Journal: The Journal of Experimental Medicine

Article Title: CD28-inducible transcription factor DEC1 is required for efficient autoreactive CD4 + T cell response

doi: 10.1084/jem.20122387

Figure Lengend Snippet: Early transcriptional regulation of activated naive T cells. (A) List of transcription factors highly correlated (P < 1 × 10 −9 ) with the transcriptome of in vitro anti-CD3/anti-CD28–activated primary human T cells, ranked in descending order of connectivity ratio. This ratio indicates the correlation between known transcriptional targets and transcript expression levels. The input gene list is composed of transcripts with a fold change of more than two by 4 h after T cell activation. (B) Volcano plot of transcripts involved in transcriptional control as annotated by GO Biological Processes. Microarray data were obtained by activating primary human T cells with anti-CD3/anti-CD28, with harvest at the times indicated. Transcription factors from the table in A with fold change greater than two and P < 0.05 are labeled in the figure. (C) Two-dimensional representation of global gene expression by PCA. 16,279 transcripts for each sample are included in the analysis. Each dot represents a sample, which is colored and shaped according to activation parameters. The combination of PC1 and PC2 summarizes 64% of the original dataset variation. (D and E) Gene expression analysis of human naive T cells by quantitative real-time PCR after 24 h of activation as indicated with or without 100 U/ml of recombinant human IL-2 and 25 µg/ml BSB. (F) DEC1 mRNA expression in human naive CD4 + T cells at different time points after activation, indexed to expression in unstimulated cells. Data are represented as mean ± SEM. P-values were calculated with an unpaired Student’s t test: *, P < 0.05; **, P < 0.01. (G) DEC1 protein expression by Western blot from 5 × 10 6 human naive CD4 + T cells 24 h after activation. Results are from at least three independent experiments. MM, molecular mass.

Article Snippet: TaqMan primer probes (Applied Biosystems) used for human experiments were IL-2 (Hs99999150_m1), BHLHE40 (Hs00186419_m1), CD25 (Hs00907777_m1), BCLXL (Hs99999146_m1), NR4A3 (Hs00545009_g1), SGPP2 (Hs00544786_m1), TNFRSF9 (Hs00155512_m1), TNFRSF14 (Hs00542476_g1), UHRF1 (Hs00273589_m1), CSF2 (Hs00929873_m1), and ZBTB20 (Hs00210321_m1); and for mouse experiments were Bhlhe40 (Mm00478593_m1), Cd25 (Mm00434261_m1), Nr4a3 (Mm01354011_m1), Il2 (Mm99999222_m1), Ifng (Mm00801778_m1), Zbtb20 (Mm00457765_m1), Bclxl (Mm00437783_m1), and csf2 (Mm01290062_m1).

Techniques: In Vitro, Expressing, Activation Assay, Control, Microarray, Labeling, Gene Expression, Real-time Polymerase Chain Reaction, Recombinant, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: CD28-inducible transcription factor DEC1 is required for efficient autoreactive CD4 + T cell response

doi: 10.1084/jem.20122387

Figure Lengend Snippet: DEC1 is necessary for efficient in vitro T cell function. (A) Jurkat T cells were infected with lentivirus containing pSICO-R vectors with shRNA complementary to human DEC1 transcript or a scrambled shRNA. RNA was harvested, and expression of DEC1 transcript was measured by quantitative real-time PCR. (B) 10 6 Jurkat cells previously infected with shRNA expression virus were seeded on day 0. The total number of cells in culture was followed for 8 d. (C) Percentage of CD69 + and CD25 + naive CD4 + T cells from DEC1 WT and DEC1 KO mice after activation and 24 h of culture. (D) IL-2 cytokine production measured by ELISA in primary CD4 + T cells from DEC1 WT and DEC1 KO animals at 4 and 25 h after activation. (E and F) Cultured primary DEC1 WT and DEC1 KO CD4 + T cells were followed for 6 d after stimulation with anti-CD3 + anti-CD28 without (E) or with (F) 100 U/ml of recombinant mouse IL-2 at day 0. Viable cells were automatically counted by video imaging (Vi-CELL). Percentage of live DEC1 KO CD4 + T cells is expressed relative to the DEC1 WT cells. (G and H) CFSE labeling analysis of viable DEC1 WT and DEC1 KO cells activated with anti-CD3 + anti-CD28 and cultured for the indicated time periods without (G) or with (H) the addition of 100 U/ml IL-2 at day 0. (I and J) Representative histogram plots of Ki67 expression after 6 d of culture with the conditions described in G and H. Cells were cultured without (I) or with (J) the addition of 100 U/ml IL-2 at day 0. Results are from at least three independent experiments. Data are represented as mean ± SEM. P-values were calculated with an unpaired Student’s t test: *, P < 0.05; **, P < 0.01.

