Journal: Nature cell biology

Article Title: Nucleated transcriptional condensates amplify gene expression.

doi: 10.1038/s41556-020-00578-6

Figure Lengend Snippet: Fig. 2 | Transcriptional condensates colocalize with and recruit additional CTD. a, THZ1 covalently binds to and inhibits the activity of CDK7, preventing phosphorylation of the CTD of Pol II and inhibiting transcription initiation. The net charge per residue (in a 5 amino-acid window) of CTD with 52 repeats of the heptad consensus amino acid sequence YSPTSPS. b, Fluorescence images of live cells expressing optoTAF15 and eGFP–CTD after blue-light illumination. Time since the start of illumination is shown with 0 s being just before illumination. c, Fluorescence intensities of cells in b, showing that optoTAF15 condensates and CTD within the condensates increase with time after blue-light illumination. Data are mean ± s.d. (n = 10 cells). AU, arbitrary units. d, Fluorescence images of cells expressing optoIDRs and eGFP–CTD after blue-light illumination. Cell nucleus is outlined by dotted line. Arrowheads point to optoIDR condensates colocalized with recruited CTD. e, CTD partition coefficients in various optoIDR condensates. OptoTAF15 condensates exhibit a nearly fourfold preferential partition of CTD, which is higher than other optoIDRs. The lines indicate the mean and error bars show s.d. derived from the number of cells as indicated in the panel. The dashed line represents a CTD partition coefficient of 1 (that is, no preferential partitioning). P values by t-test. f, OptoTAF15(10R>Q/10D>S) has a higher saturation concentration (C*) than optoTAF15 in nucleoplasm (Nuc). Inhibition of CTD phosphorylation with THZ1 leads to a decrease in the nucleoplasmic saturation concentration of optoTAF15 and optoTAF15(10R>Q/10D>S), but no significant (P > 0.05) change in the cytoplasm (Cyto). Data are mean ± s.d. derived from the number of cells as indicated in the panel. P values from pairwise one-sided Fisher’s exact tests in e,f. g, The ratio of cytoplasmic to nucleoplasmic saturation concentration correlates with nuclear CTD partitioning into optoIDR condensates. Data in e–g, are mean ± s.d. (sample sizes as shown in e and Extended Data Fig. 2a). The dashed line represents the correlation curve from a second-order polynomial fit, with R2 value shown. The number of cells (n) analysed in each panel was pooled from three independent experiments. Statistical source data are provided in Source Data Fig. 2.

Article Snippet: For CDK7 inhibition experiments, THZ1 (Med Chem Express) was dissolved in DMSO at 1 mM and further diluted with growth medium to a final concentration of 1 μM before being applied to NIH 3T3 cells followed by a 1.5 h incubation before imaging.

Techniques: Activity Assay, Phospho-proteomics, Residue, Sequencing, Fluorescence, Expressing, Derivative Assay, Concentration Assay, Inhibition

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Novel covalent CDK7 inhibitor potently induces apoptosis in acute myeloid leukemia and synergizes with Venetoclax.

doi: 10.1186/s13046-023-02750-w

Figure Lengend Snippet: Fig. 1 Efficacy of XL102 and its effects on downstream targets of CDK7. A Kinase activity of CDK7 in the presence of XL102 was measured using the LANCE TR-FRET in vitro kinase assay by incubating both CDK7 and XL102 followed by addition of ATP and U-light-MBP peptide substrate. Time-resolved fluorescence (excitation, 320 nm; emission donor, 615 nm; emission acceptor, 665 nm) was monitored by using 2030 multilabel reader Victor5 (PerkinElmer). The IC50 values were derived by fitting a sigmoidal dose–response curve to a plot of assay readout over inhibitor concentration, computed with the Graph Pad Prism. B The pull-down assay using bio-THZ1 show a dose dependent target engagement in AML cells harvested after 3 h of XL102 treatment followed by washing to remove any unbound XL102 (time point-0 h). C XL102 inhibited CTD phosphorylation of conserved residues of RPII in AML cells in dose dependent manner at 6 h and 24 h along with quantification of Western blot data. Results are the mean ± SD of three independent experiments

Article Snippet: Antibodies used against various proteins were CDK7 (Bethyl Laboratories, Massachusetts, US) and Beta actin (Santa Cruz Biotechnology, Texas, US).

