Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Design of the MICA/B vaccine. pAbs, polyclonal serum IgG. b, MICB-specific serum antibody (Ab) titres quantified by flow cytometry (n = 4 mice per group) in MICB-transgenic mice immunized with Ctrl-vax (blue) or MICB-vax (red). MFI, median fluorescence intensity. c, MICB-specific CD4+ T cell responses following immunization with MICB-vax or Ctrl-vax; CFSE dilution of splenocytes stimulated with MICB or control protein (ovalbumin (OVA)); shown are representative flow cytometry plots (left) and quantification for three mice per group (right). d, Cell-surface levels of MICB on B16F10 (MICB) tumours from mice immunized with MICB-vax or Ctrl-vax (n = 4 mice per group); staining of tumour cells with isotype-control monoclonal antibody (grey) or anti-MICA/B monoclonal antibody (specific for the α1–α2 domains, monoclonal antibody not blocked by vaccine-induced antibodies). e, Therapeutic efficacy of MICB-vax (red) or Ctrl-vax (blue) in mice with established B16F10 (MICB) tumours immunized at the indicated time points (n = 7 mice per group). Vax, vaccination. *P = 0.0137, ****P <0.0001; NS (not significant), P > 0.999. f, g, Vaccine efficacy in two models of spontaneous metastasis. Mice were immunized with Ctrl-vax (blue) or MICB-vax (red) following surgical removal of primary tumours using the B16-B6 melanoma (f; 10 mice per group) or 4T1 breast cancer (g; 13 mice per group) models. Shown are the size of primary tumours at the time of surgery (left), representative images of lung metastases (middle) and quantification of the total number of lung surface metastases (right). D, day; s.c., subcutaneous. h, i, Immunogenicity of the rhesus MICA/B α3 domain vaccine in the rhesus macaque model. h, Timeline of vaccination; blood was drawn 24 h before indicated immunization or boost. i, Serum titres of antibody to rhesus MICA/B for animal ID 9312. Representative data are shown from at least three (b) or two (c–g) independent experiments. Data from a single experiment with technical replicates for each time point are shown in i. Statistical significance was assessed by two-tailed unpaired Student’s t test (b), two-way ANOVA with Sidak’s multiple-comparison test (c), one-way ANOVA with Tukey’s multiple-comparison test (d), two-way ANOVA with Bonferroni’s post hoc test (left) and the log-rank (Mantel–Cox) test (right) (e), two-tailed Mann–Whitney test (f, g) and two-way ANOVA with Tukey’s multiple-comparison test (i). Data are depicted as the mean ± s.e.m. (b–g) or mean±s.d. (i).

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Flow Cytometry, Transgenic Assay, Fluorescence, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, HPLC gel filtration analysis of affinity-purified ferritin (immunogen for Ctrl-vax) and MICB-ferritin (immunogen for MICB-vax) proteins. The proteins formed nanoparticles of ~957 kDa (MICB-ferritin) and ~468 kDa (ferritin); molecular weight standards are indicated. b, SDS-PAGE analysis of purified ferritin and MICB-ferritin proteins under reducing conditions. c, Electron microscopy image showing MICB-ferritin protein assembled into nanoparticles (98,000x magnification). d, Identification of MICB transgenic (Tg) mice by PCR amplification using genomic DNA extracted from tail biopsies (lanes 2,6, MICB Tg; lane 7, positive control amplification from plasmid with the transgene cassette; lane 8, negative control reaction). e, Quantitative RT-PCR analysis of luciferase and MICB mRNA in the prostate of WT (grey) and MICB-tg (orange) mice (n = 2 WT, n = 3 MICB-Tg mice) normalized to mouse Hprt mRNA. f, Quantification of day 14 MICB-specific serum Ab titers (fluorescence-based immunoassay, DELFIA) in WT and MICB-tg mice immunized with Ctrl-vax (n = 4 mice/group) (grey) (120μg ferritin protein, 100μg CpG, 1μg GM-CSF) or MICB-vax (n = 5 mice/group) (200μg MICB-ferritin, 100μg CpG, 1μg GM-CSF) without MSR (left) or with MSR (right) scaffold. g, Quantification of MICB-specific serum Ab titers (fluorescence-based immunoassay, DELFIA) in mice immunized with Ctrl-vax (grey) or MICB-vax using differing protein doses, 50μg (yellow), 100μg (blue) or 200μg (red) (n = 2 mice/group). h, Representative flow cytometry plots showing binding of polyclonal serum Abs from mice immunized with Ctrl-vax (blue) or MICB-vax (red) to cell surface MICB on B16F10 (MICB) tumor cells; serum dilutions are indicated for each condition. i, Titers of MICB Ab isotypes assessed by ELISA (n = 5 mice/group) in MICB transgenic mice immunized with Ctrl-vax (blue) or MICB-vax (red). j, Analysis of MICB-specific CD8 T-cell responses in the spleen of mice immunized with Ctrl-vax (blue) or MICB-vax (red). Intracellular cytokine staining (IFNγ) and CFSE dilution is shown in representative flow cytometry plots (left); data are quantified for T-cells from both vaccine groups (3 mice/group, right). k, Analysis of human NKG2D dimer (left) and mouse NKG2D dimer (right) binding to cell surface MICB on B16F10 (MICB-ZsGreen) tumor cells pre-incubated with sera (5μl) from Ctrl-vax or MICB-vax mice. l, Representative flow cytometry plots showing binding of anti-human MICA/B antibody (6D4, specific for MICA/B α1-α2 domains) to cell surface MICB on B16F10 (MICB) tumor cells pre-incubated with sera from mice immunized with Ctrl-vax (blue) or MICB-vax (red). Representative data from >3 independent experiments (a, b). Data from a single experiment with technical replicates (c). Representative data from three experiments (d, e, g, h, i). Representative data from two experiments (j, k, l). Two-tailed unpaired Student’s t-test (e); Two-way ANOVA with Tukey’s multiple comparison test (f, g); Two-way ANOVA with Sidak’s multiple comparison test (i, j). Data depict mean +/− SD (e,g) or mean +/− SEM (f, i,j).