Article Snippet: TaqMan primer probes (Applied Biosystems) used for human experiments were IL-2 (Hs99999150_m1), BHLHE40 (Hs00186419_m1), CD25 (Hs00907777_m1), BCLXL (Hs99999146_m1), NR4A3 (Hs00545009_g1), SGPP2 (Hs00544786_m1), TNFRSF9 (Hs00155512_m1), TNFRSF14 (Hs00542476_g1), UHRF1 (Hs00273589_m1), CSF2 (Hs00929873_m1), and ZBTB20 (Hs00210321_m1); and for mouse experiments were Bhlhe40 (Mm00478593_m1), Cd25 (Mm00434261_m1), Nr4a3 (Mm01354011_m1), Il2 (Mm99999222_m1), Ifng (Mm00801778_m1), Zbtb20 (Mm00457765_m1), Bclxl (Mm00437783_m1), and csf2 (Mm01290062_m1).

Techniques: In Vitro, Cell Function Assay, Infection, shRNA, Expressing, Real-time Polymerase Chain Reaction, Virus, Activation Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Imaging, Labeling

Journal: The Journal of Experimental Medicine

Article Title: CD28-inducible transcription factor DEC1 is required for efficient autoreactive CD4 + T cell response

doi: 10.1084/jem.20122387

Figure Lengend Snippet: DEC1 deficiency protects mice from EAE. EAE was induced by CFA and MOG 35–55 peptide. (A and B) Clinical disease score (A) and incidence (B). Number of animals per group: WT n = 20, het n = 11, and KO n = 18. (C) Spinal cord tissue sections were stained with H&E from DEC1 WT and DEC1 KO mice at day 15 after MOG 35–55 immunization (peak disease for WT mice). Bars, 100 µm. (D) Single cell suspensions from cerebellum and spinal cord of DEC1 WT and DEC1 KO mice at day 15 after immunization were used for quantification of CD4 + T cells among CNS cells. (E) Flow cytometric analysis of MHC class II expression in CNS-infiltrating myeloid cells and microglia cells from unprimed mice and DEC1 WT or DEC1 KO mice at day 15 after MOG 35–55 immunization. (F) Production of IFN-γ, GM-CSF, and IL-17 by CNS-infiltrating cells of unprimed, DEC1 WT , and DEC1 KO mice at day 15 after priming (gated on CD45 hi CD11b − CD4 + ). (G and H) RAG KO mice were transferred with 2 × 10 6 CD4 + T cells isolated from DEC1 WT or DEC1 KO mice. 7 d after the transfer, EAE was induced by MOG 35–55 immunization. EAE clinical score was determined every other day. Data are representative of at least three different independent experiments. Number of animals per group: WT n = 7 and KO n = 8. Data are represented as mean ± SEM. P-values were calculated with an unpaired Student’s t test: *, P < 0.05.

