Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: AP39 inhibits ferroptosis by inhibiting mitochondrial autophagy through the PINK1/parkin pathway to improve myocardial fibrosis with myocardial infarction.

doi: 10.1016/j.biopha.2023.115195

Figure Lengend Snippet: Fig. 3. AP39 improves myocardial fibrosis in rats with myocardial infarction. (A): Masson’s staining of myocardial tissue in Control, ISO, ISO+AP39, ISO +AP39 +PAG, and AP39 groups of rats, 10 × 10 and 10 × 40 magnification (blue staining indicates collagen fibers), n = 3. (B): Immunohistochemical detection of Collagen III protein expression in myocardial tissue of each group of rats, 10 × 40 magnification, n = 3. (C-E): Western blot analysis of the expression changes of α-SMA and Collagen III in myocardial tissue of each group of rats. (F) Collagen volume fraction determined by Masson’s staining. (G) Collagen III volume fraction determined by immunohistochemistry, n = 3, * P < 0.05 vs Control; #P < 0.05 vs ISO; $P < 0.05 vs ISO+AP39.

Article Snippet: AP39 (HY-126124) and RSL3 were obtained from MCE.

Techniques: Staining, Control, Immunohistochemical staining, Expressing, Western Blot, Immunohistochemistry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: AP39 inhibits ferroptosis by inhibiting mitochondrial autophagy through the PINK1/parkin pathway to improve myocardial fibrosis with myocardial infarction.

doi: 10.1016/j.biopha.2023.115195

Figure Lengend Snippet: Fig. 4. AP39 increases CSE levels and mitochondrial membrane potential in myocardial tissue and cardiomyocytes of rats with myocardial infarction, while reducing ROS levels in cardiomyocytes. (A): Changes in the viability of H9c2 cardiomyocytes induced by different concentrations of CoCl2 as measured by the CCK8 assay. (B): Western blot analysis of CSE expression in H9c2 cardiomyocytes. (C): Western blot analysis of CSE expression in myocardial tissue of different groups of rats. n = 3, * P < 0.05 vs Control; #P < 0.05 vs ISO; $P < 0.05 vs ISO+AP39. (D): Mean gray value (IntDen/Area) determined by JC-1, n = 3, * P < 0.05 vs Control; #P < 0.05 vs CoCl2; $P < 0.05 vs CoCl2 +AP39. (E): First and Second line: Detection of H2S levels in mitochondria of H9c2 cardiomyocytes using a mitochondrial H2S probe (Mito- HS); Third line: Detection of ROS levels in H9c2 cardiomyocytes using a ROS fluorescence probe; Fourth and Fifth line: Assessment of mitochondrial membrane potential changes in H9c2 cardiomyocytes using a mitochondrial membrane potential assay kit (JC-1).

Article Snippet: AP39 (HY-126124) and RSL3 were obtained from MCE.

Techniques: Membrane, CCK-8 Assay, Western Blot, Expressing, Control, Fluorescence

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: AP39 inhibits ferroptosis by inhibiting mitochondrial autophagy through the PINK1/parkin pathway to improve myocardial fibrosis with myocardial infarction.

doi: 10.1016/j.biopha.2023.115195

Figure Lengend Snippet: Fig. 5. AP39 inhibits mitochondrial autophagy in hypoxic H9c2 cardiomyocytes. (A-B): RT-qPCR analysis of PINK1 and P62 mRNA changes in H9c2 cardiomyocytes of each group. n = 3, * P < 0.05 vs Control; #P < 0.05 vs CoCl2; $P < 0.05 vs CoCl2 +AP39. (C-G): Western blot analysis of PINK1, Parkin, P62, and LC3 protein expression changes in H9c2 cardiomyocytes. n = 3, * P < 0.05 vs Control; #P < 0.05 vs CoCl2; $P < 0.05 vs CoCl2 +AP39.

Article Snippet: AP39 (HY-126124) and RSL3 were obtained from MCE.

Techniques: Quantitative RT-PCR, Control, Western Blot, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: AP39 inhibits ferroptosis by inhibiting mitochondrial autophagy through the PINK1/parkin pathway to improve myocardial fibrosis with myocardial infarction.

doi: 10.1016/j.biopha.2023.115195

Figure Lengend Snippet: Fig. 6. AP39 increases mitochondrial membrane potential, while inhibiting the expression of PINK1 and Parkin in hypoxic primary rat cardiomyocytes. First and Second line: Assessment of mitochondrial membrane potential changes in primary rat cardiomyocytes using a mitochondrial membrane potential assay kit (JC-1). Third to Sixth line: The expression of PINK1 and Parkin in primary rat cardiomyocytes using Immunofluorescence Staining. (A-B): Mean gray value(IntDen/Area) determined by JC-1 and Immunofluorescence Staining, n = 3, *P < 0.05 vs Control; #P < 0.05 vs CoCl2; $P < 0.05 vs CoCl2 +AP39.

