t47d wt cell line  (ATCC)

Journal: Cell Death and Differentiation

Article Title: Ripk1 is critical for preserving effector regulatory T cells and the suppressive transcriptional program in regulatory T cells

doi: 10.1038/s41418-025-01550-3

Figure Lengend Snippet: A Immunoblot analysis of Ripk1 in FACS-sorted Tcon (CD4 + YFP – ) and Treg (CD4 + YFP + ) cells from Ripk1 Δ Foxp3 and Foxp3 Cre (control) mice. Pictures of cervical ( B ) and inguinal ( C ) area of 9-week-old Ripk1 Δ Foxp3 and Foxp3 Cre mice. Arrows indicate lymph nodes. D Representative histological sections of paraffin-embedded tissue of the indicated organs of Ripk1 Δ Foxp3 and Foxp3 Cre mice stained with hematoxylin and eosin. E Heat map of histological score of the indicated organs of Ripk1 Δ Foxp3 and Foxp3 Cre mice ( n = 5, each group). F Heat map of bone marrow erythropoiesis and granulopoiesis of Ripk1 Δ Foxp3 and Foxp3 Cre mice ( n = 5, each group).

Article Snippet: Naïve CD4 + T cells were isolated from pooled spleen and lymph node single-cell suspensions from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 mice using Naive CD4 + T Cell Isolation kit (130-104-453, Miltenyi Biotec) according to manufacturer ́s instructions.

Techniques: Western Blot, Control, Staining

Journal: Cell Death and Differentiation

Article Title: Ripk1 is critical for preserving effector regulatory T cells and the suppressive transcriptional program in regulatory T cells

doi: 10.1038/s41418-025-01550-3

Figure Lengend Snippet: A Representative dot plots of Foxp3-expressing cells within CD4 + cells of Ripk1 Δ Foxp3 and Foxp3 Cre mice. Percentages (left) and absolute cell number (right) of CD4 + Foxp3 + cells ( B ), CD4 + cells ( C ), CD8 + cells ( D ), and B cells ( E ) of spleen and lymph nodes (LN) from Ripk1 Δ Foxp3 and Foxp3 Cre mice. F Concentration of the indicated cytokines in sera from Ripk1 Δ Foxp3 and Foxp3 Cre mice determined by Luminex assay ( Ripk1 Δ Foxp3 n = 13 , Foxp3 Cre n = 5). Bar graphs represent the mean ± SEM. Statistical analyses were performed by two-tailed Mann–Whitney tests. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significant. G Representative zebra plots (left) and frequencies (right) of Treg cell activation markers CD62L and CD44 in CD4 + Foxp3 + cells from spleen and lymph nodes (LN) of Ripk1 Δ Foxp3 and Foxp3 Cre mice. B – E , G Mean ± SEM are given. Each symbol represents a single mouse in the scatter plots. H Representative histograms and geometric mean fluorescence intensity (gMFI) bar graphs of ICOS (upper) and TIGIT (lower) expression in CD4 + Foxp3 + cells from spleen and lymph nodes (LN) of Ripk1 Δ Foxp3 and Foxp3 Cre mice ( n = 2, each). I Representative histograms and gMFI bar graphs of Ki67 expression in CD4 + Foxp3 + cells from spleen and lymph nodes (LN) of Ripk1 Δ Foxp3 and Foxp3 Cre mice ( n = 2, each). J Representative histograms (top) and summary bar graph (bottom) of active caspase-3/7 in CD4 + YFP + cells from spleen and lymph nodes (LN) of Ripk1 Δ Foxp3 and Foxp3 Cre mice ( n = 5, each). B – G , J Statistical analyses were performed by two-tailed Mann–Whitney tests. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significant.

Article Snippet: Naïve CD4 + T cells were isolated from pooled spleen and lymph node single-cell suspensions from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 mice using Naive CD4 + T Cell Isolation kit (130-104-453, Miltenyi Biotec) according to manufacturer ́s instructions.

