Journal: Frontiers in Veterinary Science

Article Title: Evaluation of the immunogenicity of a Crimean-Congo hemorrhagic fever virus vaccine candidate in mice developed based on a baculovirus Zera nanoparticle delivery system

doi: 10.3389/fvets.2023.1126785

Figure Lengend Snippet: Analysis of immunogenicity in mice. Analysis of induced IgG responses in mice by indirect ELISA for the Gn recombinant protein (A) and the Np recombinant protein (B) . The y-axis represents the OD450 nm of serum samples collected at 0, 14, 28, 35 and 42 d after immunization (DAI) for each group, *** p < 0.001, with significant differences from PBS and rvAc-dual (Bonferroni test). (C) Results of lymphocyte proliferation experiments. The y-axis represents the stimulation index of splenic lymphocyte samples collected at 35 and 42 DAI. ns: no significant difference; *** p < 0.001, significant difference (Bonferroni test). Quantitative analysis of IL-4 (D) and TNF-α (E) levels in the serum of immunized mice. All analyses were performed in triplicate, and error bars show the standard deviation (SD).

Article Snippet: TNF-α and IL-4 cytokines were detected in mouse sera by a commercial ELISA kit (Mouse IFN-γ/IL-4 ELISA BASIC kit Boster), and all sera to be tested were made in three replicates.

Techniques: Immunopeptidomics, Indirect ELISA, Recombinant, Standard Deviation

Journal: Oncotarget

Article Title: Stimulation of dendritic cells by DAMPs in ALA-PDT treated SCC tumor cells

doi:

Figure Lengend Snippet: Secretion of IFN-γ A. and IL-12 B. from DCs after incubation with ALA-PDT-treated PECA cells was quantified in cell culture supernatants using ELISA assay, with or without HSP70, HMGB1 or CRT neutralizing antibodies in the co-incubation medium. Unstimulated, immature DCs were used for negative control and DCs incubated with LPS were used for positive control. ALA-PDT-treated PECA cells induced the secretion of IFN-γ and IL-12 markedly higher than that induced by untreated PECA cells ( p < 0.05). When HSP70, HMGB1 and CRT were blocked individually, or in combination, IFN-γ secreted from DCs was markedly reduced ( p < 0.05). When all of three antibodies were included in the co-incubation medium, IL-12 secreted from DCs significantly decreased when compared with PDT-DCs ( p < 0.05). Statistical analysis was performed by t -test; * p < 0.05 and ** p < 0.01 with respect to the PDT-DCs (unless otherwise specified); ns = not statistically significant. Means ± SD are shown ( n = 5 from 3 independent experiments.)

Article Snippet: In addition, we used mouse HSP70 ELISA Kit (ENEO, USA), mouse HMGB1 ELISA Kit (SHINO-TEST, Japan), mouse IFN-γ and IL-12 ELISA Kit (Boster, China), rabbit anti-Mouse CD80-PE, rabbit anti-Mouse CD86-PE, rabbit anti-Mouse MHC-II-PE, and rat IgG2a K Isotype Control PE (Biolegend, USA).

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control

Journal: The EMBO Journal

Article Title: Role of PCIF1 ‐mediated 5′‐cap N6 ‐methyladeonsine mRNA methylation in colorectal cancer and anti‐PD ‐1 immunotherapy

doi: 10.15252/embj.2022111673

Figure Lengend Snippet: A Flow cytometric analysis of the percentage of MDSCs and NK cells in extracts of control and Pcif1‐depleted MC38 tumors. Each spot represents individual mice. Data are the mean ± SEM of the indicated number of mice. ** P < 0.01 by Student's t ‐test. B IHC staining of Pcif1 and Fos protein expression in control and Pcif1‐depleted CT26 tumors after anti‐PD‐1 antibody injection. n = 3 mice/group. Scale bars, 25 μm. C qRT‐PCR analysis of Tgfb1‐3 mRNA in control and Pcif1‐depleted CT26 tumors of the indicated number of mice. Data are the mean ± SEM of n = 5 mice/group. * P < 0.05, *** P < 0.001 by Student's t ‐test. D Quantification of Tgfb1‐3 secretion levels in serum extracts from BALB/c mice carrying control and Pcif1‐depleted CT26 tumors by ELISA. Data are the mean ± SEM of n = 5. Symbols represent individual mice. E Fos expression was validated in the indicated cells transfected with a Fos overexpression vector or empty vector by western blot. F, G The indicated mRNA expression was assessed using qRT‐PCR in Pcif1‐depleted and control CT26 (F) and MC38 tumors (G). Data are the mean ± SEM of the indicated number of mice. * P < 0.05, ** P < 0.01 by Student's t ‐test. H BALB/c mice bearing Pcif1‐deficient and control tumors were treated with PD‐1 antibody and IFN‐γ‐blocking antibody as indicated. Tumor volume was recorded over time. n , the numbers of mice. Data are mean ± SEM of the indicated number of mice. ** P < 0.01 by Student's t ‐tests. I The production of IFN‐γ and TNFα in tumor (left) and serum (right) extracts from BALB/c mice injected with control and Pcif1‐depleted CT26 cells via ELISA. Data are the mean ± SEM of the indicated number of mice. Each spot represents individual mice. * P < 0.05, ** P < 0.01 by Student's t ‐test.

