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

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Histone Methyltransferase G9a Activates Autophagy and Provides Protection in Dry Eye Disease

doi: 10.1167/iovs.66.14.52

Figure Lengend Snippet: Validation of the mTOR–G9a–ATG7 axis in an in vitro DED model. ( A ) Western blot analysis of p-mTOR and total mTOR expression in control and HO groups (β-actin adjusted, n = 3). ( B ) The effect of mTOR activation on G9a mRNA expression in cells was measured by qPCR ( n = 3). ( C ) Western blot analysis of p-mTOR, mTOR, G9a, and H3K9me2 protein levels, with quantification of relative protein levels (β-actin adjusted, n = 3). ( D ) Western blot analysis of ATG7 expression and quantification in different treatment groups (β-actin adjusted, n = 3). ( E ) ATG7 mRNA levels in vitro for control, HO, and UNC0642 groups were measured by qPCR ( n = 3). ( F ) Changes in H3K9me2 binding at the ATG7 promoter after UNC0642 intervention were analyzed through ChIP experiments ( n = 3). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The experimental design involved three pharmacological interventions: (1) G9a inhibition, 18-hour pretreatment with UNC0642 (T4166; TargetMol, Boston, MA, USA); (2) autophagy blockade, 1-hour treatment with chloroquine (CQ; Sigma-Aldrich, St. Louis, MO, USA); and (3) mammalian target of rapamycin (mTOR) activation, 30-minute pretreatment with MHY1485 (T1908; TargetMol).

Techniques: Biomarker Discovery, In Vitro, Western Blot, Expressing, Control, Activation Assay, Binding Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Histone Methyltransferase G9a Activates Autophagy and Provides Protection in Dry Eye Disease

doi: 10.1167/iovs.66.14.52

Figure Lengend Snippet: Schematic diagram of the G9a-mediated epigenetic mechanism of autophagy in DED. Under hyperosmotic stress in HCECs, mTOR-mediated histone methyltransferase G9a negatively regulates autophagy by catalyzing the formation of inhibitory H3K9me2 marks in the ATG7 promoter region. The use of UNC0642 can counteract this modification and restore the inflammatory, apoptotic, and oxidative stress-induced damage of HCECs by activating autophagy.

Article Snippet: The experimental design involved three pharmacological interventions: (1) G9a inhibition, 18-hour pretreatment with UNC0642 (T4166; TargetMol, Boston, MA, USA); (2) autophagy blockade, 1-hour treatment with chloroquine (CQ; Sigma-Aldrich, St. Louis, MO, USA); and (3) mammalian target of rapamycin (mTOR) activation, 30-minute pretreatment with MHY1485 (T1908; TargetMol).

Techniques: Modification

Journal: Neural Regeneration Research

Article Title: The compound ( E )-2-(3,4-dihydroxystyryl)-3-hydroxy-4H-pyran-4-one alleviates neuroinflammation and cognitive impairment in a mouse model of Alzheimer’s disease

doi: 10.4103/NRR.NRR-D-23-01890

Figure Lengend Snippet: Effects of D30 on PI3K-AKT pathway signaling and Gal-3 expression in vitro. (A, B) GO enrichment analysis (bubble map; A) and KEGG pathway analysis (histogram; B) performed as part of the pharmacological network analysis. (C) PI3K, p-AKT, AKT, p-mTOR, and mTOR expression in the hippocampus, as measured by immunoblot, after two fAβ injections. (D) Band densities are expressed as ratios of PI3K, p-AKT, AKT, p-mTOR, and mTOR to β-actin. (E) PI3K , AKT , mTOR , and Gal-3 mRNA expression levels in primary microglial cells stimulated with fAβ, as measured by qPCR. Values are expressed as the mean ± SEM ( n = 3–4). * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with Tukey’s multiple comparisons post hoc test). AKT: Protein kinase B; D30: ( E )-2-(3,4-dihydroxystyryl)-3-hydroxy-4H-pyran-4-one; fAβ: fibrillar amyloid-β; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; mTOR: mammalian target of rapamycin; p-AKT: phosphorylated protein kinase B; p-mTOR: phosphorylated mammalian target of rapamycin; PI3K: phosphatidylinositol-3-hydroxykinase; qPCR: quantitative polymerase chain reaction.

Article Snippet: Next, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-glial fibrillary acidic protein (GFAP; rabbit, 1:8000, Proteintech, Wuhan, China, Cat# 16825-1-AP, RRID: AB_2109646), anti-Iba1 (goat, 1:1000, Abcam, Cat# ab5076, RRID: AB_2224402), anti-NeuN (rabbit, 1:1000, CST, Danvers, MA, USA, Cat# 24307, RRID: AB_2651140), anti-inducible nitric oxide synthase (iNOS; rabbit, 1:2000, Proteintech, Cat# 22226-1-AP, RRID: AB_2879038), anti-Aβ (rabbit, 1:1000, CST, Cat# 8243, RRID: AB_2797642), anti-phosphatidylinositol-3-hydroxykinase (PI3K; rabbit, 1:1000, CST, Cat# 4249, RRID: AB_2165248), anti-phosphorylated protein kinase B (p- AKT; rabbit, 1:2000, CST, Cat# 4060, RRID: AB_2315049), anti-AKT (rabbit, 1:2000, CST, Cat# 4691, RRID: AB_915783), anti-phosphorylated mammalian target of rapamycin (p-mTOR; rabbit, 1:1000, CST, Cat# 5536, RRID:AB_10691552), anti-mammalian target of rapamycin (mTOR; rabbit, 1:1000, CST, Cat# 2983, RRID: AB_2105622), anti-synaptophysin (rabbit, 1:5000, Abcam, Cat# ab32127, RRID: AB_2286949), anti-postsynaptic density protein 95 (PSD95; mouse, 1:3000, Abcam, Cat# ab13552, RRID: AB_300453), anti-Gal-3 (rabbit, 1:4000, Proteintech, Cat# 14979-1-AP, RRID: AB_2136768), anti-CD68 (rabbit, 1:2000, Proteintech, Cat# 28058-1-AP, RRID: AB_2881049), anti-C3 (rabbit, 1:3000, Abcam, Cat# ab97462, RRID: AB_10679468), anti-TNF-α (rabbit, 1:2000, Proteintech, Cat# 26405-1-AP, RRID: AB_2918102), anti-vesicular γ-aminobutyric acid transporter (VGAT; guinea pig, 1:2000, SYSY, Göttingen, Germany, Cat# 131-004, RRID: AB_887873), anti-β-actin (mouse, 1:6000, Proteintech, Cat# 66009-1-lg, RRID: AB_2782959), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; rabbit, 1:1000, CST, Cat# 5174, RRID: AB_10622025).

Techniques: Expressing, In Vitro, Western Blot, Real-time Polymerase Chain Reaction