Techniques: Expressing, Concentration Assay, Luminex, Two Tailed Test, MANN-WHITNEY, Activation Assay, Fluorescence

Journal: Cell Death and Differentiation

Article Title: Ripk1 is critical for preserving effector regulatory T cells and the suppressive transcriptional program in regulatory T cells

doi: 10.1038/s41418-025-01550-3

Figure Lengend Snippet: A Immunoblot analysis of Ripk1 in purified Treg cells from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 (control) mice treated with tamoxifen or vehicle for the indicated time points. B Representative dot plots, C Foxp3 + hCD2 + percentages, and D Foxp3 geometric mean fluorescence intensity (gMFI) of events within live CD4 + cells of MACS-sorted naïve CD4 + T cells from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 ( Ripk1 fl/fl ; n = 4) and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 ( Ripk1 wt/wt control; n = 4) mice treated with tamoxifen for 72 h and subsequently with Treg cell polarizing conditions. Data was obtained from four independent experiments. E Representative dot plots (left) and bar graphs (right) of viability analysis determined by CellEvent Caspase-3/7 and LIVE/DEAD Fixable Blue staining of MACS-sorted CD4 + hCD2 + cells from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 ( Ripk1 fl/fl ; n = 5) and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 ( Ripk1 wt/wt control; n = 5) mice and treated in vitro with Tamoxifen for 72 h. Data was obtained from five independent experiments. F Frequencies of viable cells determined by LIVE/DEAD Fixable Blue staining of MACS-sorted CD4 + hCD2 + cells from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 ( Ripk1 fl/fl ; n = 8) and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 ( Ripk1 wt/wt control; n = 8) mice and treated in vitro with Tamoxifen for 72 h and, subsequently with TNF (50 ng/ml), anti-CD3 and anti-CD28, combination of anti-CD3, anti-CD28 and TNF (50 ng/ml) or left untreated for 16 additional hours. C – F Data in bar graphs are represented as mean ± SEM. Statistical analyses were performed by two-tailed Mann–Whitney tests. * p < 0.05, n.s. not significant.

Article Snippet: Naïve CD4 + T cells were isolated from pooled spleen and lymph node single-cell suspensions from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 mice using Naive CD4 + T Cell Isolation kit (130-104-453, Miltenyi Biotec) according to manufacturer ́s instructions.

Techniques: Western Blot, Purification, Control, Fluorescence, Staining, In Vitro, Two Tailed Test, MANN-WHITNEY

Journal: Cell Death and Differentiation

Article Title: Ripk1 is critical for preserving effector regulatory T cells and the suppressive transcriptional program in regulatory T cells

doi: 10.1038/s41418-025-01550-3

Figure Lengend Snippet: A Distribution of the percentages of YFP - and YFP + cells within the CD4 + Foxp3 + population of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 11). B Representative dot plots of Foxp3-expressing cells within CD4 + cells (upper) and YFP - expressing cells within Foxp3 + cells (lower) from spleen, lymph nodes (LN) and thymus of Ripk1 fl/fl Foxp3 Cre/wt mice. C Ki67 positive events in YFP - and YFP + cells within CD4 + CD25 + cells from spleen and lymph nodes (LN) of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 11). D Proliferation represented by inverse geometric mean fluorescence intensity (gMFI) of CTV dilution from FACS sorted CD4 + CD25 + YFP – and CD4 + CD25 + YFP + cells of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 4) stimulated with anti-CD3 (1 µg/ml), anti-CD28 (2 µg/ml) and IL-2 (10 ng/ml) for 72 h. E Active caspase-3/7 positive events in YFP - and YFP + cells within CD4 + CD25 + cells from spleen and lymph nodes (LN) of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 11). F Viable cell frequency of FACS sorted CD4 + CD25 + YFP - and CD4 + CD25 + YFP + cells of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 6) stimulated with anti-CD3 (1 µg/ml), anti-CD28 (2 g/ml) and IL-2 (10 ng/ml) for 16 h. G Representative zebra plots (left) and distribution in frequencies (right) of resting (rTreg; CD62L + CD44 - ) and effector (eTreg; CD62L - CD44 + ) Treg cells within CD4 + Foxp3 + cells from spleen and lymph nodes (LN) of Ripk1 wt/wt Foxp3 Cre/wt mice ( n = 6) and Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 7). Data in bar graphs are represented as mean ± SEM. Statistical analyses were performed by Kruskal-Wallis test followed by Dunn´s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, otherwise not significant. H Geometric mean fluorescence intensity (gMFI) of Foxp3 in CD4 + CD25 + cells from spleen and lymph nodes (LN) of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 6) and Ripk1 wt/wt Foxp3 Cre/wt mice ( n = 4). I Fold change of average percentages of YFP + vs. YFP - events within eTreg cells from spleen and lymph nodes (LN) of Ripk1 wt/wt Foxp3 Cre/wt mice and Ripk1 fl/fl Foxp3 Cre/wt mice. J Frequency of Foxp3 + cells in CD4 + cells in lamina propria (LP) and in the intraepithelial lymphocyte compartment (IEL) of small intestine of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 4). K Distribution in frequency of YFP - and YFP + within CD4 + Foxp3 + cells and their respective Helios ( L ) and RORγt expression ( M ) in lamina propria (LP) and in the intraepithelial lymphocyte (IEL) compartment of small intestine of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 4). ( A , C – F , H and J – M ) Data in bar graphs are represented as mean ± SEM. Statistical analyses were performed by two-tailed Mann–Whitney tests. * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant.