Techniques: Activity Assay, In Vitro, Kinase Assay, Fluorescence, Derivative Assay, Concentration Assay, Pull Down Assay, Drug discovery, Phospho-proteomics, Western Blot

Journal: Molecular carcinogenesis

Article Title: EEF1E1 promotes glioma proliferation by regulating cell cycle through PTEN/AKT signaling pathway.

doi: 10.1002/mc.23611

Figure Lengend Snippet: FIGURE 3 EEF1E1 interacts with the downstream PTEN/AKT pathway. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology analysis of EEF1E1‐related genes obtained from the STRING database reveals a relationship between EEF1E1 and the cell cycle. (B) Single gene expression heatmaps suggest a relationship between EEF1E1 and AKT3, PTEN, p53, CDK2, CDK4, and CDK7. (C, D) Pathway correlation analysis using theThe Cancer Genome Atlas database demonstrates that EEF1E1 is correlated with the phosphatidylinositol‐3‐kinase (PI3K)/AKT signaling pathway and the cell cycle pathway (p < 0.01). (E) Western blot analysis verification of cell cycle‐related proteins. (F) Immunofluorescence double staining confirms the colocalization of EEF1E1 and PTEN in cells. (G) Colocalization of EEF1E1 and PTEN in glioma tissue using immunofluorescence double staining. (H) Co‐ immunoprecipitation (IP) experiment verifies the protein interaction between EEF1E1 and PTEN. CDK, cyclin‐dependent kinase; DAPI, 4′,6‐diamidino‐2‐ phenylindole; TPM, transcripts per million.

Article Snippet: Proteins were then separated by 10% sodium dodecyl‐sulfate polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and blocked for 1 h. Membranes were incubated with primary antibodies EEF1E1 (Abcam), PTEN, CDK2, CDK4 (Servicebio), CDK7 (Protein), AKT, P‐AKT (Cell Signaling), and P‐PTEN (Abclonal), and β‐Tubulin (Protein) was selected as the internal control at 4°C overnight.

Techniques: Gene Expression, Western Blot, Immunofluorescence, Double Staining, Immunoprecipitation

Journal: Molecular carcinogenesis

Article Title: EEF1E1 promotes glioma proliferation by regulating cell cycle through PTEN/AKT signaling pathway.

doi: 10.1002/mc.23611

Figure Lengend Snippet: FIGURE 6 EEF1E1 promotes the expression of cell cycle proteins cyclin‐dependent kinases (CDK2, CDK4, and CDK7) through interaction with the PTEN/AKT pathway, thereby affecting the G1 and S phases of the cell cycle and promoting glioma cell proliferation. PI3K, phosphatidylinositol‐3‐kinase.

Article Snippet: Proteins were then separated by 10% sodium dodecyl‐sulfate polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and blocked for 1 h. Membranes were incubated with primary antibodies EEF1E1 (Abcam), PTEN, CDK2, CDK4 (Servicebio), CDK7 (Protein), AKT, P‐AKT (Cell Signaling), and P‐PTEN (Abclonal), and β‐Tubulin (Protein) was selected as the internal control at 4°C overnight.

Techniques: Expressing

Journal: Molecular Cancer Therapeutics

Article Title: Triptolide Induces Cell Killing in Multidrug-Resistant Tumor Cells via CDK7/RPB1 Rather than XPB or p44

doi: 10.1158/1535-7163.mct-15-0753

Figure Lengend Snippet: Figure 3. Triptolide induces RPB1 degradation in both parental and MDR cell lines and RPB1 phosphorylation at Ser1878. A, MES/SA, MES-SA/MX5, KB, and KB/VCR cells were treated as shown in Materials and Methods and immunoblotted for RPB1. B, SK-OV-3, KB, and KB/VCR cells were pretreated with a CDK7- specific inhibitor and then assayed for RPB1. C, SK-OV-3 cells were treated as indicated and immunoblotted for phosphorylated RPB1 at Ser5 and phosphorylated CDK7 at Thr170. D, KB and KB/VCR cells were treated as indicated and immunoblotted for phosphorylated RPB1 at Ser5 and phosphorylated CDK7 at Thr170. E, cells were cultured as depicted in Materials and Methods, and IC50 values were measured using a Cell Counting Kit-8 (CCK-8) assay. F, KB and KB/VCR cells were cultured as indicated and immunoblotted for RPB1. G, SK-OV-3 cells were treated as indicated and immunoprecipitated RPB1 and separated with SDS-PAGE. Gel bands were excised, digested, and assessed using LC/MS-MS to identify the phosphorylation site of RPB1. m/z, mass-to-charge ratio.