Article Snippet: AP39 (HY-126124) and RSL3 were obtained from MCE.

Techniques: Membrane, Expressing, Immunofluorescence, Staining, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: AP39 inhibits ferroptosis by inhibiting mitochondrial autophagy through the PINK1/parkin pathway to improve myocardial fibrosis with myocardial infarction.

doi: 10.1016/j.biopha.2023.115195

Figure Lengend Snippet: Fig. 7. AP39 inhibits mitochondrial autophagy in rat myocardial infarction heart tissue. (A-E): Western blot analysis of the expression changes of PINK1, Parkin, P62, and LC3 in rat myocardial tissue. n = 3, * P < 0.05 vs Control; #P < 0.05 vs ISO; $P < 0.05 vs ISO+AP39. (F): Transmission electron microscopy observation of mitochondrial morphology and mitochondrial autophagy in rat myocardial infarction heart tissue.

Article Snippet: AP39 (HY-126124) and RSL3 were obtained from MCE.

Techniques: Western Blot, Expressing, Control, Transmission Assay, Electron Microscopy

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: AP39 inhibits ferroptosis by inhibiting mitochondrial autophagy through the PINK1/parkin pathway to improve myocardial fibrosis with myocardial infarction.

doi: 10.1016/j.biopha.2023.115195

Figure Lengend Snippet: Fig. 8. AP39 inhibits myocardial ferroptosis in rat myocardial tissue and CoCl2-induced hypoxic damage in H9c2 myocardial cells. (A-C): Western blot analysis of the expression changes of GPX4 and SLC7a11 in rat myocardial tissue. n = 3, * P < 0.05 vs Control; #P < 0.05 vs ISO; $P < 0.05 vs ISO+AP39. (D-F): Western blot analysis of the expression changes of GPX4 and SLC7a11 in H9c2 myocardial cells. n = 3, * P < 0.05 vs Control; #P < 0.05 vs CoCl2; $P < 0.05 vs CoCl2 +AP39. (G-H): RT-qPCR analysis of the mRNA level changes of GPX4 and SLC7a11 in H9c2 myocardial cells. n = 3, * P < 0.05 vs Control; #P < 0.05 vs CoCl2; $P < 0.05 vs CoCl2 +AP39. (I): Detection of changes in the content of free Fe2+ in myocardial cell mitochondria using the mitochondrial Fe2+ fluorescent probe (Mito-FerroGreen).

Article Snippet: AP39 (HY-126124) and RSL3 were obtained from MCE.

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: AP39 inhibits ferroptosis by inhibiting mitochondrial autophagy through the PINK1/parkin pathway to improve myocardial fibrosis with myocardial infarction.

doi: 10.1016/j.biopha.2023.115195

Figure Lengend Snippet: Fig. 9. AP39 can antagonize ferroptosis in hypoxic myocardial cells by inhibiting mitochondrial autophagy through the PINK1/Parkin pathway. (A-E): Western blot analysis of the expression changes of PINK1, Parkin, GPX4, and SLC7a11 in H9c2 myocardial cells. n = 3, * P < 0.05 vs Control; #P < 0.05 vs CoCl2; $P < 0.05 vs CoCl2 +AP39.

Article Snippet: AP39 (HY-126124) and RSL3 were obtained from MCE.

Techniques: Western Blot, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: AP39 inhibits ferroptosis by inhibiting mitochondrial autophagy through the PINK1/parkin pathway to improve myocardial fibrosis with myocardial infarction.

doi: 10.1016/j.biopha.2023.115195

Figure Lengend Snippet: Fig. 10. AP39 inhibits ferroptosis and downregulates the expression of fibrosis-related proteins in hypoxic myocardial cells. (A-F): Western blot analysis of the expression changes of GPX4, SLC7a11, Collagen I, TGF-β, and α-SMA in H9c2 myocardial cells. n = 3, * P < 0.05 vs Control; #P < 0.05 vs CoCl2; $P < 0.05 vs CoCl2 +AP39.

Article Snippet: AP39 (HY-126124) and RSL3 were obtained from MCE.