Article Snippet: Naïve CD4 + T cells were isolated from pooled spleen and lymph node single-cell suspensions from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 mice using Naive CD4 + T Cell Isolation kit (130-104-453, Miltenyi Biotec) according to manufacturer ́s instructions.

Techniques: Expressing, Fluorescence, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Cell Death and Differentiation

Article Title: Ripk1 is critical for preserving effector regulatory T cells and the suppressive transcriptional program in regulatory T cells

doi: 10.1038/s41418-025-01550-3

Figure Lengend Snippet: A Chord plot of differentially expressed genes in FACS sorted CD4 + CD25 + YFP - (Ripk1 +/+ ) and CD4 + CD25 + YFP + (Ripk1 –/– ) cells from Ripk1 fl/fl Foxp3 Cre/wt mice. Upregulated genes in CD4 + CD25 + YFP + (Ripk1 –/– ) cells are labeled in red (positive logFC) and downregulated genes are labeled in blue (negative logFC). Chord plot shows a detailed relationship between the log-fold change of differentially expressed genes (DEG) (left semicircle) and their enriched gene ontology (GO) terms (right semicircle). B Heat map analysis of Treg gene signature in FACS sorted CD4 + CD25 + YFP – (Treg Ripk1 +/+ ) and CD4 + CD25 + YFP + (Treg Ripk1 –/– ) cells from Ripk1 fl/fl Foxp3 Cre/wt mice. C Representative CNS2/TSDR methylation patterns of FACS sorted CD4 + CD25 + YFP – and CD4 + CD25 + YFP + cells from Ripk1 fl/fl Foxp3 Cre/wt mice. Amplicons are vertically arranged, each representing a single CpG-motif. Color code indicates the percentage of CNS2/TSDR methylation in the purified populations ( n = 4). Data was obtained from two independent experiments. D Seurat clustering of scRNA-seq of FACS-sorted CD4 + CD25 hi cells from spleen and lymph nodes of Ripk1 fl/fl Foxp3 Cre/wt mice. UMAP projections represent clusters of pre-labeled YFP - (left) and YFP + (right) cells. E scRNASeq volcano plot showing changes in gene expression of CD4 + CD25 hi YFP + (Treg Ripk1 –/– ) cells vs. CD4 + CD25 hi YFP - (Treg Ripk1 +/+ ) cells. Heat map analysis of ( F ) Bcl-2 family members, ( G ) NF-κB pathway components, ( H ) Top20 downregulated NF-κB target genes in YFP + cells and ( I ) NF-κB target genes of cytokines/chemokines in FACS sorted CD4 + CD25 + YFP - (Treg Ripk1 +/+ ) and CD4 + CD25 + YFP + (Treg Ripk1 –/– ) cells from Ripk1 fl/fl Foxp3 Cre/wt mice.

Article Snippet: Naïve CD4 + T cells were isolated from pooled spleen and lymph node single-cell suspensions from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 mice using Naive CD4 + T Cell Isolation kit (130-104-453, Miltenyi Biotec) according to manufacturer ́s instructions.

Techniques: Labeling, Methylation, Purification, Gene Expression