Article Snippet: TaqMan primer probes (Applied Biosystems) used for human experiments were IL-2 (Hs99999150_m1), BHLHE40 (Hs00186419_m1), CD25 (Hs00907777_m1), BCLXL (Hs99999146_m1), NR4A3 (Hs00545009_g1), SGPP2 (Hs00544786_m1), TNFRSF9 (Hs00155512_m1), TNFRSF14 (Hs00542476_g1), UHRF1 (Hs00273589_m1), CSF2 (Hs00929873_m1), and ZBTB20 (Hs00210321_m1); and for mouse experiments were Bhlhe40 (Mm00478593_m1), Cd25 (Mm00434261_m1), Nr4a3 (Mm01354011_m1), Il2 (Mm99999222_m1), Ifng (Mm00801778_m1), Zbtb20 (Mm00457765_m1), Bclxl (Mm00437783_m1), and csf2 (Mm01290062_m1).

Techniques: Staining, Expressing, Isolation

Journal: The Journal of Experimental Medicine

Article Title: CD28-inducible transcription factor DEC1 is required for efficient autoreactive CD4 + T cell response

doi: 10.1084/jem.20122387

Figure Lengend Snippet: DEC1 is required for efficient T cell priming during EAE. (A and B) Total cellularity (A) and CD4 + T cell quantification (B) from pooled spleen and lymph nodes of DEC1 WT or DEC1 KO mice at day 7 after MOG 35–55 immunization. (C and D) Flow cytometric analysis (C) and quantification (D) of MOG 38–49 tetramer staining of CD4 + T cells subpopulations from spleen and lymph nodes of DEC1 WT or DEC1 KO mice at day 7 after MOG 35–55 immunization. Data are representative of at least six independent experiments. (E) Flow cytometric analysis (left) and CD25 quantification (right) of MOG 38–49 tetramer–stained CD4 + T cell subpopulations. MFI, mean fluorescence intensity. (F) Flow cytometric analysis (left) and Ki67 quantification (right) of MOG 38–49 tetramer–stained CD4 + T cell subpopulations. (G and H) Spleen and lymph node cells collected from immunized DEC1 WT or DEC1 KO mice or from unimmunized control mice were cultured in the presence of the indicated concentrations of MOG 35–55 peptide for 3 d. Tritiated thymidine was added to restimulated cultures for 14 h before harvest. Cytokine production was measured by ELISA after 48 h of culture. Results are from at least three independent experiments. Data are represented as mean ± SEM. P-values were calculated with an unpaired Student’s t test: *, P < 0.05.

Article Snippet: TaqMan primer probes (Applied Biosystems) used for human experiments were IL-2 (Hs99999150_m1), BHLHE40 (Hs00186419_m1), CD25 (Hs00907777_m1), BCLXL (Hs99999146_m1), NR4A3 (Hs00545009_g1), SGPP2 (Hs00544786_m1), TNFRSF9 (Hs00155512_m1), TNFRSF14 (Hs00542476_g1), UHRF1 (Hs00273589_m1), CSF2 (Hs00929873_m1), and ZBTB20 (Hs00210321_m1); and for mouse experiments were Bhlhe40 (Mm00478593_m1), Cd25 (Mm00434261_m1), Nr4a3 (Mm01354011_m1), Il2 (Mm99999222_m1), Ifng (Mm00801778_m1), Zbtb20 (Mm00457765_m1), Bclxl (Mm00437783_m1), and csf2 (Mm01290062_m1).

Techniques: Staining, Fluorescence, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: CD28-inducible transcription factor DEC1 is required for efficient autoreactive CD4 + T cell response

