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: 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: Virology

Article Title: Molecular pathways executing the "trophic sentinel" response in HPV-16 E7-expressing normal human diploid fibroblasts upon growth factor deprivation.

doi: 10.1016/j.virol.2003.11.008

Figure Lengend Snippet: Fig. 1. (A) The trophic sentinel response in HPV E7-expressing cells induced by serum starvation is p53 dependent. Induction of apoptosis in the different cell lines as determined by fluorescence microscopic analysis of nuclear abnormalities (left panel) and nucleosomal DNA fragmentation using an ELISA-based quantitative DNA laddering assay (right panel). The values shown were calculated by determining the relative increases of apoptosis and DNA fragmentation, respectively, for each cell population in the absence or presence of fetal bovine serum for 48 h derived from a single experiment. Similar results were obtained in multiple independent experiments. (B) Immunoblot analysis of the steady state levels of p53, p21CIP1, and HPV-16 E7 in the different cell populations in response to DNA damage induced by actinomycin D (actD) treatment. GAPDH expression is shown as a loading control. See Materials and methods for details. (C) Analysis of p53 phosphorylation by immunoblot analysis with various phospho-specific antibodies in control (LXSN) and HPV-16 E7-expressing (E7) IMR-90 cells in the absence (0%) or presence (10%) of fetal bovine serum (FBS). UV-irradiated cos7 and control (LXSN) and HPV-16 E7-expressing (E7) IMR-90 cells were used as positive controls. GAPDH expression is shown to document equal loading. (D) DNA binding capacity of p53 in control (LXSN) and HPV-16 E7-expressing (E7) IMR-90 cells grown in the absence (0%) or presence (10%) of fetal bovine serum (FBS) by EMSA. Wild-type or mutant oligonucleotide competitors or a p53-specific antibody were added to demonstrate the specificity of the shifted band; ns denotes a nonspecific band. Lysates from the human Raji cell line were supplied as a positive control by the manufacturer of the kit (GENEKA).

Article Snippet: The following antibodies were used according to the recommendations of their suppliers: p53 (Ab-6, Calbiochem), p21CIP1 (Ab-1, Oncogene Science), Noxa (Pharmingen), GAPDH (CHEMICON), HPV-16 E7 (mixture of 8C9, 1:100, Zymed and ED17, 1:1000, Santa Cruz), InB (Rabbit polyclonal, Santa Cruz), IGFBP-1, 2,3,4,5,6 (goat polyclonal, Santa Cruz), p50 (Rockland), p65 (Santa Cruz).

Techniques: Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay, DNA Laddering, Derivative Assay, Western Blot, Control, Phospho-proteomics, Irradiation, Binding Assay, Mutagenesis, Positive Control

Journal: Journal of Biological Chemistry

Article Title: Mammary Epithelial Cell Polarity Is Regulated Differentially by p73 Isoforms via Epithelial-to-mesenchymal Transition

doi: 10.1074/jbc.m112.358143

Figure Lengend Snippet: FIGURE 4. p73-KD and TAp73-KD, but not Np73-KD induce EMT pheno- types in MCF10A cells. Western blots were prepared using extracts from MCF10A cells (lane 1), and MCF10A cells with p73-KD (lane 2), with Np73-KD (lane3),orwithTAp73-KD(lane4).MCF10AcellsweregrowninMatrigelfor20 days.TheblotswereprobedwithantibodiesagainstE-cadherin(A),-catenin (B), laminin V (B), Snail (B), Slug (B), Twist (B), p21 (C), PUMA (C), and actin (A–C), respectively. The basal levels of each gene were arbitrarily set at 1.0, and fold change is shown below each lane.

Article Snippet: Antibodies used were purchased from Calbiochem (anti-p73 (ab-1)-ER-13, anti-p73 (ab-2)-ER-15), Millipore (anti-laminin 2), Santa Cruz Biotechnology (anti- - catenin (E-5), anti-Snail-1, anti-Twist, p21, and anti-GAPDH), Cell Signaling (anti-Slug), BDTransduction Laboratories (antiE-cadherin), Prosci (PUMA), Sigma (anti-actin), and Bio-Rad (secondary antibodies against rabbit or mouse IgG conjugated with HRP).

Techniques: Western Blot

Journal: Cancer Medicine

Article Title: The GNL3L ‐ MDM2 Interaction Drives Esophageal Squamous Cell Carcinoma Progression

doi: 10.1002/cam4.71146

Figure Lengend Snippet: GNL3L positively regulated MDM2 expression and interacted with MDM2. (a) After GNL3L knockdown, qRT‐PCR, and western blot (WB) were performed to determine MDM2 expression in TE‐1 cells. (b) Differences in the expression of p53 and p21 in TE‐1 cells after the knockdown of GNL3L. (c) Co‐IP of GNL3L and MDM2 from ESCC cell lines. The input represents the total protein extract used in the IP. The GNL3L protein expression was normalized to that of GAPDH in ESCC cells. (IP, immunoprecipitation; IgG, negative control). (d) qRT‐PCR analysis of MDM2 and GNL3L expression in TE‐1 cells in different groups. (e) Protein expression of MDM2 and GNL3L of TE‐1 in different groups was determined by WB. (f) Cell proliferation was analyzed in different groups using the CCK8 assay for TE‐1 cell lines. (g) Proliferation was analyzed using an EdU assay. (** p < 0.01, ## p < 0.01).