Article Snippet: The secretions of Tgfb1, Tgfb2, Tgfb3, IFN‐γ, and TNFα were quantified using a TGF beta‐1 Human/Mouse ELISA Kit (Fisher Scientific, 88‐8350‐22), a mouse TGFB2 ELISA Kit (Aviva Systems Biology, OKEH00309), a Tgfb3 mouse ELISA Kit (Boster Biological Technology, EK1104), an IFN‐γ mouse ELISA Kit (Fisher Scientific, 88‐7314‐22), and a TNFα mouse ELISA Kit (Fisher Scientific, 88‐7324‐22), respectively, according to the manufacturers' protocols.

Techniques: Control, Immunohistochemistry, Expressing, Injection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Over Expression, Plasmid Preparation, Western Blot, Blocking Assay

Journal: The EMBO Journal

Article Title: Role of PCIF1 ‐mediated 5′‐cap N6 ‐methyladeonsine mRNA methylation in colorectal cancer and anti‐PD ‐1 immunotherapy

doi: 10.15252/embj.2022111673

Figure Lengend Snippet: qRT‐PCR analysis of Fos mRNA expression in control and Pcif1‐depleted CT26 tumors after anti‐PD‐1 antibody injection. Data are the mean ± SEM of n = 5. ** P < 0.01 by Student's t ‐test. m 6 Am‐exo‐qPCR analysis of m 6 Am enrichment in Fos mRNA in control and Pcif1‐depleted CT26 tumors. Gapdh served as a negative control. Data are the mean ± SEM of n = 3. ** P < 0.01 by Student's t ‐test. ELISA quantification of Tgfb1‐3 secretion levels in tumor extracts from BALB/c mice injected with control and Pcif1‐depleted CT26 cells. Data are the mean ± SEM of n = 5. Symbols represent individual mice. * P < 0.05, *** P < 0.001 by Student's t ‐test. Tumor growth with the indicated gene edited CT26 cells in BALB/c mice treated with anti‐PD‐1 antibody. n , mouse number. Data are the mean ± SEM of the indicated mice in each group. * P < 0.05, ** P < 0.01 by Student's t ‐test. Production of Tgfb1‐3 was measured by ELISA from the indicated tumors. Data are the mean ± SEM of n = 5. Symbols represent individual mice. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t ‐test. Volcano plot of differentially expression genes analyzed using DESeq2 in Pcif1‐depleted tumors compared with control. Significantly up or downregulated genes are plotted in red and blue points, respectively. n.s, nonsignificant. Meta‐enrichment analysis summary for the significant up and down‐regulated genes identified as Pcif1 targets in CT26 tumor with PD‐1 treatment. m 6 Am enrichment in the indicated mRNA was assessed in control and Pcif1‐depleted CT26 tumors by m 6 Am‐exo‐qPCR. Data are the mean ± SEM of n = 3. * P < 0.05 by Student's t ‐test.

Article Snippet: The secretions of Tgfb1, Tgfb2, Tgfb3, IFN‐γ, and TNFα were quantified using a TGF beta‐1 Human/Mouse ELISA Kit (Fisher Scientific, 88‐8350‐22), a mouse TGFB2 ELISA Kit (Aviva Systems Biology, OKEH00309), a Tgfb3 mouse ELISA Kit (Boster Biological Technology, EK1104), an IFN‐γ mouse ELISA Kit (Fisher Scientific, 88‐7314‐22), and a TNFα mouse ELISA Kit (Fisher Scientific, 88‐7324‐22), respectively, according to the manufacturers' protocols.

Techniques: Quantitative RT-PCR, Expressing, Control, Injection, Negative Control, Enzyme-linked Immunosorbent Assay