doi: 10.1084/jem.20122387

Figure Lengend Snippet: Exogenous survival signals can restore DEC1-deficient T cell expansion during EAE priming phase. (A) Total cellularity from pooled spleen and lymph nodes of DEC1 WT or DEC1 KO mice at day 7 after MOG 35–55 immunization with or without IL-2 complex treatment (days 1, 3, and 5). (B) Quantification of MOG 38–49 tetramer–positive CD4 + T cells of the DEC1 WT or DEC1 KO cells described in A. (C) Development of EAE clinical disease in DEC1 WT or DEC1 KO mice treated with IL-2 complex during priming phase (treated on days 1, 3, and 5) or during EAE onset (labeled “ONSET,” treated on days 6–8). (D) Spleen and lymph nodes cells collected at day 7 after immunization from DEC1 WT or DEC1 KO mice treated with IL-2 complex or untreated were cultured in the presence of the indicated concentrations of MOG 35–55 peptide for 3 d. Tritiated thymidine was added to restimulated cultures for 14 h before harvest. (E and F) Bone marrow cells from DEC1 WT and DEC1 KO mice were mixed and transferred to mice lacking endogenous T cells. Mixed bone marrow chimeras were immunized with MOG 35–55 peptide at least 7 wk after cell transfer. Splenocytes and CNS-infiltrating lymphocytes were harvested and analyzed at day 15 after immunization (peak of disease). Flow cytometry (E) was used for quantification (F) of MOG 38–49 tetramer staining of CD4 + T cells subpopulations from chimera splenocytes. (G) CD25 staining and quantification of T reg and T conv cells from mixed bone marrow chimera splenocytes. MFI, mean fluorescence intensity. (H) Ki67 staining and quantification of T reg and T conv cells from mixed bone marrow chimera splenocytes. Results are from at least three independent experiments. Data are represented as mean ± SEM. P-values were calculated with an unpaired Student’s t test: *, P < 0.05.

Article Snippet: TaqMan primer probes (Applied Biosystems) used for human experiments were IL-2 (Hs99999150_m1), BHLHE40 (Hs00186419_m1), CD25 (Hs00907777_m1), BCLXL (Hs99999146_m1), NR4A3 (Hs00545009_g1), SGPP2 (Hs00544786_m1), TNFRSF9 (Hs00155512_m1), TNFRSF14 (Hs00542476_g1), UHRF1 (Hs00273589_m1), CSF2 (Hs00929873_m1), and ZBTB20 (Hs00210321_m1); and for mouse experiments were Bhlhe40 (Mm00478593_m1), Cd25 (Mm00434261_m1), Nr4a3 (Mm01354011_m1), Il2 (Mm99999222_m1), Ifng (Mm00801778_m1), Zbtb20 (Mm00457765_m1), Bclxl (Mm00437783_m1), and csf2 (Mm01290062_m1).

Techniques: Labeling, Cell Culture, Flow Cytometry, Staining, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: CD28-inducible transcription factor DEC1 is required for efficient autoreactive CD4 + T cell response

doi: 10.1084/jem.20122387

Figure Lengend Snippet: DEC1 is required for proinflammatory cytokine production in a T cell–intrinsic manner. (A) Percentage of DEC1 WT CD4 + T cells during EAE peak disease in spleen and CNS of DEC1 WT/KO mixed bone marrow chimeras compared with DEC1 KO CD4 + T cells. (B) Flow cytometry and analysis of CD4 + MOG 38–49 tetramer T cells from CNS-infiltrating cells. (C) Production of IFN-γ, GM-CSF, IL17, and IL2 by CNS-infiltrating DEC1 WT and DEC1 KO cells (gated on CD45 hi CD11b − CD4 + ). Results are from at least three independent experiments. Data are represented as mean ± SEM. P-values were calculated with an unpaired Student’s t test: *, P < 0.05.

Article Snippet: TaqMan primer probes (Applied Biosystems) used for human experiments were IL-2 (Hs99999150_m1), BHLHE40 (Hs00186419_m1), CD25 (Hs00907777_m1), BCLXL (Hs99999146_m1), NR4A3 (Hs00545009_g1), SGPP2 (Hs00544786_m1), TNFRSF9 (Hs00155512_m1), TNFRSF14 (Hs00542476_g1), UHRF1 (Hs00273589_m1), CSF2 (Hs00929873_m1), and ZBTB20 (Hs00210321_m1); and for mouse experiments were Bhlhe40 (Mm00478593_m1), Cd25 (Mm00434261_m1), Nr4a3 (Mm01354011_m1), Il2 (Mm99999222_m1), Ifng (Mm00801778_m1), Zbtb20 (Mm00457765_m1), Bclxl (Mm00437783_m1), and csf2 (Mm01290062_m1).

