Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type I IFN-producing CD4 Valpha14i NKT cells facilitate priming of IL-10-producing CD8 T cells by hepatocytes.

doi: 10.4049/jimmunol.178.4.2083

Figure Lengend Snippet: FIGURE 6. The cytokine response of OT-I CD8 T cells primed by HC with or without NKT cell help. A, OT-I CD8 T cells (105/well) were cocul- tured with GalCer- and SIINFEKL peptide-pulsed HC (104/well) with () or without () NKT cells (4 104/well). Cells were harvested after a 72-h culture, incubated for 4 h with ionomycin (750 ng/ml) and PMA (50 ng/ml), washed, and tested for IL-10 secretion in a 3-h capture assay. Cells were labeled with a PE-conjugated anti-IL-10 mAb and an anti-CD8 mAb. Data from a representative of five independent experiments are shown. B, OT-I CD8 T cells (105/well) were cocultured for 72 h with GalCer-/ SIINFEKL peptide-pulsed HC (104/well) with () or without () NKT cells (4 104/well). CD8 T blasts were harvested, purified, and restimu- lated in vitro for 48 h with peptide-pulsed DC. IFN-, IL-2, and IL-10 in 48-h supernatants of these secondary cultures were determined by ELISA. Mean values of triplicates (SEM) of a representative of four independent experiments are shown.

Article Snippet: IL-10-producing CD8 T cells were identified by a cytokine capture assay (catalog no. 130-090-489; Miltenyi Biotec) combined with surface staining with the allophycocyanin-conjugated anti-CD8 mAb 53-6.7 (catalog no. 553035; BD Biosciences).

Techniques: Incubation, Labeling, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: SIRT1-mediated deacetylation of FOXO3 enhances mitophagy and drives hormone resistance in endometrial cancer.

doi: 10.1186/s10020-024-00915-7

Figure Lengend Snippet: Fig. 3 Effects of silencing or overexpressing SIRT1 on EC cell growth, proliferation, migration, and invasion. A Schematic diagram of the cell experiments; B Viability changes of RL95-2 cells after silencing or overexpressing SIRT1 at 12, 24, 36, and 48 h measured using the CCK-8 assay; C Proliferation capacity of RL95-2 cells after silencing or overexpressing SIRT1 detected using the EdU assay, where EdU-positive cells appear pink, and EdU-negative cells appear blue; D Colony formation assay to measure the colony formation ability of RL95-2 cells after silencing or overexpressing SIRT1; E Transwell assay to evaluate the migration and invasion capacity of RL95-2 cells after silencing or overexpressing SIRT1; F Wound healing assay to assess the migration of RL95-2 cells after silencing or overexpressing SIRT1; G Flow cytometry analysis to detect apoptosis of RL95-2 cells after silencing or overexpressing SIRT1. Data are presented as mean ± SD, and each cell experiment was repeated three times. *p < 0.05 compared to the sh-NC group; #p < 0.05 compared to the oe-NC group

Article Snippet: Apoptosis in EC cells was detected using the Annexin V-FITC/PI apoptosis detection kit (C1062L, Beyotime).

Techniques: Migration, CCK-8 Assay, EdU Assay, Colony Assay, Transwell Assay, Wound Healing Assay, Flow Cytometry

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: SIRT1-mediated deacetylation of FOXO3 enhances mitophagy and drives hormone resistance in endometrial cancer.

doi: 10.1186/s10020-024-00915-7

Figure Lengend Snippet: Fig. 5 Impact of SIRT1 overexpression on EC cell survival via FOXO3. A Schematic diagram of the experimental procedure; B CCK-8 assay to measure the viability changes of EC cells in different intervention groups at 12, 24, 36, and 48 h; C EdU assay to assess the proliferation ability of EC cells in different intervention groups, with EdU-positive cells shown in pink and EdU-negative cells shown in blue; D Colony formation assay to evaluate the colony-forming ability of EC cells in different intervention groups; E Transwell assay to investigate the migration and invasion ability of EC cells in different intervention groups; F Wound healing assay to examine the migration of EC cells in different intervention groups; G Flow cytometry analysis to detect the apoptosis of EC cells in different intervention groups; H JC-1 staining experiment to assess the change in MMP of EC cells in different intervention groups. Data are presented as mean ± SD, with each cellular experiment repeated 3 times. *p < 0.05 compared to the oe-NC + sh-NC group; #p < 0.05 compared to the oe-SIRT1 + sh-NC group