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Filtration, Affinity Purification, Molecular Weight, SDS Page, Purification, Electron Microscopy, Transgenic Assay, Amplification, Positive Control, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Luciferase, Fluorescence, Flow Cytometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Incubation, Two Tailed Test

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, b, Inhibition of MICA/B shedding and surface stabilization by vaccine-induced Abs. Flow cytometric analysis of cell surface MICA/B levels (a) and shed MICA/B in supernatants (b) for human A375 melanoma, A549 lung carcinoma, HCT116 colon carcinoma and K562 myelogenous leukemia cell lines 24 h following incubation with 10 μl of sera from Ctrl-vax (blue) or MICB-vax (red) immunized mice; isotype control Ab staining shown in grey (a). c, d, Inhibition of MICB shedding and surface stabilization by vaccine-induced Abs on mouse tumor cell lines. Flow cytometric analysis of cell surface MICB levels (c) and shed MICB in supernatants (d) for mouse B16F10 (MICB) melanoma, EL4 (MICB) lymphoma and 4T1 (MICB) triple negative breast cancer cell lines 24 h following incubation with 10 μl of sera from Ctrl-vax (blue) or MICB-vax (red) immunized mice. Isotype control Ab staining shown in grey (c). e–g. Vaccine efficacy at different MICB expression levels induced in tumor cells using a doxycycline (dox) inducible promoter. Representative flow cytometry histograms showing MICB surface expression levels on B16F10 (MICB-dox) tumors in vivo in mice treated with PBS (blue histogram) or different concentrations of doxycycline (dox): low dox (2.5mg/kg, orange), medium dox (5mg/kg, red) or high dox (10mg/kg, green) or control B16F10 (Ctrl-dox) tumors treated with high dox (10mg/kg, grey) (e). Analysis of B16F10 (MICB-dox) tumor growth kinetics at different MICB expression levels by tumor cells. Mice received Ctrl-vax (grey, blue, black) or MICB-vax (orange, red, green) on day 0 and a boost on day 14. B16F10 (MICB-dox) tumor cells were implanted on day 21, and MICB expression was induced on tumor cells on day 25 when tumors were palpable by treating mice with different concentrations of doxycycline as indicated (f) (n=7 mice/group). Quantification of serum levels of shed MICB in mice immunized with Ctrl-vax (grey, blue, black) or MICB-vax (orange, red, green) 96 h post dox-mediated induction of MICB on B16F10 (MICB-dox) tumor cells. Serum levels of shed MICB were analyzed in 5 randomly selected mice in each group (g). h–i, Representative flow cytometry histogram showing surface MICB levels on B16F10 (MICB) clone G12 (red) or indicated pooled clones (gradient of turquoise). Grey histogram represents isotype antibody staining of B16F10 (MICB) clone G12 (h). Assessment of therapeutic efficacy of MICB-vax (red, green) or Ctrl-vax (blue, grey) in mice with tumors established with B16F10 (MICB) clone G12 or pooled clones (B3, A3, C6, B1, G1) (n=8 mice/group) (i). j–n, Assessment of vaccine efficacy targeting MICA or MICB α3 domains. The MSR scaffold was formulated with antigens, GM-CSF and CpG. Mice received one or two doses of Ctrl-vax, MICB-vax (j–m) or MICA-vax (n) and were then challenged with B16F10 tumor cells expressing MICB (allele 005) (j, k) or MICA (allele 009) (m, n) or EL4 tumor cells expressing MICB (allele 005) (l). Vaccination and tumor challenge schedule is illustrated above each experiment; n = 7 mice/group (j–l), n = 6 mice in Ctrl-vax, n = 8 mice in MICB-vax (m) and n = 6 mice/group (n). For experiments shown in (j), tumor-free mice were rechallenged on day 120 using the same dose of B16F10 (MICB) tumor cells as in the initial inoculation. Representative data from two experiments (a–d, j–n). Data from single experiment (e–g, h–i). Two-tailed unpaired Student’s t-test (b, d); one-way ANOVA with Tukey’s multiple comparison test (g); two-way ANOVA with Bonferroni’s post hoc test (f, i (left), j (left), k. l, m, n); log-rank (Mantel-Cox) test (i (right), j (right). Data depict mean +/− SD (b, d) or mean +/− SEM (f, g, i–n).