Techniques: Expressing, Western Blot, Control

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: Oxidative Medicine and Cellular Longevity

Article Title: AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons

doi: 10.1155/2016/8360738

Figure Lengend Snippet: AP39 generates H 2 S in WT neurons, primarily in the mitochondria. (a) The contribution of AP39 to H 2 S production in neurons. Neurons from WT mice were treated with various concentrations of AP39 for 2 h, and H 2 S production was detected by methylene blue assay. (b) Neurons were treated with different concentrations of AP39 for 2 h, and intracellular H 2 S was detected using the fluorescent probe AzMC. DAPI was used to stain nuclei, and MitoTracker was used to stain mitochondria. The colocalization of H 2 S with mitochondria was indicated by the overlapping of red (mitochondria) and green (H 2 S) fluorescence in the merged image. Note the concentration-dependent increase in the H 2 S signal in response to AP39 treatment. ∗ P < 0.05, ∗∗ P < 0.01 compared with control treatment (no AP39).

Article Snippet: AP39, a novel mitochondria-targeted H 2 S donor, was designed and synthesized by Medicilon Inc., as described previously [ , ].

Techniques: Staining, Fluorescence, Concentration Assay, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons

doi: 10.1155/2016/8360738

Figure Lengend Snippet: Biphasic effects of AP39 on the cellular bioenergetics of the WT neurons. Neurons from WT mice were incubated in AP39 (25-250 nM) for 2 h, and bioenergetic parameters were measured using an Extracellular Flux Analyzer. (a) Representative tracings. (b) The calculated bioenergetic parameters. # and ## indicate a significant enhancement in the bioenergetic parameter, compared to the control group (no AP39) ( P < 0.05 and P < 0.01, resp.); ∗ indicates a significant reduction in the bioenergetic parameter, compared to the control group ( P < 0.05).

Article Snippet: AP39, a novel mitochondria-targeted H 2 S donor, was designed and synthesized by Medicilon Inc., as described previously [ , ].

Techniques: Incubation, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons

doi: 10.1155/2016/8360738

Figure Lengend Snippet: Cytoprotective effects of AP39 in APP/PS1 neurons. (a) A time course shows that increasing levels of A β 42 are released into the culture media of neurons from transgenic mice but not their WT littermates. (b) A β was overexpressed in neurons from APP/PS1 mice compared to A β expression in neurons from their WT littermates. (c) Representative of A β blots is shown with quantification. (d) The effects of AP39 (25–250 nM) treatment for 24 h on cell viability in WT neurons. AP39 alone did not affect MTT conversion. (e) The effects of AP39 (25–250 nM) on MTT conversion in APP/PS1 neurons. There was a decrease in MTT conversion in the APP/PS1 neurons compared to the WT neurons; these effects were attenuated by AP39. (f) After treatment of WT neurons with AP39 for 24 h, AP39 alone did not affect LDH release in the cellular medium. (g) The effects of AP39 on LDH release from APP/PS1 neurons. ∗ P < 0.05, versus the APP/PS1 group; # P < 0.05, the APP/PS1 group versus the WT group.

Article Snippet: AP39, a novel mitochondria-targeted H 2 S donor, was designed and synthesized by Medicilon Inc., as described previously [ , ].

Techniques: Transgenic Assay, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons

doi: 10.1155/2016/8360738

Figure Lengend Snippet: Protective effects of AP39 on the cellular bioenergetics of APP/PS1 neurons. APP/PS1 neurons were incubated in AP39 (100 nM) for 24 h, and bioenergetic parameters were measured using the Extracellular Flux Analyzer. (a) Representative tracings are shown. (b) The calculated bioenergetic parameters are shown. ∗ indicates a significant enhancement of the bioenergetic parameter compared to the control (H 2 O) ( P < 0.05); # indicates a significant reduction in the bioenergetic parameter compared to the control (WT + H 2 O) ( P < 0.05).

Article Snippet: AP39, a novel mitochondria-targeted H 2 S donor, was designed and synthesized by Medicilon Inc., as described previously [ , ].

Techniques: Incubation, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons

doi: 10.1155/2016/8360738

Figure Lengend Snippet: AP39 protected mitochondrial function in APP/PS1 neurons. (a) AP39 increased the cellular ATP levels. (b) AP39 protected mtDNA integrity. (c) ROS levels were detected by flow cytometry. (d) AP39 reduced ROS levels. ## P < 0.01, compared with the WT group; ∗∗ P < 0.01, compared with the control group (H 2 O).

Article Snippet: AP39, a novel mitochondria-targeted H 2 S donor, was designed and synthesized by Medicilon Inc., as described previously [ , ].

