Journal: Nature Communications

Article Title: Exploiting loss of heterozygosity for allele-selective colorectal cancer chemotherapy

doi: 10.1038/s41467-020-15111-4

Figure Lengend Snippet: a NAT2 mediates APA acetylation. The velocity of NAPA formation was determined by LC-MS/MS in slow (green), rapid (yellow) NAT2 clones, vector control (blue), and parental RKO cells (black) after incubation with APA at indicated concentrations. b APA is selectively metabolized by NAT2. Enzyme kinetics of NAPA formation by human recombinant NAT1 (black) and NAT2 (red) proteins. c , d siRNA-mediated gene silencing of protein kinases with differential inhibition by APA and NAPA expressed in RKO CRC cells. Relative growth of RKO ( c ) and DLD-1 ( d ) cells transfected with a pool of siRNA targeting DYRK1A , AURKA , CDK7 , non-target RNA control and mock transfection as percentage of the growth of untransfected cells. Mean and S.D. (error bars) of three independent experiments is shown. Data were analyzed by one-way ANOVA. * P = 0.0435, ** P = 0.0013, *** P = 0.0004. e APA (2 µM) reduces the phospho-Thr288 Aurora kinase A (pAURKA) staining in NAT2-deficient cells but not in rapid NAT2 clones. Mitotic spindles were visualized by α-tubulin staining (green) and pAURKA levels (red). Representative images of three independent repeats are shown for each cell type. Scale bar, 10 µm. f Quantification of pAURKA signal in parental DLD-1 and slow and rapid NAT2 clones after APA treatment (2 µM). The pAURKA levels are shown as a fold of the DMSO control group. Mean and S.D. (error bars) of three independent experiments. Data were analyzed using two-way ANOVA. n.s., P = 0.0619 and **** P < 0.0001. g APA (2 µM), similarly to the AURKA selective inhibitor S1451 (10 µM) reduces the total protein level of AURKA in contrast to nocodazole (100 ng mL −1 ). DLD-1 cells in different treatments were subjected to immunoblot analysis with different antibodies for detection of total AURKA, PCNA (loading control), histone H3 pSer10, and total histone H3. Representative immunoblot from at least two independent experiments is shown.

Article Snippet: The expression of NAT2 , DYRK1A , AURKA , or CDK7 transcripts was detected by qPCR using TaqMan probes against NAT2 (Hs04194721_s1), DYRK1A (Hs00176369_m1), AURKA (Hs01582072_m1), or CDK7 (Hs00361486_m1) with ACTB (Hs01060665_g1) as an internal reference gene.

Techniques: Liquid Chromatography with Mass Spectroscopy, Clone Assay, Plasmid Preparation, Control, Incubation, Recombinant, Inhibition, Transfection, Staining, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: Protein expression and subcellular localization of AURKA during meiotic maturation. (A) Immunoblot blot analysis of AURKA in cumulus-free oocytes (200 oocytes per lane) cultured in vitro to various stages—GV (0 h), GVBD (1 h), MI (7 h), MII (18 h). The amount of AURKA protein increased slightly around GVBD. (B) Quantification of immunoblots. The experiment was performed three times and the data are expressed as mean ±SEM. Statistical differences (p < 0.05) in comparison to GV-stage are marked (*) (C) Phase-contrast and fluorescence microscopy images of oocytes after co-injection of Gfp-Aurka mRNA (2–5 pl of 200 ng/μl) and mCherry-H2B (2–5 pl of 25 ng/μl) into GV-stage. Total AURKA was mainly present at MTOC at GV-stage oocytes, at MI and MII AURKA was mainly associated with spindle with a clear concentration on the spindle poles and cytoplasmic MTOCs (arrowheads).