Article Snippet: The primary antibodies were GAPDH polyclonal antibody (1:3000, Affinity, AF7021, 36 kDa), MDM2 polyclonal antibody (1:1000, Affinity, AF0208, 90 kDa), GNL3L antibody (1:1000, Affinity, DF4113, 66 kDa), p53 antibody (1:1000, Affinity, AF0879, 53 kDa), and p21 antibody (1:1000, MedChemExpress, HY‐ P80774 , 18 kDa).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, CCK-8 Assay, EdU Assay

Journal: Cancer Medicine

Article Title: The GNL3L ‐ MDM2 Interaction Drives Esophageal Squamous Cell Carcinoma Progression

doi: 10.1002/cam4.71146

Figure Lengend Snippet: GNL3L knockdown reversed the increased malignant behaviors of ESCC cells induced by MDM2 overexpression. (a) Wound healing assay results in TE‐1 cells from different groups. (b) Invasion and migration of TE‐1 cells were assessed using Transwell assays. (c) Distribution of the cell phases of TE‐1 in the different groups. (d) In the different groups, apoptosis was measured in TE‐1 cells using flow cytometry. (e) Expression of P53 and P21 in TE‐1 cells in different groups. (** p < 0.01, ## p < 0.01).

Article Snippet: The primary antibodies were GAPDH polyclonal antibody (1:3000, Affinity, AF7021, 36 kDa), MDM2 polyclonal antibody (1:1000, Affinity, AF0208, 90 kDa), GNL3L antibody (1:1000, Affinity, DF4113, 66 kDa), p53 antibody (1:1000, Affinity, AF0879, 53 kDa), and p21 antibody (1:1000, MedChemExpress, HY‐ P80774 , 18 kDa).

Techniques: Knockdown, Over Expression, Wound Healing Assay, Migration, Flow Cytometry, Expressing

Journal: Cancer Medicine

Article Title: The GNL3L ‐ MDM2 Interaction Drives Esophageal Squamous Cell Carcinoma Progression

doi: 10.1002/cam4.71146

Figure Lengend Snippet: GNL3L overexpression reversed the decreased malignant behavior of ESCC cells induced by MDM2 knockdown. (a) Protein expression of MDM2 and GNL3L in TE‐1 cells in different groups was determined by western blot (WB). (b) Cell proliferation was analyzed in different groups using an EdU assay. (c) Migration and invasion of TE‐1 cells were assessed using Transwell assays. (d) In the different groups, apoptosis was measured in TE‐1 cells using flow cytometry. (e) Expression of P53 and P21 in TE‐1 cells in different groups. (** p < 0.01, ## p < 0.01).

Article Snippet: The primary antibodies were GAPDH polyclonal antibody (1:3000, Affinity, AF7021, 36 kDa), MDM2 polyclonal antibody (1:1000, Affinity, AF0208, 90 kDa), GNL3L antibody (1:1000, Affinity, DF4113, 66 kDa), p53 antibody (1:1000, Affinity, AF0879, 53 kDa), and p21 antibody (1:1000, MedChemExpress, HY‐ P80774 , 18 kDa).

Techniques: Over Expression, Knockdown, Expressing, Western Blot, EdU Assay, Migration, Flow Cytometry

Journal: PeerJ

Article Title: Dieting alleviates hyperuricemia and organ injuries in uricase-deficient rats via down-regulating cell cycle pathway

doi: 10.7717/peerj.15999

Figure Lengend Snippet: (A) Casp3, Cdk1 and Ki67 in the liver was evaluated at mRNA level, and a high value of FPKM means a high expression; (B) Casp3, Cdk1 and Ki67 in the liver was evaluated by Western blot; C, Results of B were quantified by relative brightness. FPKM, Fragments Per Kilobase of exon model per Million mapped fragments. Control, control group, KDY rats were given free approach to food and water; 75% dieting, 75% dieting group, rats were given 75% food of control group at the same age in days and given free access to water. * P < 0.05 vs control group, Student’s t -test, two-tailed.

Article Snippet: Hematoxylin-eosin (HE) staining kits, mouse anti- β -actin antibody (BM0627), rabbit anti-CASP3 antibody (BA2142), rabbit anti-CDK1 antibody (PB9533), rabbit anit-Ki67 antibody (M00254-4), and horseradish peroxidase (HRP) conjugated goat anti-rabbit/mouse IgG (H+L) (BA1056) were all purchased from Boster Biological Engineering Co., Ltd (Wuhan, China).