Techniques: Flow Cytometry, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons

doi: 10.1155/2016/8360738

Figure Lengend Snippet: AP39 reduced the expression of proteins involved in mitochondrial fusion but increased the expression of a mitochondrial fission protein. (a) Protein samples were probed for Drp1, Fis1, Mfn1, Mfn2, OPA-1, and VDAC expression. (b–f) Representative blots are shown with quantification. ## P < 0.01 AD mice receiving water compared to WT mice receiving water; ∗∗ P < 0.01 mice receiving AP39 compared with mice receiving water.

Article Snippet: AP39, a novel mitochondria-targeted H 2 S donor, was designed and synthesized by Medicilon Inc., as described previously [ , ].

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons

doi: 10.1155/2016/8360738

Figure Lengend Snippet: AP39 generates H 2 S in WT mice and AP39 increased H 2 S levels in APP/PS1 mice. (a) The contribution of AP39 to H 2 S production in the cortex and hippocampus of WT mice. (b) AP39 increased H 2 S levels in APP/PS1 mice. ## P < 0.01, compared with the WT group; ∗∗ P < 0.01, compared with the control group (H 2 O).

Article Snippet: AP39, a novel mitochondria-targeted H 2 S donor, was designed and synthesized by Medicilon Inc., as described previously [ , ].

Techniques: Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons

doi: 10.1155/2016/8360738

Figure Lengend Snippet: AP39 ameliorated the learning and memory deficits of APP/PS1 mice. WT or AD mice were treated with water or AP39 (100 nM/kg) for 6 weeks, followed by assessment using the Morris water maze and novel object recognition task tests. (a) Spatial learning and memory in AD mice are scored as the latency to locate a hidden platform. (b) After 24 h, a 60-s probe trial was performed. (c) Spatial learning was tested as the latency to locate a hidden platform for the 25 nM/kg AP39-treated mice and scored. (d) Effects of AP39 on the memory performance of 12-month WT or AD mice treated with water or AP39 were tested in the NORT test.

Article Snippet: AP39, a novel mitochondria-targeted H 2 S donor, was designed and synthesized by Medicilon Inc., as described previously [ , ].

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons

doi: 10.1155/2016/8360738

Figure Lengend Snippet: AP39 alleviated the brain atrophy of APP/PS1 mice as observed by brain magnetic resonance imaging (MRI). Brain MRI data were collected at different coronal and axial sections in 12-month-old mice receiving different treatments. Notes: panels (a), (c), and (e) show coronal sections, and panels (b), (d), and (f) show axial sections. The highlighted white regions indicate cerebrospinal fluid (CSF) in the ventricle, and the hippocampus is adjacent to the ventricle. Representative images from brain MRI depict slices of the T2-weighted morphologic images of 12-month-old WT mice treated with water (a)-(b) or AD mice treated with water (c)-(d) or AP39 (e)-(f). (g) ADC value determination of the mouse brains.

Article Snippet: AP39, a novel mitochondria-targeted H 2 S donor, was designed and synthesized by Medicilon Inc., as described previously [ , ].

Techniques: Magnetic Resonance Imaging

Journal: Oxidative Medicine and Cellular Longevity

Article Title: AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons

doi: 10.1155/2016/8360738

Figure Lengend Snippet: AP39 reduced A β production and A β deposition in APP/PS1 mice. (a)-(b) After intraperitoneal injection of AP39 for 6 weeks, A β 40 and A β 42 levels in the mouse brains were decreased from 925 pg/mL and 532 pg/mL to 531 pg/mL and 365 pg/mL, respectively. (c) A β plaques were detected in the cortex and the hippocampus of the WT and APP/PS1 mouse brains based on 6E10 immunostaining. The statistical results show the area of the A β plaques. Original magnification: –40x; scale bars = 100 μ m.

Article Snippet: AP39, a novel mitochondria-targeted H 2 S donor, was designed and synthesized by Medicilon Inc., as described previously [ , ].

Techniques: Injection, Immunostaining

Journal: Experimental Animals

Article Title: AP39 ameliorates high fat diet-induced liver injury in young rats via alleviation of oxidative stress and mitochondrial impairment

doi: 10.1538/expanim.21-0056

Figure Lengend Snippet: Effect of AP39 on obesity. High-fat-diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) rats were intravenously injected with 0.05 or 0.1 mg/kg AP39 once a day for 7 weeks. (A) Schematic abstract of the experimental process. (B, C) Body weight and food intake of young rats was recorded once a week for 7 weeks. ** P< 0.01 vs. Control; ^ P< 0.05, ^^ P< 0.01 vs. HFD.