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Expressing, Western Blot, Cell Culture, In Vitro, Comparison, Fluorescence, Microscopy, Injection, Concentration Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: PI3K-PKB and CDK1 independent AURKA activation precedes resumption of meiosis. Localization of pT288 AURKA (active form) during meiotic maturation (A). MTOC-associated AURKA is phosphorylated on T288 before GVBD (bGVBD), then during prometaphase I (PMI) and metaphase I (MI) pT288 AURKA remains associated with MTOCs. Green—pT288 AURKA, red—pericentrin (MTOCs). (B) Inhibition of CDK1 activity does not prevent activation of AURKA and multiplication of MTOCs. Oocytes, cultured for 90 min in the presence of Roscovitine, showed phosphorylation of AURKA on T288 at amplified MTOCs or within the nucleus. (C) Ability of LY-294002 or SH-6 oocytes to resume meiosis. The experiment was performed four times and around 200 oocytes were counted for each group. Error bars show confidence intervals. Statistical differences (p < 0.05) in comparison to control are marked (*) (D) PI3K-PKB signaling pathway is not involved in AURKA activation. Oocytes were cultured in medium supplemented with LY-294002 or SH-6 for 90 min and then GV-stage oocytes were used for imunofluorescence staining. Inhibition of PI3K or PKB did not block phosphorylation of AURKA, multiplication of MTOC and induced presence of pT288 AURKA within the nucleus.

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Activation Assay, Inhibition, Activity Assay, Cell Culture, Phospho-proteomics, Amplification, Comparison, Control, Staining, Blocking Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: Overexpression of AURKA triggers MTOCs multiplication in GV-arrested oocytes. Gfp-Aurka. mRNA was microinjected into GV-blocked oocytes (2–5 pl of 250 ng/μl); Egfp mRNA was used as a control. (A) Exogenous AURKA caused multiplication of MTOCs in the presence of an intact GV after overnight incubation. GFP-AURKA was located on all MTOCs, (B) but 2 h after injection was non-phosphorylated. Overnight incubation led to phosphorylation of exogenous GFP-AURKA on T288. Pericentrin was used as a MTOC marker. (C) Endogenous AURKA is not phosphorylated during overnight culture in IBMX-containing medium (D) Activity of CDK1 (arbitrary units) was assayed on extracts from 10 oocytes per sample. The experiment was done 3 times and activity is expressed as mean ± SEM. Statistical difference (p < 0.05) in comparisons to GV-stage control is marked (*).

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Over Expression, Control, Incubation, Injection, Phospho-proteomics, Marker, Activity Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: MI spindle defects induced by AURKA overexpression. (A) Aurka mRNA (2–5 pl, 350 ng/ul) injected oocytes, cultured for 18 h, were evaluated. Overexpression of AURKA blocks oocytes in PMI/MI. Altogether 100 GFP (control) and 99 GFP-AURKA oocytes were evaluated in three independent experiments. Error bars show confidence intervals. Statistical differences (p < 0.05) in individual cell cycle stages (MI and MII) are marked (*), difference in GV stage is not significant (B) Morphological defects such as formation of abnormally long spindle, absence of congression of tetrads or formation of unipolar (arrow) and multipolar spindles (arrowheads) were analyzed by immunocytochemistry. The 83 injected oocytes from three independent experiments were evaluated and morphologically analyzed. Red—acetylated α-tubulin, blue—DAPI.

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Over Expression, Injection, Cell Culture, Control, Immunocytochemistry