Article Snippet: All antibodies were commercially available: RPB1 [carboxy-terminal domain (CTD) repeat], p-S5-RPB1, XPB, OCT-4, SOX-2, NANOG, ubiquitin, and p-Thr170-CDK7were fromAbcam,GAPDHand IgGwere from Beyotime Institute of Biotechnology (Haimen, China), CDK7 and p44 were from Santa Cruz Biotechnology, and c-MYC was from BD Biosciences.

Techniques: Phospho-proteomics, Cell Culture, Cell Counting, CCK-8 Assay, Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 1 The protective efficacy of the Ad-AURKA/CDK7 vaccine on various tumors in the preventive model. (A) Schematic diagram of subcutaneous tumors inoculation (Renca, RM-1, MC38 or Hepa1-6) after immunization with Ad-Ctrl or Ad-AURKA/ CDK7 vaccine (n=5 mice per group). (B, F, J and N) Average tumor volumes of each group in the Renca, RM-1, MC38, or Hepa1-6 subcutaneous tumors were measured twice a week. The tumor volume was statistically analyzed 35 days after tumor inoculation. (C, G, K and O) The tumor volume of an individual mouse in each group of Renca, RM-1, MC38, or Hepa1-6 subcutaneous tumors was plotted. (D, H, L and P) Tumor weights were measured at the end of the experiment. (E, I, M and Q) Tumor inhibition rate in the Renca, RM-1, MC38, or Hepa1-6 subcutaneous tumors was, respectively, calculated by (D, H, L and P). The two-tailed independent Student’s t-test was used to analyze two-group comparisons. The data showed as means±SD. The statistical significance levels were set as *p<0.05, **p<0.01 and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; i.m, intramuscular; s.c, subcutaneous.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Inhibition, Two Tailed Test

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 2 Antitumor efficacy of Ad-AURKA/CDK7 immunization in the Renca subcutaneous tumor model. (A) Schematic diagram showed the overall design of Renca therapeutic subcutaneous tumor (n=5 mice per group). (B) Tumor growth volume of each group after Renca tumor inoculation was measured twice a week. The tumor volume was statistically analyzed 35 days after tumor inoculation. (C) The final tumor volume of different treatment groups on day 35 after Renca tumor implantation. (D) Tumor weights were measured at the end of the experiment. (E) The tumor inhibition rate in (D) is shown. (F, G) The percentages of CD3+ T cells, CD4+ T cells, CD8+ T cells, DCs, NK, Mφ, MDSC, or regulatory T cells (Treg) were from spleens and tumors in each group. One-way analysis of variance was used for comparisons among multiple groups. Data are means±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001 and not significant (ns). Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin- dependent kinase 7; DCs, dendritic cells; i.m, intramuscular; MDSC, myeloid-derived suppressor cells; NK, natural killer; s.c, subcutaneous; TIL, tumor-infiltrating leukocyte.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Tumor Implantation, Inhibition, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 3 The maturation and differentiation of DC subgroups induced by Ad-AURKA/CDK7 vaccine in vivo. Different DC subgroups in spleens were analyzed by flow cytometry at the end of the experiments. (A) The percentages of CD8+CD11c+ DC subsets in immunized mice of each group (n=5), a typical flow cytometry data is displayed from each group. (B, C) Statistical analysis of the ratio of CD11c+ DCs or CD8+CD11c+ DCs. (D) The representative flow cytometry data of the percentage of CD80+CD11c+, CD86+CD11c+, MHC-II+CD11c+, or CD40+CD11c+ DC subsets in spleens of each group. (E–H) Statistical analysis of different DC subgroups in (D). One-way analysis of variance was used for comparisons among multiple groups. Data are means±SD. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin- dependent kinase 7; DCs, dendritic cells.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: In Vivo, Flow Cytometry