Techniques: Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: CD28-inducible transcription factor DEC1 is required for efficient autoreactive CD4 + T cell response

doi: 10.1084/jem.20122387

Figure Lengend Snippet: Overlap in CD28- and DEC1-dependent transcriptional profiles after T cell activation. (A) Functional analysis performed by IPA based on genes 1.4-fold differentially expressed between DEC1 WT and DEC1 KO naive T cells activated with anti-CD3 plus anti-CD28 for 24 h. The significance of the association of the gene expression pattern with a biological function is calculated using right-tailed Fisher’s exact test. (B, top) CD28 co-stimulation–dependent signature described as a scatter plot summarizing the gene expression differences between DEC1 WT naive T cells activated with anti-CD3 versus anti-CD3 plus anti-CD28. (bottom) Quantification of genes significantly up- and down-regulated by the effect of CD28 signaling (P < 0.05). (C, top) Red dots represent significantly up-regulated genes between DEC1 WT naive T cells activated with anti-CD3 plus anti-CD28 and DEC1 KO naive T cells activated with anti-CD3 plus anti-CD28. (bottom) Pie graph indicates the percentage of a set of DEC1 up-regulated genes whose expression was regulated by CD28 co-stimulation, in the direction indicated. (D, top) Green dots represent significantly down-regulated genes between DEC1 WT naive T cells activated with anti-CD3 plus anti-CD28 and DEC1 KO naive T cells activated with anti-CD3 plus anti-CD28. (bottom) Pie graph indicates the percentage of a set of DEC1 down-regulated genes whose expression was regulated by CD28 co-stimulation, in the direction indicated. P-values were calculated by χ 2 test. (E) PCA of DEC1-dependent genes in DEC1 WT and DEC1 KO naive T cells activated with anti-CD3 alone or anti-CD3 plus anti-CD28 at 24 h. The DEC1-regulated genes include genes differentially expressed between CD28–co-stimulated DEC1 WT and DEC1 KO T cells (594 genes; P < 0.05). Each dot represents a sample, which is colored and shaped according to activation parameters. The combination of PC1 and PC2 summarizes 84% of the dataset variation. (F) mRNA expression of the indicated genes at 24 h after activation of DEC1 WT and DEC1 KO naive T cells with anti-CD3 alone or anti-CD3 plus anti-CD28. (G) Gene expression analysis as in F with or without 100 U/ml of recombinant IL-2 or 25 µg/ml anti-CD25. Results are from at least three independent experiments. Data are represented as mean ± SEM.

Article Snippet: TaqMan primer probes (Applied Biosystems) used for human experiments were IL-2 (Hs99999150_m1), BHLHE40 (Hs00186419_m1), CD25 (Hs00907777_m1), BCLXL (Hs99999146_m1), NR4A3 (Hs00545009_g1), SGPP2 (Hs00544786_m1), TNFRSF9 (Hs00155512_m1), TNFRSF14 (Hs00542476_g1), UHRF1 (Hs00273589_m1), CSF2 (Hs00929873_m1), and ZBTB20 (Hs00210321_m1); and for mouse experiments were Bhlhe40 (Mm00478593_m1), Cd25 (Mm00434261_m1), Nr4a3 (Mm01354011_m1), Il2 (Mm99999222_m1), Ifng (Mm00801778_m1), Zbtb20 (Mm00457765_m1), Bclxl (Mm00437783_m1), and csf2 (Mm01290062_m1).