Article Snippet: To investigate the impact of AP39 on HFD rats, the rats in HFD+L-AP39 and HFD+H-AP39 groups were injected daily by tail vein with AP39 (APExBIO, Houston, TX, USA) at the dose of 0.05 mg/kg or 0.1 mg/kg, respectively [ ].

Techniques: Injection, Control

Journal: Experimental Animals

Article Title: AP39 ameliorates high fat diet-induced liver injury in young rats via alleviation of oxidative stress and mitochondrial impairment

doi: 10.1538/expanim.21-0056

Figure Lengend Snippet: Effect of AP39 on high-fat-diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD). (A) At the end of the experiment, the liver tissues were harvested. Gross morphology of liver tissue was observed. (B) Liver weight index was calculated according to the formula liver wet weight / body weight × 100%. (C, D) The histological alternations of liver tissues were visualized with Oil red O staining (×200) and hematoxylin and eosin (H&E) staining (×200), and the representative photomicrographs were shown. (E) NAFLD activity score, a histological scoring system for NAFLD, was counted. ** P< 0.01 vs. Control; ^ P< 0.05, ^^ P< 0.01 vs. HFD.

Article Snippet: To investigate the impact of AP39 on HFD rats, the rats in HFD+L-AP39 and HFD+H-AP39 groups were injected daily by tail vein with AP39 (APExBIO, Houston, TX, USA) at the dose of 0.05 mg/kg or 0.1 mg/kg, respectively [ ].

Techniques: Staining, Activity Assay, Control

Journal: Experimental Animals

Article Title: AP39 ameliorates high fat diet-induced liver injury in young rats via alleviation of oxidative stress and mitochondrial impairment

doi: 10.1538/expanim.21-0056

Figure Lengend Snippet: Effect of AP39 on lipid profiles in serum of rats. (A–E) Relative levels of serum ALT, AST, TC, TG, LDL-C and HDL-C were determined using the corresponding kits. ALT, alanine transaminase; AST, aspartate transaminase; TC, total cholesterol; TG, total triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol. ** P< 0.01 vs. Control; ^ P< 0.05, ^^ P< 0.01 vs. HFD.

Article Snippet: To investigate the impact of AP39 on HFD rats, the rats in HFD+L-AP39 and HFD+H-AP39 groups were injected daily by tail vein with AP39 (APExBIO, Houston, TX, USA) at the dose of 0.05 mg/kg or 0.1 mg/kg, respectively [ ].

Techniques: Control

Journal: Experimental Animals

Article Title: AP39 ameliorates high fat diet-induced liver injury in young rats via alleviation of oxidative stress and mitochondrial impairment

doi: 10.1538/expanim.21-0056

Figure Lengend Snippet: Effect of AP39 on high-fat diet (HFD)-induced oxidative stress. (A) DHE staining (×400) was used for ROS detection in liver. (B–D) Levels of markers of oxidative stress, MDA, GSH and SOD, were measured with commercial kits. ROS, reactive oxygen species; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase. ** P< 0.01 vs. Control; ^ P< 0.05, ^^ P< 0.01 vs. HFD.

Article Snippet: To investigate the impact of AP39 on HFD rats, the rats in HFD+L-AP39 and HFD+H-AP39 groups were injected daily by tail vein with AP39 (APExBIO, Houston, TX, USA) at the dose of 0.05 mg/kg or 0.1 mg/kg, respectively [ ].

Techniques: Staining, Control

Journal: Experimental Animals

Article Title: AP39 ameliorates high fat diet-induced liver injury in young rats via alleviation of oxidative stress and mitochondrial impairment

doi: 10.1538/expanim.21-0056

Figure Lengend Snippet: Effect of AP39 on H 2 S level and mitochondrial function. (A) Hepatic H 2 S level was detected using the H 2 S determination kit. (B) mtDNA copy number in the liver was measured by quantitative real-time polymerase chain reaction (RT-qPCR). (C) The degree of mitochondrial swelling was analyzed at 520 nm wavelength on a spectrophotometer. (D) Mitochondrial membrane potential (MMP) changes were detected using JC-1 probe. (E) Hypoxia-inducible factor-1α (HIF-1α) expression at mRNA and protein levels was determined by RT-qPCR and western blot, respectively. ** P< 0.01 vs. Control; ^ P< 0.05, ^^ P< 0.01 vs. high-fat diet (HFD).

Article Snippet: To investigate the impact of AP39 on HFD rats, the rats in HFD+L-AP39 and HFD+H-AP39 groups were injected daily by tail vein with AP39 (APExBIO, Houston, TX, USA) at the dose of 0.05 mg/kg or 0.1 mg/kg, respectively [ ].

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Spectrophotometry, Membrane, Expressing, Western Blot, Control