Article Snippet: Apoptosis in EC cells was detected using the Annexin V-FITC/PI apoptosis detection kit (C1062L, Beyotime).

Techniques: Over Expression, CCK-8 Assay, EdU Assay, Colony Assay, Transwell Assay, Migration, Wound Healing Assay, Flow Cytometry, Staining

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: SIRT1-mediated deacetylation of FOXO3 enhances mitophagy and drives hormone resistance in endometrial cancer.

doi: 10.1186/s10020-024-00915-7

Figure Lengend Snippet: Fig. 8 Overexpression of SIRT1 enhances the intracellular growth of EC cells. A Diagram illustrating the procedure of animal experiments (n = 6); B Line graph showing the tumor volume increase in subcutaneous tumor model mice from day 8 to 44 (n = 6); C Dissection of subcutaneous transplanted tumors in mice from each group on day 44 (n = 6); D Statistical analysis of tumor weight in subcutaneous tumor model mice on day 44 (n = 6); E TUNEL staining for apoptosis in mouse tumors from each group (n = 6); F MitoSOX immunofluorescence staining for ROS production in mouse tumor tissues from each group (n = 6). Data are presented as mean mean ± SD, with 6 nude mice in each group. *p < 0.05 compared to sh-NC group; #p < 0.05 compared to oe-NC group

Article Snippet: Apoptosis in EC cells was detected using the Annexin V-FITC/PI apoptosis detection kit (C1062L, Beyotime).

Techniques: Over Expression, Dissection, TUNEL Assay, Staining, Immunofluorescence

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: SIRT1-mediated deacetylation of FOXO3 enhances mitophagy and drives hormone resistance in endometrial cancer.

doi: 10.1186/s10020-024-00915-7

Figure Lengend Snippet: Fig. 10 Overexpression of SIRT1 enhances tumor growth by inducing cell autophagy. A Schematic diagram of the animal experiment process (n = 6); B Line graph showing the growth of subcutaneously transplanted tumors in mice from day 8 to day 36 (n = 6); C Anatomical diagram of subcutaneously transplanted tumors in mice on day 36 (n = 6); D Statistics of tumor weight in mice with subcutaneously transplanted tumors on day 36 (n = 6); E TUNEL staining to detect apoptosis of tumor cells in mice in each group (n = 6); F MitoSOX immunofluorescence staining to detect the generation of ROS in tumor tissues of mice in each group (n = 6). Data are presented as mean ± SD, with 6 nude mice in each group. *p < 0.05 compared to the PBS group; #p < 0.05 between the two groups

Article Snippet: Apoptosis in EC cells was detected using the Annexin V-FITC/PI apoptosis detection kit (C1062L, Beyotime).

Techniques: Over Expression, TUNEL Assay, Staining, Immunofluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Identification and analysis of islet antigen specific CD8+ T cells with T cell libraries