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 4 Ad-AURKA/CDK7 treatment induced memory CD8+ T-cell immune response. (A, B) The proliferation capability of antigen-specific CD8+ T cells from mice spleens in each group was detected by the EdU assay after the continuous stimulation with AURKA/CDK7 antigens. (C, D) The number of IFN-γ-secreting T lymphocytes was observed using the ELISpot assay. (E, F) The representative flow cytometry data of CD8+ T cells secreting IFN-γ, TNF-α, or IL-2 in each group after antigen stimulation. (G, H) The co-culture experiment was detected to assess CTL-specific killing ability. (I, J) The proportions of effector memory T cells or central memory T cells in spleens were measured, and typical flow cytometry data was selected from each group. (K) The tumor volume of a single mouse in the Renca subcutaneous tumors was measured twice a week after tumor rechallenge (n=5 mice per group). (L) The survival curve of the Ad-AURKA/CDK7 or Ad-Ctrl group was observed after re-implantation of the Renca tumor (n=10 mice per group). One-way analysis of variance was used for comparisons among multiple groups. Survival analysis was performed using the log-rank (Mantel-Cox) test. Data are means±SD. **p<0.01, ***p<0.001, ***p<0.001 and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; CTL, cytotoxic T lymphocytes; ELISpot, enzyme-linked immunosorbent spot; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: EdU Assay, Enzyme-linked Immunospot, Flow Cytometry, Co-Culture Assay, ELISpot Assay

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 5 Multifunctional CD8+ T cells exerted an indispensable role in the antitumor effects of Ad-AURKA/CDK7 vaccine. (A–D) The percentages of multifunctional CD8+ T cells secreting TNF-α+IFN-γ+, TNF-α+IL-2+, IFN-γ+IL-2+, or TNF-α+IFN-γ+IL-2+ in spleens of each group were detected by flow cytometry after continuous stimulation with AURKA/CDK7 antigens for 96 hours. (E–H) The proportions of tumor-infiltrating multifunctional CD8+ T lymphocytes secreting TNF-α+IFN-γ+, TNF-α+IL-2+, IFN- γ+IL-2+ or TNF-α+IFN-γ+IL-2+ in tumor tissues of each group were analyzed on day 35 after Renca tumor inoculation. (I) The tumor weights of mice in each group were measured at the end of CD8 depletion. (J) Tumor inhibition rates in (I). (K, L) The percentages of CD8+ T cells or CD8+CD11c+ DCs were detected in spleens and tumor tissues of each group in the CD8+ T-cell depletion assay. The two-tailed independent Student’s t-test was used to analyze two-group comparisons. One-way analysis of variance was used for comparisons among multiple groups. The data are shown as means±SD, with n=5 mice per group. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; IFN, interferon; IL, interleukin; TIL, tumor-infiltrating leukocytes; TNF, tumor necrosis factor.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Flow Cytometry, Inhibition, Depletion Assay, Two Tailed Test

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 6 Ad-AURKA/CDK7 treatment suppressed tumor metastases by activating multifunctional CD8+ T cells. (A) A schematic diagram displayed the design and treatment of lung metastasis. (B) The representative image of lung metastasis nodules in each group. (C) The survival curve of mice in different groups after Renca tumor lung metastasis (n=10 mice per group). (D) The number of metastatic nodules was counted on the lung surface of immunized mice (n=5 mice per group). (E) Typical immunohistochemistry image of the lung-infiltrating CD8+ T cells in each group immunized with different vaccines. The scale was 200 µm. (F, G) The proportion of CD8+ T cells and CD8+CD11c+ DCs in the lungs of treated mice in each group. (H, I) Percentages of multifunctional CD8+ T lymphocytes producing IFN-γ, TNF-α, or IL-2 in spleens and tumor tissues of different groups. (J) The EdU assay was used to detect the proliferation of CD8+ T cells. (K) The number of IFN-γ-secreting T lymphocytes was counted using ELISpot assay. (L) The percentages of tumor-specific killing capability of CTL in each group. One-way analysis of variance was used for comparisons among multiple groups. Survival analysis was performed using the log-rank (Mantel-Cox) test. The data expressed as means±SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; CTL, cytotoxic T lymphocyte; DC, dendritic cell; ELISpot, enzyme- linked immunosorbent spot; IFN, interferon; IL, interleukin; i.m, intramuscular; i.v, intravenous; TNF, tumor necrosis factor.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Immunohistochemistry, Vaccines, EdU Assay, Enzyme-linked Immunospot, ELISpot Assay