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Inhibition, Incubation, Staining, Expressing, Flow Cytometry, In Vivo, Clone Assay, Two Tailed Test

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Strategy for characterization of tumour-infiltrating immune cells by flow cytometry (FACS) and scRNA-seq. Mice received two immunizations (days 0 and 14), B16F10 (MICB-dox) tumour cells were implanted (day 21), MICB expression was induced on tumour cells by doxycycline treatment (day 28) and tumour-infiltrating immune cells were analysed 7 d later. b–d, Tumour-infiltrating T cell populations following immunization with Ctrl-vax (blue) or MICB-vax (red) (Ctrl-vax, n = 9 mice per group; MICB-vax, n = 10 mice per group; n = 7 mice per group for the γδ T cell panel). Treg cells, regulatory T cells. e, Tumour-infiltrating NK cells (n = 8 mice per group). f, g, Quantification of IFNγ-positive CD4+ and CD8+ T cells (Ctrl-vax, n = 9 mice per group; MICB-vax, n = 10 mice per group). h, i, UMAP representation of all T cell clusters from scRNA-seq data (h) and the fraction of each T cell subpopulation among total CD45+ cells from the experimental group (MICB-vax + doxycycline; red) and three combined control groups (Ctrl-vax±doxycycline and MICB-vax without doxycycline; blue) (i). DN, double negative. j, k, UMAP representation of NK and ILC1 cells from all experimental groups (j) and the fraction of these subpopulations among total CD45+ cells from experimental (red) and combined control (blue) groups (k). l, Fraction of T cells representing expanded clones based on TCR sequence analysis for the experimental group (red) and combined control groups (blue). m, Contribution of CD4+ T cells, NK cells and NKG2D receptor to vaccine efficacy. Mice were first immunized with MICB-vax (M) or Ctrl-vax (C) (days 0 and 14) and treated with isotype-control monoclonal antibody (iso), depleting monoclonal antibody (targeting CD4+ T cells or NK cells (αCD4 and αNK1.1, respectively)) or NKG2D receptor-blocking monoclonal antibody (αNKG2D) starting on day 21, followed by implantation of B16F10 (MICB) tumour cells (n = 7 mice per group). Representative data from three independent experiments are shown in b–g. scRNA-seq data from a single experiment with sorted CD45+ cells pooled from five mice per group are shown in h–l. Representative data from two independent experiments are shown in m. Statistical significance was assessed by two-tailed Mann–Whitney test (b–g) or log-rank (Mantel–Cox) test (m). Data are depicted as the mean±s.e.m.

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Flow Cytometry, Expressing, Clone Assay, Sequencing, Blocking Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Effect of doxycycline on tumor growth in vaccinated mice. Mice received Ctrl-vax (blue, grey) or MICB-vax (green, magenta, red) on day 0 and a boost on day 14. B16F10 cells transduced with Ctrl-dox or MICB-dox lentiviral vectors as indicated were implanted on day 21, and mice were treated with doxycycline (or PBS as control) starting on day 25 to induce MICB expression on tumor cells (n=5 mice/group). b–k, Analysis of tumor-infiltrating T-cell populations. Tumor-infiltrating T-cells were analyzed 7 days following induction of MICB expression by tumor cells (n = 7 mice/group). b–c, Analysis of CD62L and CD44 expression by tumor-infiltrating CD4 (b) and CD8 (c) T-cells. d–e, Representative flow plots (left) and quantification (right) of NKG2D receptor expression by CD4 (d) and CD8 (e) T-cells. f–g, Representative flow plots and quantification of CXCR6 receptor expression by CD4 (f) and CD8 (g) T-cells. h–i, Quantification of proliferating Ki67+ CD4 (h) and CD8 (i) T-cells. j–k, Quantification of TNFα positive CD4 (j) and CD8 (k) T-cells. Two-way ANOVA with Bonferroni’s post hoc test (a); two-way ANOVA with Tukey’s multiple comparison test (b–c); two-tailed Mann Whitney test (d–k). Representative data from two independent experiments (b–i); representative data from three independent experiments (j–k). Data depict mean+/− SEM.

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Functional Assay, Transduction, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a–b, Representative histograms (a) and quantification (b) of PD-1, CTLA-4, Tim-3, Tigit and Lag3 expression by tumor-infiltrating CD8 T-cells from Ctrl-vax (blue) or MICB-vax (red) mice (n = 9 mice/group). c–d, Comparison of T-cell and NK cell populations in B16F10 (MICB) tumors following treatment with a MICA/B mAb or the MICB vaccine. In the vaccine arm, mice received Ctrl-vax (C-vax) (n = 9 mice) or MICB-vax (M-vax) (n = 10 mice) on days 0 and 14, while mice in the mAb treatment group (n = 8 mice/ group) received two buffer injections. Mice were implanted with B16F10 (MICB-dox) tumor cells on day 21. MICB expression was induced on tumor cells by doxycycline treatment starting on day 28, and mice in the mAb treatment group received either mouse IgG2a isotype control mAb (iso) or MICA/B mAb (mAb) treatment every 48 h starting on day 28. Tumor-infiltrating immune cells were analyzed in all groups on day 35. Total numbers of tumor-infiltrating CD4+ T-cells, CD8+ T-cells and NK cells were quantified by flow cytometry (c), and intracellular staining was performed for IFNγ (d) in all four treatment groups. e–g, scRNA-seq analysis of changes in tumor-infiltrating immune cells induced by the vaccine. CD45+ immune cells in B16F10 (MICB-dox) tumors were investigated by scRNA-seq under four experimental conditions: the MICB-vax (+dox) experimental group and the three control groups, Ctrl-vax (−dox), Ctrl-vax (+dox) and MICB-vax (−dox). Doxycycline was administered to mice for seven days prior to scRNA-seq analysis to induce MICB expression on tumor cells in two of these groups (+dox). For each of the four groups, CD45+ immune cells were pooled from five mice to reduce variation from individual tumors. e, UMAP projection of CD45+ immune cells combined from all experimental groups. Major immune cell populations are annotated based on differentially expressed genes. f, Comparison of immune subpopulations across all clusters for the experimental MICB-vax (+dox) group (red) versus the three combined control groups (blue). g, Distribution of CD45+ cells across individual clusters (color-coded as in e) for the experimental MICB-vax (+dox) group (MICB) and the three combined control groups (Ctrl). Representative data of two experimental repeats (a–b). Data from a single experiment (c–d). ScRNA-seq data from a single experiment with sorted CD45+ cells pooled from 5 mice/group (e–g). Two-tailed Mann Whitney test (b); one-way ANOVA with Tukey’s multiple comparison test (c–d). Data depict mean+/− SEM.