doi: 10.4049/jimmunol.1800267

Figure Lengend Snippet: Libraries were prepared from CD8+CD45RO+ cells sorted from the peripheral blood as described from 29 patients with T1D and 15 HC. Each well was expanded with irradiated allogeneic PBMC+IL-2, IL-7, and IL-15 as described in Materials and Methods. After 10 days the wells were washed and stimulated with K562 cells that had been pulsed with 6 diabetes peptides, peptides from EBV/flu, or treated with DMSO alone. IFNγ levels in the supernatants were measured by ELISA after 6 days. Data from a representative patient and HC subject are shown in Supplemental Figure 2B. The frequencies of positive wells that were above the threshold, which was mean+3SD of DMSO wells were calculated for each subject. There was a significantly greater proportion of positive wells from CD45RO+ cells from patients with T1D reactive with islet antigens (A)(*p=0.028, t-test with Welch’s correction) compared to HC, but not to peptides from EBV/flu (B). CD45RA+CD8+ cells were sorted from 25 and 13 of the T1D patients and HC respectively. (C,D) There was not a significant difference between the frequency of islet antigen-reactive (C) or EBV/flu reactive cells (D). The relationship between frequency of CD45RO+ CD8+ islet antigen-reactive cells and diabetes duration (E) or age (F) are shown (p=ns, Spearman corr). The orange and green dots represent samples from concordant identical triplets and twins respectively.

Article Snippet: To identify the TCRs ex vivo , PBMCs were stimulated with the ZnT8 186–194 peptide for 6 hours and IFNγ+ and – CD8+ cells, identified with anti-CD8 mAb(HIT8a, PerCP-Cy5.5, Biolegend) and an anti-IFNγ antibody (130–054–201, Miltenyi), were sorted by a flow cytometer.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Identification and analysis of islet antigen specific CD8+ T cells with T cell libraries

doi: 10.4049/jimmunol.1800267

Figure Lengend Snippet: (A) Positive wells from the CD8+ T cell libraries from 4 patients with T1D and 3 HC subjects were further expanded with cytokines and challenged with K562 cells pulsed with each individual peptide used in the original pool. The levels of IFNγ were measured after 6 days. The data are from 11 wells from the 3 patients described in the text (Pt 116, Pt 69, and Pt 63) with T1D and 3 HC subjects (HC1023, 19, PC24). Each graph represents the analysis of positive wells from an individual patient. The bars (black and grey) represent the cytokine responses of different positive wells to the peptides. There were positive responses to ZnT8186–194 in wells from the patients with T1D but responses to IGRP228–236, PPI34–42, PPI15–24, and ZnT8186–194 in the HC subjects. (B) The CD45RO+ cells from one library well without and two library wells with an IFNγ response to the peptide pulsed K562 cells from Pt 63 were expanded in cytokines and stained with tetramers loaded with ZnT8186–194 peptide or control tetramer and analyzed by flow cytometry. The percentages refer to the frequency of tetramer+ cells in the CD8+ gate.

Article Snippet: To identify the TCRs ex vivo , PBMCs were stimulated with the ZnT8 186–194 peptide for 6 hours and IFNγ+ and – CD8+ cells, identified with anti-CD8 mAb(HIT8a, PerCP-Cy5.5, Biolegend) and an anti-IFNγ antibody (130–054–201, Miltenyi), were sorted by a flow cytometer.

Techniques: Staining, Control, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Identification and analysis of islet antigen specific CD8+ T cells with T cell libraries

doi: 10.4049/jimmunol.1800267

Figure Lengend Snippet: (A) The Vα and Vβ sequences, identified in 5C above from pt 63, were detected, using PCR for the TCR chains, in positive (+) but not negative (−) library wells (selected for IFNγ responses). (DW=control well) There were multiple wells in the CD45RO libraries but a single positive well from the CD45RA+ libraries in which the Vα and Vβ sequences were identified. (B) PBMC isolated in a repeat draw from the same patient were cultured with ZnT8186–194 peptide. IFNγ+CD8+ and IFNγ−CD8+ T cells were identified with a capture assay and sorted. The presence of the TCR Vα and Vβ chains were again identified by PCR (arrow) in the IFNγ+ cells.

Article Snippet: To identify the TCRs ex vivo , PBMCs were stimulated with the ZnT8 186–194 peptide for 6 hours and IFNγ+ and – CD8+ cells, identified with anti-CD8 mAb(HIT8a, PerCP-Cy5.5, Biolegend) and an anti-IFNγ antibody (130–054–201, Miltenyi), were sorted by a flow cytometer.

Techniques: Control, Isolation, Cell Culture