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 7 Antitumor efficacy of Ad-AURKA/CDK7 vaccine requires for multi-functional CD8+ T cells in the Renca orthotopic model. (A) Schematic diagram illustrating the design of the Renca orthotopic model and vaccination. (B) Typical image of renal orthotopic tumors in the different vaccine-treated groups at the endpoint of the experiment (n=5 mice per group). (C) The survival curve of each group in the orthotopic model (n=10 mice per group). (D) Tumor weights were calculated by the formula: Tumor mass (g)=left kidney mass − right kidney mass. (E) Tumor inhibition rate in (D). (F) Tumor-infiltrating CD8+ T lymphocytes were captured by immunohistochemical staining in the left kidney and the representative images were shown. The scale was 200 µm. (G, H) Statistical analysis of the proportion of CD8+ T cells or CD8+CD11c+ DCs in renal tumor tissues of each group. (I, J) The percentages of multifunctional CD8+ T lymphocytes secreting IFN-γ+, TNF-α+, IL-2+, TNF-α+IFN-γ+, TNF-α+IL-2+, IFN- γ+IL-2+, or TNF-α+IFN-γ+IL-2+ were detected by flow cytometry in spleens and tumors in each group. (K) The antigen-stimulated proliferation of CD8+ T cells in different groups. (L) The tumor-specific killing capability of CTL was detected by the co-culture experiment. One-way analysis of variance was used for comparisons among multiple groups. Survival analysis was performed using the log-rank (Mantel-Cox) test. The data are shown as means±SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; CTL, cytotoxic T lymphocyte; DC, dendritic cell; IFN, interferon; IL, interleukin; i.m, intramuscular; s.c, subcutaneous; TNF, tumor necrosis factor.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Functional Assay, Inhibition, Immunohistochemical staining, Staining, Flow Cytometry, Co-Culture Assay

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 8 The therapeutic effect induced by Ad-AURKA/CDK7 vaccine in the humanized mice model. (A) Schematic diagram explaining the establishment of the humanized mice model and experiment design. (B) Final tumor volumes were measured in each group on day 35 after OSRC-2 tumor inoculation. (C) Tumor weights of each group at the end of the experiment. (D) Tumor inhibition rate in (C). (E) The representative flow cytometry data of CD8+CD11c+, CD103+CD11c+, CD80+CD11c+, CD86+CD11c+, and HLA-A2+CD11c+ DC subgroups in spleens of each group. (F–J) Statistical analysis of DC subsets in (E) in the spleens of humanized mice. (K–O) The proportions of tumor-infiltrating DC subsets in different groups were detected. (P) The typical flow cytometry image of multi-functional CD8+ T lymphocytes secreting IFN-γ+, TNF-α+, or IL-2+ in spleens in the Ad-Ctrl or Ad-hAURKA/CDK7 treatment group. (Q, S) The percentages of multifunctional CD8+ T cells in (P) in spleens. (R, T) Statistical analysis of the proportion of IFN-γ+CD8+, TNF-α+CD8+, IL-2+CD8+, TNF-α+IFN-γ+CD8+, TNF-α+IL-2+CD8+, IFN-γ+IL-2+CD8+, or TNF-α+IFN-γ+IL-2+CD8+ in antigen-specific CD8+ T lymphocytes of tumor tissues per group. The two-tailed independent Student’s t-test was used to analyze two-group comparisons. One-way analysis of variance was used for comparisons among multiple groups. The data are shown as means±SD, with n=5 mice per group. **p<0.01, ***p<0.001, and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; DC, dendritic cell; IFN, interferon; IL, interleukin; i.m, intramuscular; TIL, tumor-infiltrating leukocytes; TNF, tumor necrosis factor..

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Inhibition, Flow Cytometry, Functional Assay, Two Tailed Test