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Expressing, Flow Cytometry, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a–d, scRNA-seq analysis of T-cell clusters among CD45+ tumor-infiltrating cells in B16F10 (MICB-dox) tumors. UMAP representation of T-cell subclusters (a) and visualization of T-cell populations for the experimental MICB-vax (+dox) group (MICB) and the three control groups (b). c, Contribution of each T-cell subcluster to the total CD45+ immune population for each of the four treatment groups. d, Quantification of expanded TCR clonotypes for CD4, Treg, CD8 effector, CD8 exhausted and CD8 proliferating clusters shown for all four treatment groups. e, f, Ranking of differentially expressed genes in scRNA-seq data from the indicated T-cell subpopulations (e) and NK cells (f) comparing cells from the experimental MICB-vax (+dox) group to cells from the three combined control groups, Ctrl-vax (−dox), Ctrl-vax (+dox) and MICB-vax (−dox). g–j, Violin plots showing expression levels of activation-related genes (g) and Th1-related genes (h) in CD4 T-cells as well as activation-related genes (i) and chemokine receptor genes (j) in CD8 T-cells from the experimental MICB-vax (+dox) group compared to cells from the three combined control groups (Ctrl). ScRNA-seq data from a single experiment with sorted CD45+ cells pooled from 5 mice/group (a–j). Pairwise Wilcoxon rank sum test (g–j).

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Expressing, Activation Assay

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Contribution of CD8 T cells to vaccine efficacy. Mice were first immunized with MICB-vax or Ctrl-vax (d0, d14), treated with either isotype control mAb, depleting mAb targeting CD8 T-cells starting on day 21, followed by implantation of B16F10 (MICB) tumor cells (n = 7 mice/group). b, Impact of IFNγ versus TNFα neutralization on the efficacy of the MICB α3 domain vaccine. MICB-transgenic mice received IFNγ or TNFα neutralizing mAbs or an isotype control mAb every 48 h, starting two days prior to subcutaneous injection of B16F10 (MICB) tumor cells on day 21 following immunization (n = 7 mice/group). Tumor growth (left) and survival analysis (right) are shown. c, Comparison of vaccine efficacy against B16F10 (MICB) wild-type tumors and tumors with resistance mutation in H2-Aa gene. Mice received MICB-vax (n=8 mice/ group) or Ctrl-vax (n = 7 mice/ group) and were then challenged with tumors of the indicated genotypes. d, Quantification of MICB-specific serum Ab titers in mice immunized with Ctrl-vax (blue) or MICB-vax followed by treatment with CD4 T-cell depletion (green) or control (red) mAbs for 3 weeks, starting on day 28 following immunization (n=5 mice/group). e, Impact of CD4 T-cell depletion on vaccine-induced NK cell infiltration into tumors. Flow cytometric quantification of the percentage of Ki67+ NK cells (top) and IFNγ+ NK cells (bottom) in WT (left) and B2m-KO (right) tumors for the following treatment groups: Ctrl-vax + isotype mAb (blue), Ctrl-vax + anti-CD4 (orange), MICB-vax + isotype mAb (red) and MICB-vax+anti-CD4 (green); (n = 7 mice/group). Representative data from two independent experiments (a-c, e). Data from a single experiment with technical triplicates (d). Log-rank (Mantel-Cox) test (a); two-way ANOVA with Bonferroni’s post hoc test (b left, c left) and Log-rank (Mantel-Cox) test (b right, c right); two-way ANOVA with Tukey’s multiple comparison test (d); one-way ANOVA with Tukey’s multiple comparison test (e).

