mtor inhibitor rapamycin  (InvivoGen)

Journal: Signal Transduction and Targeted Therapy

Article Title: Tissue-adapted Tregs harness inflammatory signals to promote intestinal repair from therapy-related injury

doi: 10.1038/s41392-025-02476-5

Figure Lengend Snippet: T reg cells promote organoid growth via mTORC1 and Myc activation in ISCs. Murine SI organoids were co-cultured with CD25 high FoxP3-GFP + T reg cells alone or in the presence of IFNγ and IL-10R blocking antibodies, or were stimulated with rIFNγ + rIL-10 for 4 days, and subsequently analyzed via scRNA-Seq. The data were pooled from 3 independent experiments. a Plots of single cells in UMAP space for all experimental conditions, colored by SingleR cell type annotation. b Venn diagrams indicating the overlap of upregulated pathways between experimental conditions and control organoids. Only gene sets/pathways significantly upregulated when controlling for an FDR of 10% were considered. c Heatmap of Pearson correlation coefficients obtained from correlating GSEA-derived NESs (normalized enrichment scores) between conditions. The values indicate correlations of regulated gene sets/pathway activation between different conditions in the indicated cell types. Only gene sets/pathways significantly up- or downregulated when controlling for an FDR of 10% were considered. d Dot plot of the GSEA results of selected pathways/gene sets for different cell types and treatments (vs. control). Dots are colored according to the negative log 10 of the GSEA q value (FDR), and the sign indicates the direction of the regulation (up positive, down negative). The size of the dots corresponds to the GSEA NES. Gene sets/pathways are derived from the Hallmark (H) and Reactome (R) gene set collections of MSigDB. e Relative organoid growth of SI WT organoids ± stimulation with 0.25 ng/mL rIFNγ and rIL-10 and ± the mTOR inhibitor rapamycin (1 µg/mL) or control (DMSO), f ± myc inhibition (compound 10058-F4, 100 µM/mL) or control (DMSO), and g ± co-cultured T reg cells ± myc inhibition ± mTOR inhibitor. The dotted line represents the growth of control organoids without stimulation. The number of biological replicates ( n ), indicating the number of separate organoid culture experiments, is shown in the figure. The data were analyzed via ordinary one-way ANOVA for multiple comparisons and are presented as the means ± S.E.M.s

Article Snippet: The mTOR inhibitor rapamycin (1 μg/mL, InvivoGen) or the c-myc inhibitor 10058-F4 (100 μM/mL, Sigma‒Aldrich) was added 6 h after organoid seeding.

Techniques: Activation Assay, Cell Culture, Blocking Assay, Control, Derivative Assay, Inhibition

Journal: Signal Transduction and Targeted Therapy

Article Title: Tissue-adapted Tregs harness inflammatory signals to promote intestinal repair from therapy-related injury

doi: 10.1038/s41392-025-02476-5

Figure Lengend Snippet: IFNγ and IL-10 compensate for the depletion of epithelial growth factors. Murine SI organoids were cultured under normal growth conditions (ENR) or EGF-depleted conditions (NR + anti-EGF antibody) and stimulated with the indicated cytokines. a Representative images, b quantification of organoid size on day 6 of culture, c relative organoid growth (number of organoids after first passage, compared with control conditions) ( n = 4 independent experiments), and d relative organoid growth during long-term culture and several passages ( n = 3 independent experiments). e Relative organoid growth of murine SI organoids as described above ± mTOR or myc inhibitors ( n = 4 independent experiments). f Human LI organoids were cultured under optimal (WENR) or EGF-depleted (WNR) conditions and stimulated with the indicated cytokines ± mTOR or myc inhibitors. The area of viable organoids per image (used as a surrogate marker for organoid size) was determined on day 6, and g ) the relative organoid growth (number of viable organoids) was determined after the first passage ( n = 4 independent experiments). h Murine SI organoids were stimulated for 16 h with the indicated cytokines (representative plots). i The cell cycle phase was analyzed to distinguish proliferating (G1/2/S/M phase) and non-proliferating (G0 phase) cells ( n = 6 independent experiments). The proportion of proliferating cells was statistically analyzed. j Murine SI organoids were stimulated for 5 days with the indicated cytokines, and the abundance of Lrg5+ ISCs among all viable epithelial cells (EpCAM + ) and k the number of proliferating (EdU + ) cells among all viable epithelial cells were determined via flow cytometry ( n = 5 independent experiments). l Human LI organoids were subjected to Wnt-depleted conditions (ENR) and stimulated with the indicated cytokines or Wnt. Organoid size on day 6 of culture ( n = 7 independent experiments). m Human LI organoids were cultured under Wnt-depleted conditions and stimulated with the indicated cytokines or Wnt. The number of viable organoids was determined on day 6 of culture ( n = 4 independent experiments). n Murine SI organoids were cultured and mechanically disrupted. One hundred organoids were seeded into culture and stimulated with the indicated cytokines immediately ( < 5 min) or after 90 min. The number of viable organoids was determined on day 6 of culture ( n = 6–15 engraftment culture wells from 3–6 independent experiments). o Analysis of Ifng +/+ and Ifng −/− mice on day 7 after starting ABI (5 × 4.5 Gy/day from day 0 until day 4). The number of Ki-67 + epithelial cells within SI epithelial crypt cells was quantified, and p representative immunohistochemistry images are shown. Data from 2 independent experiments with n = 9 Ifng +/+ mice and n = 9 Ifng −/− mice were pooled, and a total of 131 crypts were analyzed. q The number of Lgr5 + (Lgr5-GFP + ) ISCs within small intestinal epithelial crypts was quantified, and r representative in situ hybridization images are shown. Data from 2 independent experiments with n = 7 Ifng +/+ mice and n = 9 Ifng −/− mice were pooled, and a total of 993 crypts were analyzed. Violin plots ( o , q ) showing the distribution of values, with medians (solid lines) and quartiles (dotted lines) indicated. All the other data are presented as the means ± S.E.M. p values were calculated via two-tailed t tests or ordinary one-way ANOVA for multiple comparisons

Article Snippet: The mTOR inhibitor rapamycin (1 μg/mL, InvivoGen) or the c-myc inhibitor 10058-F4 (100 μM/mL, Sigma‒Aldrich) was added 6 h after organoid seeding.

Techniques: Cell Culture, Control, Marker, Flow Cytometry, Immunohistochemistry, In Situ Hybridization, Two Tailed Test