Techniques: Activation Assay, Functional Assay, Gene Expression, Expressing, Recombinant

Journal: Nature Communications

Article Title: Chlorella-derived natural photosynthetic system for in situ energy metabolism enhancement in cardiomyocytes

doi: 10.1038/s41467-025-63749-9

Figure Lengend Snippet: A Bright-field and fluorescence-field images of C. pyre in HAMA, scale bar, 5 μm. B Activity of C. pyre . C Dissolved O 2 level changes of the solution containing HCU with various treatments. D Flow chart of induced hypoxia in cardiomyocytes. E Fluorescence images of H9c2 cells staining with RDPP probe after various treatments. F Fluorescence images of H9c2 cells staining with HIF-1α antibody (red) and DAPI (blue) after various treatments, scale bar: 50 μm. G Quantitative fluorescence analysis of RDPP probes ( n = 5, n represent the number of independent experiments, mean ± s.d.). H Quantitative fluorescence analysis of HIF-1α ( n = 5, n represent the number of independent experiments, mean ± s.d.). I The levels of proteins including HIF-1α, Bcl-2, C-Caspase 3 and Cyt c in H9c2 cells analyzed by western blotting. J – M Corresponding quantitative histograms of protein expression ( n = 5, n represent the number of independent experiments, mean ± s.d.). N Anti-apoptosis analysis of H9c2 cells by flow cytometry after various treatments. O Corresponding quantitative histograms of anti-apoptosis rate ( n = 3, n represent the number of independent experiments, mean ± s.d.). Statistical analysis was performed using unpaired two-tailed Student’s t test. Independent experiments were performed ( n = 3) for ( A ) with similar results. Source data are provided as a Source Data file.

Article Snippet: The antibodies used are listed below: HIF-1α (1:1000, BOSTER, China, Cat No. ZP1229BP29, Lot No. PB9253); Cyt c (1:5000, Zenbio, China, Cat No. R22867 , Lot No. N19AP7P); C-Caspase3 (1:1000, Zenbio, China, Cat No. 341034, Lot No. N10AP01); Bcl-2 (1:1000, BOSTER, China, Cat No. BA0412, Lot No. 24 F 11 0305C19); β-Actin (1:1000, BOSTER, China, Cat No. BM0627, Lot No. 17K15B27).

Techniques: Fluorescence, Activity Assay, Staining, Western Blot, Expressing, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Chlorella-derived natural photosynthetic system for in situ energy metabolism enhancement in cardiomyocytes

doi: 10.1038/s41467-025-63749-9

Figure Lengend Snippet: A Schematic illustration of the animal treatment protocol in the MI model. B ECG of mice before (left) and after (right) induction. C Echocardiography of mice in different treatment groups after treatment for 7 days. D – G Quantitative analysis of the EF, FS, LVESV and LVESD at different periods ( n = 6, n represent biologically independent animal samples in each group, mean ± s.d.). H Representative images of the mouse ischemic cardiac detected by Color Doppler Flow Imaging. I TTC staining of hearts from mice in different groups. J Masson trichrome staining displayed the fibrous tissue (blue) and myocardium (red) and HE trichrome staining of sections of hearts from mice in different groups, scale bar: 100 μm. K HIF-1α (red) and DAPI immunostaining (blue) of myocardium in different groups, scale bar: 50 μm. L – N Quantitative analysis of collagen area, fibrosis area, infarct area ( n = 6, n represent biologically independent animal samples in each group, mean ± s.d.). Statistical analysis was performed using unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.

Article Snippet: The antibodies used are listed below: HIF-1α (1:1000, BOSTER, China, Cat No. ZP1229BP29, Lot No. PB9253); Cyt c (1:5000, Zenbio, China, Cat No. R22867 , Lot No. N19AP7P); C-Caspase3 (1:1000, Zenbio, China, Cat No. 341034, Lot No. N10AP01); Bcl-2 (1:1000, BOSTER, China, Cat No. BA0412, Lot No. 24 F 11 0305C19); β-Actin (1:1000, BOSTER, China, Cat No. BM0627, Lot No. 17K15B27).

Techniques: Imaging, Staining, Immunostaining, Two Tailed Test