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Expressing, Neutralization, Transgenic Assay, Injection, Mutagenesis

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, b, Comparison of vaccine efficacy against B16F10 (MICB) WT tumours and tumours with resistance mutations in the B2m (a) or Ifngr1 (b) gene. Mice received MICB-vax or Ctrl-vax and were then challenged with tumours of the indicated genotype (n = 7 mice per group). KO, knockout. c, Effect of CD4+ T cell and NK cell depletion on immunity to B2m-knockout tumours. Mice were immunized with MICB-vax or Ctrl-vax; treatment with depleting or isotype-control monoclonal antibody was started 2 d before injection of B2m-knockout B16F10 (MICB) tumour cells (n = 7 mice per group). d, Contribution of vaccine-induced anti-MICB antibodies to NK cell-mediated cytotoxicity against B2m-knockout B16F10 (MICB) tumour cells. CFSE-labelled B2m-knockout B16F10 (MICB) tumour cells were pre-incubated with 10 μg per well of purified serum IgG from mice immunized with MICB-vax or Ctrl-vax before the addition of NK cells at different effector to target (E:T) ratios as indicated. The percentage of dead target cells was assessed by flow cytometry. e, Effect of CD4+ T cell depletion on vaccine-induced NK cell infiltration into tumours. Flow cytometry quantification of total NK cell numbers is shown in WT (left) and B2m-knockout (right) tumours for the following treatment groups: Ctrl-vax+isotype-control monoclonal antibody (blue), Ctrl-vax + anti-CD4 (orange), MICB-vax+isotype-control monoclonal antibody (red) and MICB-vax + anti-CD4 (green) (n = 7 mice per group). Representative data from two independent experiments are shown in a–e. Statistical significance was assessed by two-way ANOVA with Bonferroni’s post hoc test (left) and log-rank (Mantel–Cox) test (right) (a–c), two-way ANOVA with Sidak’s multiple-comparison test (d) and one-way ANOVA with Tukey’s multiple-comparison test (e). Data are depicted as the mean±s.e.m.

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Knock-Out, Injection, Incubation, Purification, Flow Cytometry

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Effect of CD4+ T cells on migratory DC populations in the tdLNs of mice immunized with MICB-vax versus Ctrl-vax. Total migratory DCs as well as cDC1 and cDC2 cells were quantified 2 d after induction of MICB expression in tumour cells by doxycycline treatment (n = 7 mice per group, except n = 6 for Ctrl-vax without anti-CD4). b, Migratory DC subsets within the tdLN of MICB-vax-immunized mice treated following immunization (days 28 + 30) with isotype-control, CD4-depleting or CD40L-blocking monoclonal antibody (n = 7 mice per group). c, Quantification of DC populations within the tumours of mice immunized with Ctrl-vax (blue) or MICB-vax (red) on day 7 following induction of MICB expression with doxycycline (n = 7 mice per group). d, Effect of cDC1 depletion on MICB vaccine-induced T cell and NK cell accumulation within tumours in Xcr1DTR mice. Mice were treated with DT or left untreated starting on day 26 following immunization with Ctrl-vax or MICB-vax (days 0 + 14) and B16F10 (MICB-dox) tumour implantation (day 21). Immune cells were analysed in tumours 7 d after induction of MICB expression on tumours with doxycycline (day 37) (n = 7 mice per group). e, Contribution of vaccine-induced anti-MICB antibodies to DC-mediated cross-presentation of tumour antigens to CD8+ T cells. Bone marrow-derived DCs (BMDCs) were pre-incubated with B2m-knockout B16F10 (MICB-OVA) tumour cells in the presence of affinity-purified serum IgG from mice immunized with Ctrl-vax or MICB-vax at the indicated concentrations. DCs were co-cultured with CFSE-labelled OT-1 CD8+ T cells with T cell proliferation as the readout. The role of activating Fc receptor (FcR) was assessed using BMDCs from Fcer1g−/− mice (orange) or pre-incubation of BMDCs with FcR-blocking antibody (yellow) before tumour cell addition. Representative data from two independent experiments are shown in a–e. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple-comparison test (a, b, d), two-tailed Mann–Whitney test (c) and two-way ANOVA with Tukey’s multiple-comparison test (e). Data are depicted as the mean ± s.e.m. (a–d) or mean ± s.d. (e).

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Expressing, Blocking Assay, Derivative Assay, Incubation, Knock-Out, Affinity Purification, Cell Culture, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Experimental outline and gating strategy for identification of migratory DC (mDC) subsets within the tumor draining lymph node (tdLN) of immunized mice by flow cytometry. b, Impact of CD4 T-cell depletion on mDC and resident (rDC) populations in tdLN of vaccinated mice. DC populations were analyzed in tdLN on day 32, two days following induction of MICB expression on tumor cells with dox (n = 7 mice per group, except in control-αCD4 ab (n = 6 mice). c, Differentially expressed genes of mDCs (bulk RNA-seq) sorted from tdLN of MICB-vax and Ctrl-vax mice treated with CD4 depleting (αCD4) or isotype control (iso) mAb. d, Upregulated genes in mDCs from the MICB-vax (isotype control mAb, iso) group compared to either MICB-vax plus CD4 T-cell depletion (αCD4) or the Ctrl-vax (isotype mAb) groups. Venn diagram and heatmap of genes with higher expression in MICB-vax (iso) compared to both other groups. e–g, Characterization of myeloid cells within the tumors of vaccinated mice. Mice were immunized (d0 and 14), B16F10 (MICB-dox) tumor cells were implanted on day 21 and MICB expression was induced on tumor cells on day 28 by doxycycline (dox) treatment. Tumor-infiltrating myeloid cells were analyzed 7 days later. e, Gating strategy used for identification of tumor-infiltrating CD103+ cDC1, CD301b+ cDC2 and F4/80+ macrophages in vaccinated mice by flow cytometry. f, Quantification of macrophage population within tumors of Ctrl-Vax (blue) and MICB-vax (red) mice on day 7 following MICB induction on tumors with dox (n = 7 mice/group). g, Impact of αCSF1R treatment on immunity to B16F10 (MICB) tumors. Mice were immunized with Ctrl-vax or MICB-vax; treatment with αCSF1R or isotype control Ab was started two days prior to subcutaneous injection of B16F10 (MICB) tumor cells; mAb treatment was continued every third day; n=7 mice/group. Data representative of two independent experiments (a–b, e–g). Data from one experiment with three biological replicates per sample. DCs from 3 mice were pooled for each biological replicate (n = 9 mice/group). Significance was determined using thresholds of −log10 >2 (adjusted P value), and log2 >1 (fold change) (c–d). One-way ANOVA with Tukey’s multiple comparison test (b); Two-tailed Mann Whitney test (f); log-rank (Mantel-Cox) test (g). Data represent mean +/− SEM.

