGAS KO cells (B16 CKO) by macrophages previously treated with IFNa at 1000 U/ml for 24 h. The conditions applied were the same as in (M). (Q) Flow cytometry for H-2Kb-SIINFEKL (OVA) on macrophages at 24 h following phagocytosis of B16-OVA cGAS KO ( n = 2 biological replicates). Error bars indicate mean ± SEM; Student’s t test and (B and C) two-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Activity Assay, Injection, Enzyme-linked Immunospot, Flow Cytometry, Irradiation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Nano-STAVs can facilitate the immunotherapeutic effects of radiation treatment in a head and neck tumor model (A–H) 2 × 10 5 MOC2 and Cal27 head and neck cell lines were treated with 1 μg/ml nano-empty, nano-STAVs, or lipofectamine +/− STAVs. (A and B) Western blot analysis of total and phosphorylated cGAS, STING, TBK1, IRF3 proteins, (C, D, F, and G) qPCR analysis of IFNb1 and CXCL10 after 6 h, and (E and H) IFNb ELISA 24 h after treatment ( n = 2 biological replicates). (I and J) MOC2 cells were irradiated with 10 Gy X-ray and then incubated for 24 h followed by treatment with nano-STAVs (1 μg/ml) for another 24 h. (I) For checking protein expression, MOC2 cells were collected and lysed with RIPA buffer and then analyzed by immunoblotting with indicated antibodies. (J) For phagocytosis assay, 2 × 10 6 MOC2 cells were phagocytosed with 2 × 10 6 dendritic cells (bone marrow-derived dendritic cells, BMDCs) for 6 h followed by isolating CD11c+ dendritic cells. Cxcl10 expression in dendritic cells was evaluated by qPCR analysis ( n = 2 biological replicates). (K and L) Immunocompetent C57BL/6J mice were subcutaneously injected with 5 × 10 5 MOC2 cells per mouse on the right flank and 2.5 × 10 5 MOC2 cells per mouse on left side. Right-side tumors were irradiated at days 4, 5 and 6 after tumor inoculation at 8 Gy (red arrows). On days 7, 10 and 13, after tumor inoculation, the mice were intratumorally injected on right side only with PBS, STAVs, nano-empty, or nano-STAVs (0.1 μg/mouse) and/or with PD1 (100 μg/mouse) (black arrows). The tumor volume was measured and calculated with the formula V = (length × width 2 )/2. (K) Schematic representation of experimental design. (L and M) (L) Immunocompetent WT ( n = 6 mice per group) or (M) STING KO C57BL/6J mice ( n = 6 mice per group). The tumor volumes are represented from day 21 or 24. (N and O) (N) Primary (right flank) and (O) abscopal effects (left flank) of the treatments ( n = 6 mice per group). Error bars indicate mean ± SEM; (C–J and O) Student’s t test and (N) two-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Irradiation, Incubation, Expressing, Phagocytosis Assay, Derivative Assay, Injection

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet:

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Control, Staining, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Knock-Out, Plasmid Preparation, Expressing, Software, Microscopy

Journal: Animals : an Open Access Journal from MDPI

Article Title: Early Pregnancy Induces Expression of STAT1, OAS1 and CXCL10 in Ovine Spleen

doi: 10.3390/ani9110882

Figure Lengend Snippet: Primers used for RT-qPCR.

Article Snippet: The membranes were incubated with a goat anti-STAT1 polyclonal antibody (Abcam, Cambridge, UK, ab230428, 1:1000), a rabbit anti-OAS1 polyclonal antibody (Abcam, ab86343, 1:1000), a mouse anti-Mx1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-166412, 1:1000), and a mouse anti-CXCL10 monoclonal antibody (Santa Cruz Biotechnology, sc-374092, 1:1000), respectively.

Techniques: Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Early Pregnancy Induces Expression of STAT1, OAS1 and CXCL10 in Ovine Spleen

doi: 10.3390/ani9110882

Figure Lengend Snippet: Relative expression values of STAT1 , OAS1 , MX1 and CXCL10 mRNA in ovine spleens measured by real-time quantitative PCR. Note: DN16 = Day 16 of the estrous cycle; DP13 = Day 13 of pregnancy; DP16 = Day 16 of pregnancy; DP25 = Day 25 of pregnancy. Significant differences ( p < 0.05) are indicated by different letters within same color column.