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: Downregulation of AURKA leads to incorrect spindle assembly and PMI/MI arrest. Oocytes injected with Aurka dsRNA or with Egfp dsRNA as a control were cultured for 24 h in IBMX-supplemented medium. (A) Aurka mRNA level in relative arbitrary units after RNAi mediated knockdown in GV-arrested oocytes. Total RNA was subsequently isolated and used for real-time PCR to quantify the level of Aurka mRNA. Egfp was used as an external standard. The decrease in the amount of Aurka mRNA was significant (p < 0.05). Data are expressed as the mean ± SEM. (B) Meiotic maturation of dsRNA injected oocytes. Oocytes after microinjection of Aurka dsRNA are arrested PMI/MI, when evaluated 18 h after IBMX release. 109 GFP RNAi and 94 AURKA RNAi oocytes were analyzed in three experiments. Statistical differences (p < 0.05) in individual cell cycle stages (GV, MI and MII) are marked (*) (C) Phenotypes of AURKA knockdown in mouse oocytes. Representative images of three experiments (approx. 100 oocytes) showing contol MI phase spindle of oocytes injected with Egfp (left) and oocytes injected with Aurka dsRNA (right) at the time 7 h after their transfer to IBMX-free medium. Red—acetylated α-tubulin, blue—DAPI for DNA staining.

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Injection, Control, Cell Culture, Knockdown, Isolation, Real-time Polymerase Chain Reaction, Microinjection, Staining

Journal: Nature communications

Article Title: Targeting AURKA-CDC25C axis to induce synthetic lethality in ARID1A-deficient colorectal cancer cells.

doi: 10.1038/s41467-018-05694-4

Figure Lengend Snippet: Fig. 4 Induction of multinucleation, G2/M arrest, and apoptosis in ARID1A−/−cells by AURKA inhibition. a Abnormal chromosomal segregation induced by AURKA silencing. ARID1A isogenic HCT116 cells were transfected with AURKA siRNA and analyzed for immunofluorescence of AURKA, α-tubulin, and nuclei/chromatin (HO33342). Scale bars, 10 µm. b Induction of multinucleation in ARID1A−/−cells by AURKA silencing. ARID1A isogenic HCT116 cells were transfected with AURKA siRNA and analyzed for immunofluorescence of α-tubulin (green) and nuclei/chromatin (blue). Images in the inlets (red square) are representative cells that were magnified and shown on the right side of each figure. Scale bars, 20 µm. c Percentage of multinuclear cells in ARID1A isogenic HCT116 cells treated with AURKA siRNA. Error bars represent s.d. **P < 0.01, Student’s t test. d Cell cycle analyses of ARID1A isogenic HCT116 cells treated with AURKAi. e Percentage of cell populations in each cell cycle phase. Error bars represent s.d. f, g Preferential induction of apoptosis in ARID1A−/−cells by AURKAi. Cells with or without AURKAi treatment were subjected to FITC–Annexin V apoptosis detection with a flow cytometer (f) and the percentage of apoptotic cells were quantitated (g). Error bars represent s.d. **P < 0.01, Student’s t test. h, i Preferential induction of apoptosis in ARID1A −/−cells by AURKA silencing (h) or AURKAi treatment (i). Active (cleaved) caspase-3 was used as a marker of apoptosis induction

Article Snippet: Each aliquot of protein sample was run on a SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane for immunoblotting with primary antibodies, including ARID1A (Santa Cruz, sc-32761, 1:1000 dilution), CDC25C (Santa Cruz, sc-327, 1:2000 dilution), CDC2 (Santa Cruz, sc-54, 1:2000 dilution), GAPDH (Santa Cruz, sc-365062, 1:5000 dilution), HSP90 (Santa Cruz, sc-69703, 1:5000 dilution), αtubulin (Santa Cruz, sc-5286, 1:5000 dilution), p-CDC25C (Cell Signaling, #9529s, 1:1000 dilution), p-CDC2 (Cell Signaling, #9111s, 1:1000 dilution), AURKA (Cell Signaling, #14475s, 1:2000 dilution), and cleaved caspase 3 (Cell Signaling, #9661s, 1:2000 dilution) antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz).