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Flow Cytometry, Expressing, RNA Sequencing Assay, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Impact of cDC1 depletion on MICB vaccine-induced T-cell and NK accumulation within tumors in Xcr1DTR mice. Mice were treated +/− diphtheria toxin (DT) starting on day 26 following immunization with Ctrl-vax (C) or MICB-vax (M) (days 0 and 14) and B16F10 (MICB-dox) tumor implantation (day 21). Immune cells were analyzed in tumors 7 days following MICB induction on tumors with dox (day 37) (n = 7 mice/group). b, Contribution of activating Fc receptors to efficacy of MICB-vax. Survival curves of FceR1g−/− MICB-Tg versus MICB-Tg mice immunized with Ctrl-vax (blue) and MICB-vax (red) (n = 7 mice/group). c, Analysis of endogenous gp100 specific CD8 T-cell responses. CD8 T-cells were isolated from tdLN of mice immunized with MICB-vax or Ctrl-vax, labeled with the CTV cell proliferation dye and then co-cultured for 72 h with DCs pulsed with control (Ova) or gp100 peptide (10μg/ml). Intracellular cytokine staining (IFNγ) and CTV dilution are shown in representative flow cytometry plots (left); data are quantified for T-cells from both vaccine groups (3 mice/group, right). d, Proliferation of transferred CD8 T-cells specific for the gp100 melanoma antigen (from Pmel-1 transgenic mice) in tumor-draining lymph nodes of mice immunized with MICB-vax compared to Ctrl-vax. Mice were vaccinated twice (days 0 and 14) and B16F10 (MICB-dox) tumor cells were implanted subcutaneously on day 21. Doxycycline treatment was initiated on day 28 to induce MICB expression on tumor cells, one day prior to transfer of Thy1.1+ Pmel-1 CD8 T-cells (2x106 cells/mouse). Proliferation of CTV-labeled Pmel-1 T-cells was analyzed in tumor-draining LN (top) and spleen (bottom, control organ) four days following T-cell transfer. Cells were gated based on CD3, CD8 and Thy1.1 markers; shown is Thy1.1 marker of transferred Pmel-1 T-cells (Y-axis) and CTV dye dilution in proliferating T-cells (X-axis). Proliferating T-cell populations are indicated in representative flow plots (left) and quantification is shown (right) across the entire cohort of Ctrl-vax (blue) versus MICB-vax (red) mice (n = 7 mice/group). e, Control experiment for (d) with CD8 T-cells of irrelevant specificity (OT-1 T-cells, n = 4 mice/group). f, Role of XCR1+ DCs in the activation of transferred gp100-specific pmel-1 CD8 T-cells. Xcr1DTR mice were immunized with MICB-vax (days 0 and 14), and B16F10 (MICB-dox) melanoma cells were implanted on day 21. XCR1+ DCs were depleted by injection of diphtheria toxin (+DT, green) or solvent as a control (−DT, red) one day prior to induction of MICB expression by tumor cells with doxycycline. Thy1.1+ Pmel-1 CD8 T-cells were transferred and proliferation of these T-cells was analyzed in tdLN four days later by dilution of the CTV dye. Top flow cytometry plots show depletion of XCR1+ cells (Venus fluorescent reporter protein) in diphtheria toxin (+DT) treated mice (right) compared to control mice (−DT, left), six days following initiation of DT treatment. Bottom flow cytometry plots show proliferation of transferred pmel-1 CD8 T-cells based on dilution of the CTV dye (X-axis); data are quantified on the right (n=6 mice/group). g, Presentation of gp100 peptide by migratory DCs from MICB-vax mice. Naïve Pmel-1 CD8 T-cells were co-cultured for 72 h with migratory DCs (mDC, CD11c+, IA/E high) isolated from tdLN or non-tumor draining LN of Ctrl-vax or MICB-vax mice (pooled from 10 mice/group) implanted with B16F10 (MICB) tumor cells. CD8 T-cell proliferation was assessed based on CTV dilution. Data are representative of two independent experiments (a-d, f-g). Data from a single experiment (e). One-way ANOVA with Tukey’s multiple comparison test (a); log rank (Mantel-Cox) test (b); two-tailed Mann Whitney test (c-f); one-way ANOVA with Dunnett’s multiple comparison test (g). Data depict mean +/− SEM (a, c–f) or mean +/− SD (g).

Article Snippet: The doxycycline-inducible B16F10 (MICB-dox) cell line was generated using Lenti-X Tet-ON 3G inducible expression vectors (631363, Clontech).