Techniques: Expressing, Western Blot, Control, Two Tailed Test

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Acetylation of Atp5f1c Mediates Cardiomyocyte Senescence via Metabolic Dysfunction in Radiation-Induced Heart Damage

doi: 10.1155/2022/4155565

Figure Lengend Snippet: Ionizing radiation causes cardiomyocyte senescence. (a) Senescence-associated β -galactosidase (SA- β gal) staining of sham-irradiated and irradiated H9C2 cells. (b)–(d) Western blotting and quantification analyses of p21 and p16 protein expression in H9C2 cells in the control group and after 6h, 12h, and 24h of irradiation ( ∗ p < 0.05, ∗∗ p < 0.01). (e) q-PCR analysis of the mRNA levels of inflammation- and fibrosis-associated factors in H9C2 cells of the control, 6h, 12h, and 24h irradiation groups ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). Bar: 100 μ m; h: hour.

Article Snippet: Total proteins from the animal heart apex or H9C2 cells were extracted using RIPA (Solarbio, Beijing, China), and protein concentration and western blotting were performed as previously described [ ] The primary antibodies used in this study were anti-acetyllysine rabbit pAb (1 : 1000; Jingjie PTM BioLabs, Inc., China), anti-Atp5f1 monoclonal antibody (1 : 1000; Proteintech, Wuhan, China), anti-p21 monoclonal antibody (1 : 1000; Boster, Wuhan, China), HA-tagged monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), 6× His-tagged monoclonal antibody (1 : 10000; Proteintech, Wuhan, China), GAPDH monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), and anti- α -tubulin monoclonal antibody (1 : 1000; Boster).

Techniques: Staining, Irradiation, Western Blot, Expressing, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Acetylation of Atp5f1c Mediates Cardiomyocyte Senescence via Metabolic Dysfunction in Radiation-Induced Heart Damage

doi: 10.1155/2022/4155565

Figure Lengend Snippet: Atp5f1c is hyperacetylated at the 55th lysine site, which leads to metabolic disorders and senescence. (a) The expression of anti-His antibody after His-tagged Atp5f1c WT, K55R, and K55Q point mutation plasmids transfection in H9C2 cells. (b) Atp5f1c acetylation level verified by Co-IP after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells. (c, d) Senescence-associated β -galactosidase (SABG) staining and statistical analysis after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; NS: not significant). (e) q-PCR analysis of the mRNA levels of inflammation- and fibrosis-associated factors after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (f)–(i) Western blotting and quantification analyses of p21 and p16 protein expression after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells; relative protein levels were normalized to GAPDH or actin expression ( ∗ p < 0.05). (j, k) ATP synthase activity and ATP production after transfection of Atp5f1c WT, K55R, and K55Q plasmids into H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; NS: not significant). Co-IP: Coimmunoprecipitation; WB: western blotting; WT: wild type.

Article Snippet: Total proteins from the animal heart apex or H9C2 cells were extracted using RIPA (Solarbio, Beijing, China), and protein concentration and western blotting were performed as previously described [ ] The primary antibodies used in this study were anti-acetyllysine rabbit pAb (1 : 1000; Jingjie PTM BioLabs, Inc., China), anti-Atp5f1 monoclonal antibody (1 : 1000; Proteintech, Wuhan, China), anti-p21 monoclonal antibody (1 : 1000; Boster, Wuhan, China), HA-tagged monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), 6× His-tagged monoclonal antibody (1 : 10000; Proteintech, Wuhan, China), GAPDH monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), and anti- α -tubulin monoclonal antibody (1 : 1000; Boster).

Techniques: Expressing, Mutagenesis, Transfection, Co-Immunoprecipitation Assay, Staining, Western Blot, Activity Assay

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 3 CLOCK interference promotes GCs proliferation. A The interference efficiency of CLOCK was measured using RT-qPCR. Data are expressed as mean ± SEM (n = 5), **P < 0.01. B Western blotting reveals the expression levels of CLOCK. C Quantification of the western blot analysis. Data are expressed as mean ± SEM (n = 3), *P < 0.05. D EdU staining was used to detect the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 5), **P < 0.01. E CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 5), *P < 0.05. F RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 5), *P < 0.05, **P < 0.01. G Western blot analysis of proliferation-related gene protein level (CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A). GAPDH as a housekeeping protein. H Quantifying the Western blot analysis of CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), *P < 0.05

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Transfection

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 6 ASB9 interference promotes GCs proliferation. A RT-qPCR detected the interference efficiency of ASB9. Data are expressed as mean ± SEM (n = 6), **P < 0.01. B Western blotting reveals the expression levels of ASB9. C Quantification of the western blot analysis. Data are expressed as mean ± SEM (n = 3), *P < 0.05. D EdU staining was used to detect the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 4), **P < 0.01. E CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 16), ****P < 0.0001. F RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 5), *P < 0.05, **P < 0.01. G Western blot analysis of proliferation-related gene protein level (ASB9, CCNB1, CCNE1, CDK4, and CDKN1A). H Quantifying the western blot analysis of CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), *P < 0.05, **P < 0.01. I RNA expression of ASB9 in GCs. ZT: zone time

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Transfection, RNA Expression