Techniques: Inhibition, Transfection, Cytometry, Marker

Journal: Nature communications

Article Title: Targeting AURKA-CDC25C axis to induce synthetic lethality in ARID1A-deficient colorectal cancer cells.

doi: 10.1038/s41467-018-05694-4

Figure Lengend Snippet: Fig. 6 AURKA–CDC25C axis is a target for synthetic lethality in ARID1A-KO colorectal cancer cells. a Immunoblots showing upregulation of AURKA and phosphorylated CDC25C at Ser198 levels and downregulation of phosphorylated CDC2 at Tyr15 level in ARID1A−/−HCT116 cells. b, c Inhibition of CDC25C phosphorylation at Ser198 and increased phosphorylation of CDC2 at Tyr15 by AURKA silencing (b) and AURKAi treatment (c). d Immunofluorescence analysis of CDC25C localization in ARID1A isogenic cells treated with or without AURKA siRNA. Scale bars, 20 µm. e Synthetic lethality in ARID1A−/−HCT116 cells by CDC25 inhibitor II. f Synthetic lethality in ARID1A−/−HCT116 cells by PLK1 siRNA (siPLK1). Error bars represent s.d. *P < 0.05; **P < 0.01, Student’s t test. g Working model of the synthetic lethality between ARID1A and AURKA. In ARID1A wild-type (WT) cells, AURKA expression is negatively regulated by ARID1A, thereby reducing the activity of AURKA downstream pathway, including PLK1 and CDC25C. In ARID1A mutant (MT) cells, AURKA-PLK1-CDC25C pathway is upregulated. In addition, CDC25C activity is negatively regulated by ARID1A–ATR–CHK (checkpoint kinase) pathway under DNA damage conditions, thereby strictly controlling the CDC25 activity in ARID1A WT cells. In ARID1A MT cells, ARID1A–ATR–CHK pathways is impaired and thus CDC25C activity is de-repressed, causing it in hyper-active state. In this condition, cells can be addicted to AURKA–CDC25C pathway for cell survival and proliferation. Therefore, AURKA–CDC25C axis becomes a target for synthetic lethality in ARID1A- deficient cells

Article Snippet: Each aliquot of protein sample was run on a SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane for immunoblotting with primary antibodies, including ARID1A (Santa Cruz, sc-32761, 1:1000 dilution), CDC25C (Santa Cruz, sc-327, 1:2000 dilution), CDC2 (Santa Cruz, sc-54, 1:2000 dilution), GAPDH (Santa Cruz, sc-365062, 1:5000 dilution), HSP90 (Santa Cruz, sc-69703, 1:5000 dilution), αtubulin (Santa Cruz, sc-5286, 1:5000 dilution), p-CDC25C (Cell Signaling, #9529s, 1:1000 dilution), p-CDC2 (Cell Signaling, #9111s, 1:1000 dilution), AURKA (Cell Signaling, #14475s, 1:2000 dilution), and cleaved caspase 3 (Cell Signaling, #9661s, 1:2000 dilution) antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz).

Techniques: Western Blot, Inhibition, Phospho-proteomics, Expressing, Activity Assay, Mutagenesis

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

Journal: The FASEB Journal

Article Title: Ganetespib limits ciliation and cystogenesis in autosomal‐dominant polycystic kidney disease (ADPKD)

doi: 10.1096/fj.201700909r

Figure Lengend Snippet: Figure 1. HSP90 is required for in vitro maintenance of cilia. A) Selected subset of defined proteins that influence ciliary dynamics or structural integrity, and are HSP90 clients or regulated by HSP90 inhibition. B–E) Representative immunofluorescence (IF) image and graph with frequency of ciliated WT or Pkd12/2 murine renal epithelial cells at 2 h (B, C) or 24 h (D, E) after treatment with vehicle (V), ganetespib (G), or alisertib (A) to inhibit AURKA, or combination (C) of alisertib and ganetespib. On IF, acetylated a-tubulin (red); g-tubulin (green); DAPI (blue). Scale bars, 5 mm; original magnification, 3240. F, G) Analysis as in B and C for human PKD1-mutant WT9-7 and WT9-12 cells or for hTERT-RPE1 (RPE1) cells, 2 h after drug treatment. H) IF for phospho-T288 (activated) AURKA (magenta, indicated by white arrowheads) at ciliary basal body (g-tubulin, green) with acetylated a-tubulin (red); images of ph-AURKA and basal body are offset. No antibodies currently available for phospho-T288 are reactive with mouse AURKA. Scale bars, 5 mm. Data are expressed as means 6 SEM; n/s, not significant. IF experiments were performed in triplicate; #750 cells were scored per genotype. *P # 0.05, ***P # 0.001, ****P = 0.0001.