Techniques: Tumor Implantation, Isolation, Labeling, Cell Culture, Staining, Flow Cytometry, Transgenic Assay, Expressing, Marker, Activation Assay, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: MCPIP1 overexpression inhibits de novo KSHV infection. (A) Schematic of KSHV infection timeline following MCPIP1 overexpression. 293T-GFP and 293T-GFP-MCPIP1 cells were stimulated with 10 ng/ml doxycycline (DOX) or DMSO (unstimulated control) for 24 h prior to KSHV infection (before infection), 6h post infection (after infection), or both prior to and following infection (before and after infection). At 96h post KSHV infection, MCPIP1 expression (C and D), viral genome copies (E), and viral gene expression (F and G) were analysed. (B) 293T-GFP and 293T-GFP-MCPIP1 cells were exposed to 10 ng/ml DOX for 0, 24h, and 48h. The levels of MCPIP1 transcripts were analysed by qPCR. Results are shown relative to GAPDH and normalized to DMSO treated control. (C) Levels of MCPIP1 transcripts was analysed by qPCR. Results are shown relative to GAPDH and normalized to unstimulated control. (D and G) MCPIP1 was induced before and after infection followed by KSHV infection in 293T-GFP-MCPIP1 cells. (D) Levels of MCPIP1 protein were analysed. β-actin (ACT) was used as a loading control. (E) Viral genome copies were measured by qPCR using primers specific to the KSHV ORF73(LANA) gene. Results are shown relative to BRCA1 and normalized to unstimulated control (F) Levels of ORF73(LANA), ORF50(RTA), ORF6, ORF60, ORF34 and K8.1 transcripts were analysed by qPCR. Results are shown relative to GAPDH and normalized to unstimulated control (G) Level of LANA protein was measured by immunoblot in nuclear and cytoplasm extracts from cells. Nuclear and cytoplasmic fractions were determined by Lamin B (nuclear marker) and HSP90 (cytoplasmic marker). All results are represented standard error of the mean from independent experiments, N = 3–4 and statistical analysis was done with a Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Over Expression, Infection, Expressing, Western Blot, Marker

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: MCPIP1 knockdown enhances viral genome accumulation. (A) Schematic of timeline of KSHV infection in HUVEC following MCPIP1 knockdown. HUVECs were stimulated with IL-1β for 2 h following transfection with negative control Locked Nucleic Acid (LNA) gapmer or LNA gapmers targeting MCPIP1 for repression, subsequently the cells were infected by KSHV. At 6 h post infection, IL-1β was treated until samples were collected. At 72 h post infection, cells were harvested. (B) At 26 h post-transfection, efficiency of MCPIP1 knockdown in HUVECs was determined by qPCR. Results are shown relative to GAPDH and normalized to negative gapmer control (C) At 98 h post transfection, MCPIP1 expression was analysed by qPCR. Results are shown relative to GAPDH and normalized to negative gapmer control (D) Viral genome copies were measured by qPCR using primers specific to the ORF73(LANA). Results are shown relative to BRCA1 and normalized to negative gapmer control. All results are represented standard error of the mean from independent experiments, N = 3 and statistical analysis was done with a Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Infection, Transfection, Negative Control, Expressing

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Determining the correlation of MCPIP1 cleavage efficiency and binding affinity. (A) Cleavage efficiency was evaluated by in vitro cleavage assay. Fluorescently labelled pre-miRNAs were incubated with recombinant MCPIP1, before separation on a denaturing urea gel. The percent of full-length pre-miRNAs remaining was quantified in the accompanying graph. Size markers (left) as well as full length and cleaved RNA products (right) are indicated. (B and C) Binding affinity was determined by electrophoretic mobility shift assay (EMSA). (B) Fluorescently labelled pre-miRNAs at 12 nM were incubated with recombinant MCPIP1 D141N (RNase dead mutant) at 0, 100, 200, 400, 600 and 800 nM (lanes 1- 6, respectively), before separation on a 0.8% agarose gel. The unbound RNA and MCPIP1 D141N bound RNA complexes are marked on the left. (C) The fraction of bound RNA was plotted against MCPIP1 concentration to calculate the equilibrium dissociation constant, Kd. (D) The relation between Kd and the percent of full-length pre-miRNAs were plotted. Correlation was analysed using Pearson's correlation coefficient (r) and linear regression line is overlaid. A P-value of <0.05 was considered significant. Each point represents results obtained from each pre-miRNA. All results are represented as means ± SD of data from independent experiments, N = 3.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Binding Assay, In Vitro, Cleavage Assay, Incubation, Recombinant, Electrophoretic Mobility Shift Assay, Mutagenesis, Agarose Gel Electrophoresis, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Comparison of binding properties and cleavage efficiency of wild type and M5 base motif mutant pre-miRNAs with MCPIP1. (A) Predicted secondary structure of kshv-mir-K12–3 WT and its M5 base motif mutants (left) and hsa-let-7c WT and its M5 base motif mutant (right) based on mfold structure predictions. M5 base motif is highlighted. Cleavage efficiency was evaluated by in vitro cleavage assay. Fluorescently labelled pre-miRNAs were incubated with recombinant MCPIP1, before separation on a denaturing urea gel. The percent of full-length pre-miRNAs remaining was quantified in the accompanying graph. Size markers as well as full length and cleaved RNA products are indicated. WT, wild type and mut, mutant. (B) Binding affinity was determined by EMSA. Fluorescently labelled kshv-mir-K12–3 WT and its M5 base motif mutants (up) or fluorescently labelled hsa-let-7c WT and its M5 base motif mutant (low) were incubated with recombinant MCPIP1 D141N at 0, 100, 200, 400, 600 and 800nM (lanes 1–6) or at 0, 25, 50, 100, 200, 400, 600 and 800nM (lanes 1–8) respectively, before separation on a 0.8% agarose gel. The unbound RNA and MCPIP1 D141N bound RNA complexes are marked on the left. The fraction of bound RNA was plotted against MCPIP1 concentration in nM to calculate the equilibrium dissociation constant, Kd. (C) The relation between Kd and the percent of full-length pre-miRNAs were plotted. Correlation was analysed using Pearson's correlation coefficient (r) and linear regression line is overlaid. A P-value of < 0.05 was considered significant. Each point represents results obtained from each pre-miRNA. All results are represented as means ± SD of data from independent experiments, N = 3–4.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Binding Assay, Mutagenesis, In Vitro, Cleavage Assay, Incubation, Recombinant, Agarose Gel Electrophoresis, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Comparison of cleavage efficiency of wild type and M5 base motif introduced mutant kshv-mir-K12–6 with MCPIP1. (A) Predicted secondary structure of kshv-mir-K12–6 WT and its M5 base motif introduced mutant based on mfold structure predictions. (B) Cleavage efficiency was evaluated by in vitro cleavage assay. Fluorescently labelled kshv-mir-K12–6 and its mutant were incubated with recombinant WT MCPIP1 or MCPIP1 D141N, before electrophoresis on a denaturing urea gel. Size markers as well as full length and cleaved RNA products are indicated. (C) Each replicate of full-length pre-miRNAs remaining following MCPIP1 treatment was quantified in the graph, corresponding to lanes 2 and 5 in panel (B).