Article Snippet: The membranes were incubated with a goat anti-STAT1 polyclonal antibody (Abcam, Cambridge, UK, ab230428, 1:1000), a rabbit anti-OAS1 polyclonal antibody (Abcam, ab86343, 1:1000), a mouse anti-Mx1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-166412, 1:1000), and a mouse anti-CXCL10 monoclonal antibody (Santa Cruz Biotechnology, sc-374092, 1:1000), respectively.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Animals : an Open Access Journal from MDPI

Article Title: Early Pregnancy Induces Expression of STAT1, OAS1 and CXCL10 in Ovine Spleen

doi: 10.3390/ani9110882

Figure Lengend Snippet: Expression of STAT1, OAS1, Mx1 and CXCL10 proteins in ovine spleens analyzed by western blot. Note: DN16 = Day 16 of the estrous cycle; DP13 = Day 13 of pregnancy; DP16 = Day 16 of pregnancy; DP25 = Day 25 of pregnancy. Significant differences ( p < 0.05) are indicated by different superscript letters within the same color column.

Article Snippet: The membranes were incubated with a goat anti-STAT1 polyclonal antibody (Abcam, Cambridge, UK, ab230428, 1:1000), a rabbit anti-OAS1 polyclonal antibody (Abcam, ab86343, 1:1000), a mouse anti-Mx1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-166412, 1:1000), and a mouse anti-CXCL10 monoclonal antibody (Santa Cruz Biotechnology, sc-374092, 1:1000), respectively.

Techniques: Expressing, Western Blot

Journal: Cell stem cell

Article Title: Chronic inflammation directs an olfactory stem cell functional switch from neuroregeneration to immune defense

doi: 10.1016/j.stem.2019.08.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used: Rabbit anti-Krt5 (1:800, PRB-160P; Covance), Rabbit anti-Ki67 (1:500, Ab16667; Abcam), Rat anti-BrdU (1:400, Ab6326; Abcam), Mouse anti-Krt14 (1:800, MA5–11599; Thermo Fisher), Mouse anti-β-Tubulin III (1:200, MAB1637; Millipore), Rabbit anti-∆NP63 (1:1,000, 619001; BioLegend), Mouse anti-P63 (1:200, sc-8431; Santa Cruz), Rabbit anti-RelA (1:200, Sc-372; Santa Cruz), Rat anti-CD45 (1:200, 14–0451-81; Ebioscience), Rat anti-F4/80(1:500, MCA497GA; Bio-Rad), Rat anti-Ly6G (1:500, 127601; Biolegend), Rat anti-CD3 (1:200, 14–0032-81; Ebioscience), Goat anti-OMP (1:1000, 544–10001; Wako), Goat anti Sox2 (1:200, sc-17320; Santa Cruz), Goat anti-CCL19 (1:50, AF880; R&D), Goat anti-CXCL10 (1:100, AF-466-NA; R&D), Mouse anti Human CCL2 (1:100, MAB2791; R&D), Rabbit anti Human-CD45 (1:400, Ab40763; Abcam), Mouse anti-CD45 (1:500, 304002; Biolegend), Mouse anti-human CD3 (1:300, 300413; Biolegend), Alexa Fluor® 488 anti-human CD3 (1:200, 300454; Biolegend), Mouse anti-Human CD4 (1: 200, 555344, BD Pharmingen), and Alexa Fluor® 488 anti-β-Tubulin III (1:1000, 801203; Biolegend).

Techniques: Immunohistochemistry, Flow Cytometry, Recombinant, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Software

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Targeting EZH2 enhances antigen presentation, antitumor immunity and circumvents anti-PD-1 resistance in head and neck cancer

doi: 10.1158/1078-0432.CCR-19-1351

Figure Lengend Snippet: A. SCC9, SCC25, CAL27, and CAL33 were treated with increasing concentrations of GSK126 or EPZ6348 (1 nM-100 μM) for 72 hours to test assess impact on cell viability. B. Human HNSCC cells were treated with GSK126 (10 μM), EPZ6438 (10 μM), or DMSO as control for 72 hours. IFNγ was added in the last 24 hours of drug incubation for all assays shown. HLA cell surface protein levels were measured by flow cytometry. The data are representative of 2 independent experiments. C. B2M and CXCL10 mRNA expression levels were quantified by qRT-PCR. Relative mRNA levels were normalized to GAPDH. *P<0.05, **P<0.01, ***P<0.001. Significance was calculated by one-way ANOVA. Data are shown as Mean± SD.