Article Snippet: Primary antibodies included anti-acetylated a-tubulin mAb [mouse, clone 6-11B-1 (T6793; Sigma-Aldrich), g-tubulin (T5192 and ab191114, rabbit; Sigma-Aldrich), anti-phospho AURKA (T288) (IHC-00067; Bethyl Laboratories, Montgomery, TX, USA), and anti-total AURKA (3092S; Cell Signaling Technology, Danvers, MA, USA).

Techniques: In Vitro, Inhibition, Mutagenesis

Journal: The FASEB Journal

Article Title: Ganetespib limits ciliation and cystogenesis in autosomal‐dominant polycystic kidney disease (ADPKD)

doi: 10.1096/fj.201700909r

Figure Lengend Snippet: Figure 2. Mechanism of ganetespib regulation of AURKA activation in ciliary disassembly. A) Immunofluorescence analysis of hTERT-RPE1 cells treated for 2 h with vehicle (Veh), ganetespib (Gan), and/or proteasomal inhibitors MG-132 and bortezomib. DAPI (blue), g-tubulin (green), acetylated a-tubulin (red), activated ph-T288-AURKA (top row, magenta) or total AURKA (bottom row, magenta). Scale bars, 5 mm; original magnification, 3240. Images of ph-AURKA or total AURKA are offset from basal body. B) Frequency of ciliated cells in populations of cells 2 h after treatment with vehicle (V), ganetespib (G), and/or MG- 132 (MG) and bortezomib (Bz). C–F) Normalized quantification and representative data for Western blots showing expression of FBXW7 (C), TCHP (D), NEK8 (E), or PP2A-B (F), 2 h after treatment with indicated drugs. LC, loading control. *P # 0.05, **P # 0.01, ***P # 0.001.

Article Snippet: Primary antibodies included anti-acetylated a-tubulin mAb [mouse, clone 6-11B-1 (T6793; Sigma-Aldrich), g-tubulin (T5192 and ab191114, rabbit; Sigma-Aldrich), anti-phospho AURKA (T288) (IHC-00067; Bethyl Laboratories, Montgomery, TX, USA), and anti-total AURKA (3092S; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: Reversine exhibits antineoplastic activity in JAK2 V617F -positive myeloproliferative neoplasms

doi: 10.1038/s41598-019-46163-2

Figure Lengend Snippet: Pharmacological inhibition of JAK2/STAT signaling reduces AURKA and AURKB expression in SET2 cells. ( A ) Gene expression heatmap from RNA-seq analysis of SET2 cells upon the treatment with ruxolitinib. Data was obtained from Meyer et al . (GEO accession GSE69827). Gene expression was expressed as fold change of the mean of normalized counts of naïve SET2 cells, which was set as 1; genes demonstrating ≥1.5-fold in either direction are included in the heatmap. The mean of fold change obtained from three experimental replicates is indicated. ( B ) qPCR analysis of AURKA and AURKB mRNA expression in SET2 cells treated with graded concentrations of ruxolitinib (vehicle, 100, 300, or 1000 nM) for 48 hours. Bar graphs represent the mean ± SD of at least four independent experiments. The p values are indicated in the graphs; * p < 0.05, *** p < 0.0001; ANOVA test and Bonferroni post-test. ( C ) Western blot analysis for p-STAT3 Y705 , p-STAT5 Y694 , AURKA, AURKB, and p-histone H3 S10 , in total cell extracts from SET2 cells treated with graded concentrations of ruxolitinib (vehicle, 100, 300, or 1000 nM) for 48 hours (Right panel) or graded time of exposure (0, 3, 6, 9, 12, 24, and 48 hours) to ruxolitinib at 300 nM (Left panel); membranes were reprobed with the antibody for the detection of the respective total protein or α-tubulin, and developed with the SuperSignal™ West Dura Extended Duration Substrate system using a Gel Doc XR+ imaging system. Cropped blots are shown and full-length blots are included in Supplementary Fig. .