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Mutagenesis, In Vitro, Cleavage Assay, Incubation, Recombinant, Electrophoresis

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Cleavage efficiency of Dicer1 3′ UTR regions was evaluated by in vitro cleavage assays. Fluorescently labelled Dicer1 3′ UTR fragment RNAs were incubated with recombinant MCPIP1 or MCPIP1 D141N, before electrophoresis on a denaturing urea gel. The percent of full-length RNAs remaining for Dicer1 3′ UTR region1 and its YRY mutant (up) and Dicer1 3′ UTR region2 and its YRY mutant were quantified in the accompanying graph. Size markers(left) as well as full length and cleaved RNA products are indicated. All results are represented as means ± SD of data from independent experiments, N = 3.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: In Vitro, Incubation, Recombinant, Electrophoresis, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Analysis of terminal loop sequences of KSHV pre-miRNAs. (A) Loop sequences of KSHV pre-miRNAs for the negative set (less degraded by MCPIP1) and the positive set (more degraded by MCPIP1) that used in MEME program for motif discovery. (B) M5 base motif was found by the MEME program within the terminal loops of KSHV pre-miRNAs that were well degraded by MCPIP1.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques:

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: Examination of Dicer expression following MCPIP1 overexpression. 293T-GFP and 293T-GFP-MCPIP1 cells were exposed to 10 ng/ml DOX for 0, 24 and 48 h (A) or for 48 h (B). (A) The levels of Dicer1 transcripts were measured by qPCR. Results are shown relative to GAPDH and normalized to DMSO treated control. (B) Levels of Dicer and GFP-MCPIP1 protein were examined by immunoblot. Results are shown relative to actin and normalized to unstimulated control. All results are represented as means ± SD of data from independent experiments, N = 3 and statistical analysis was done with a Student's t test. *P < 0.05.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Expressing, Over Expression, Western Blot

Journal: Nucleic Acids Research

Article Title: Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

doi: 10.1093/nar/gkab215

Figure Lengend Snippet: A schematic model depicting the MCPIP1 mediated inhibition of KSHV infection. Upon KSHV infection, positive miRNA biogenesis factors, Dicer expression levels are enhanced while the expression of the negative miRNA biogenesis factor, MCPIP1, is suppressed to provide favourable environment for viral miRNA biogenesis. However, the human host induces the expression of inflammatory cytokines upon infection, which upregulates MCPIP1 expression. MCPIP1 expression inhibits KSHV miRNA biogenesis by directly degrading KSHV miRNAs and by suppressing the expression of a positive miRNA biogenesis factor, Dicer. Furthermore, MCPIP1 inhibits KSHV infection by suppressing the expression of Dicer that positively regulates KSHV infection.

Article Snippet: Doxycycline (DOX)-inducible GFP-MCPIP1 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% tetracycline-free fetal bovine serum (FBS) and a penicillin and streptomycin and glutamine solution (Life Technologies) and maintained with 1 μg/ml puromycin and 200 μg/ml G418 as described previously ( 18 ).

Techniques: Inhibition, Infection, Expressing

Journal: eLife

Article Title: Overcoming the cytoplasmic retention of GDOWN1 modulates global transcription and facilitates stress adaptation

doi: 10.7554/eLife.79116

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , Doxycycline (Dox) , Biogems, California, USA , Cat# 2431450 , .

Techniques: Recombinant, Plasmid Preparation, Bimolecular Fluorescence Complementation Assay, Expressing, CRISPR, Generated, Marker, Purification, Imaging, Software, Microscopy, Methylation, Transfection