Article Snippet: TaqMan Real-time PCR Assays specific for mouse B2m (Mm00437762_m1), H2-K1 (Mm01612247_mH), Cxcl10 (Mm00445235_m1), Gapdh (Mm99999915_g1), human B2M (Hs00187842_m1), HLA-A (Hs01058806_g1), HLA-B (Hs00818803_g1), HLA-C (Hs00740298_g1), CXCL10 (Hs00171042_m1), and GAPDH (Hs02786624_g1) were purchased from Thermo Fisher and assays were performed on an ABI Step One Plus for quantifying gene expression levels.

Techniques: Control, Incubation, Flow Cytometry, Expressing, Quantitative RT-PCR

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Targeting EZH2 enhances antigen presentation, antitumor immunity and circumvents anti-PD-1 resistance in head and neck cancer

doi: 10.1158/1078-0432.CCR-19-1351

Figure Lengend Snippet: A. MOC1-esc1 cells were treated with GSK126 (10 μM), EPZ6438 (10 μM), or DMSO as control for 72 hours. H3K27me3 and EZH2 protein levels were determined by western blot. Total H3 was used as loading control. B. The mRNA expression levels of B2M, H2-K1, and CXCL10 were measured by qRT-PCR in MOC1-esc1 cells treated with EZH2 inhibitors and IFNγ. Relative mRNA levels were normalized to GAPDH. C, D. Chromatin immunoprecipitation (CHIP) for EZH2, H3K27me3, and IgG, and subsequent qPCR in B2M promoter using two independent primer sets. *P<0.05, **P<0.01, ***P<0.001. Significance was calculated by one-way ANOVA and Student’s t test. Data are shown as Mean± SD.

Article Snippet: TaqMan Real-time PCR Assays specific for mouse B2m (Mm00437762_m1), H2-K1 (Mm01612247_mH), Cxcl10 (Mm00445235_m1), Gapdh (Mm99999915_g1), human B2M (Hs00187842_m1), HLA-A (Hs01058806_g1), HLA-B (Hs00818803_g1), HLA-C (Hs00740298_g1), CXCL10 (Hs00171042_m1), and GAPDH (Hs02786624_g1) were purchased from Thermo Fisher and assays were performed on an ABI Step One Plus for quantifying gene expression levels.

Techniques: Control, Western Blot, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation

Journal: Cell death and differentiation

Article Title: RIG-I-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by CD8(+) T cells.

doi: 10.1038/cdd.2014.96

Figure Lengend Snippet: Figure 1 RLH activation induces secretion of proinflammatory cytokines and induction of apoptosis in murine pancreatic cancer cells. (a) Panc02 cells were stimulated with indicated amounts of ppp-RNA, poly(I:C) or left untreated. OH-RNA served as transfection control. IFN-b levels were analyzed with qRT-PCR relative to HPRT and secretion of CXCL10 or IL-6 was measured with ELISA; (b) Panc02 cells were stimulated with RNA (24 h for poly(I:C) and 48 h for ppp-RNA) and viability was assessed by FACS analysis using annexin V/PI staining; (c) Panc02 cells were incubated with siRNA specific for RIG-I or MDA5 for 24 h and subsequently stimulated with ppp-RNA or poly(I:C). Induction of apoptosis was measured by annexin V/PI staining. Silencing efficacy, as assessed by western blot, is shown; (d) activated caspase-9 (green) was visualized using green FLICA caspase-9 assay kit. Cell membranes were costained with cholera toxin B subunit (red) and nuclei with DAPI (blue); (e and f) Panc02 cells were treated as indicated for 48 h. Full length PARP-1 (116 kDa) and the cleaved large fragment of PARP-1 (89 kDa) (e) as well as the autophagy markers LC3B-I and LC3B-II (f) were analyzed by western blot. Results are representative of at least three independent experiments

Article Snippet: After fixation with paraformaldehyde and permeabilization with saponin (both Sigma), DCs were stained for IL-6 (PE, clone MP5-20F3; BioLegend) or goat anti-mouse CXCL10 (R&D Systems).

Techniques: Activation Assay, Transfection, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Incubation, Western Blot