Article Snippet: Quantitative PCR (qPCR) was performed with an ABI 7500 Sequence Detector System using the TaqMan system for AURKA (Hs00269212.m1), AURKB (Hs00177782.m1), ATCB (4310881E-1008027), and GUS (43108881E-0905024) genes (Life Technologies) or SyberGreen System for AURKA , AURKB , BCL2 , BCL2L1 , BIRC5 , BNIP3 , BNIP3L , and BIK (Supplementary Table ).

Techniques: Inhibition, Expressing, Gene Expression, RNA Sequencing, Western Blot, Imaging

Journal: Scientific Reports

Article Title: Reversine exhibits antineoplastic activity in JAK2 V617F -positive myeloproliferative neoplasms

doi: 10.1038/s41598-019-46163-2

Figure Lengend Snippet: Reversine induces DNA damage and apoptosis markers in JAK2 V617F -positive cell lines. ( A ) Western blot analysis for AURKA, AURKB, phospho(p)-histone H3 S10 , p-histone H2A.X S139 ( γ -H2A.X) and Caspase 3 (total and cleaved) in total cell extracts from SET2 and HEL cells treated with graded concentrations of reversine (vehicle, 1, 2.5, 5, 10 or 25 μM) for 48 hours; membranes were reprobed with the antibody for the detection of the respective total protein or α-tubulin, and developed with the SuperSignal™ West Dura Extended Duration Substrate system using a Gel Doc XR+ imaging system. Cropped blots are shown and full-length blots are included in Supplementary Fig. . ( B ) Immunofluorescence analysis of SET2 and HEL cells treated or not with 5 μM reversine for 24 hours, displaying α-tubulin (green) and DAPI (blue) staining. Blank and red arrows illustrate mitotic catastrophe and nuclear fragmentation, respectively. Scale bars are shown in the figure (50 μm). Bar graphs represent the quantification of morphological findings of at least 200 cells for each condition. ( C ) Gene expression heatmap from qPCR array analysis of SET2 cells treated with reversine (5 μM). The mRNA levels are normalized to those of vehicle-treated SET2 cells and calculated as fold change in expression; genes demonstrating ≥1.5-fold in either direction compared to vehicle-treated cells upon the treatment with reversine are included in the heatmap. Two independent experiments of each condition were used for the analysis; green indicates repressed mRNA levels and red elevated mRNA levels. ( D ) qPCR analysis of BCL2 , BCL2L1 , BIRC5 , BNIP3 , BNIP3L , and BIK mRNA expression in SET2 and HEL cells treated with reversine (5 μM). The dashed line represents the mean gene expression in vehicle-treated cells and bars represent the fold change in gene expression in SET2 and HEL cells treated with reversine (5 μM) compared to their respective untreated cells. The p values and cell lines are indicated in the graphs. ** p < 0.01, ** p < 0.001; Student t test.

Article Snippet: Quantitative PCR (qPCR) was performed with an ABI 7500 Sequence Detector System using the TaqMan system for AURKA (Hs00269212.m1), AURKB (Hs00177782.m1), ATCB (4310881E-1008027), and GUS (43108881E-0905024) genes (Life Technologies) or SyberGreen System for AURKA , AURKB , BCL2 , BCL2L1 , BIRC5 , BNIP3 , BNIP3L , and BIK (Supplementary Table ).

Techniques: Western Blot, Imaging, Immunofluorescence, Staining, Gene Expression, Expressing