Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a-c , CD8 + T cells were treated with EPZ004777 for 48 hours. Western Blot showed H3K79me2 in CD8 + T cells ( a ). FACS demonstrated CD8 + T cell apoptosis ( b ) and cytokines ( c ). d , Western Blot showed H3K79me2 in Dot1l f/f and Dot1l −/− CD8 + T cells. e, f , Effect of DOT1L deletion on CD8 + T cells apoptosis ( e ) and cytokines ( f ). g-i , Effect of T cell DOT1L deficiency on MC38 growth ( g, h ) and T cell viability ( i ). j, k , Effect of methionine supplementation on Dot1l f/f and Dot1l −/− CD8 + T cells apoptosis ( j ) and cytokines ( k ). l , GSEA plot showed enriched apoptotic pathway in Dot1l −/− CD8 + T cells. m , Heat map showed Jak-Stats mRNA levels in mouse Dot1l −/− vs Dot1l f/f CD8 + T cells. n , Western blot showed STAT5 and p-STAT5 in Dot1l f/f ( f/f ) and Dot1l −/− (−/−) CD8 + T cells. o , Western blot showed STAT5 and p-STAT5 in CD8 + T cells cultured with fresh medium (FM), Supernatant (Sup), and Sup supplemented with different metabolites (Sup+). p , ChIP assay showed H3K79me2 occupancy on the Stat5b promoter in CD8 + T cells. q , ChIP assay showed H3K79me2 occupancy on the Stat5b promoter in CD8 + T cells cultured with FM, Sup, or Sup+Met. Data are mean ± s.e.m. Sample sizes (n), P values, statistical tests and number of times experiments were replicated are listed in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Western Blot, Cell Culture

Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a, b , H3K79me2 ( a ) and STAT5 ( b ) levels in CD8 + T cells from tumor draining lymph node (dLN) and tumor in B16F10 bearing mice. c, d , H3K79me2 ( c ) and STAT5 ( d ) levels in CD8 + T cells from spleen and tumor ascites in ID8 bearing mice. e , H3K79me2 levels in CD8 + T cells from healthy peripheral blood and human ovarian cancers ascites. f, g , H3K79me2 ( f ) and STAT5 ( g ) levels in CD8 + T cells from healthy human blood and human ovarian cancer omentum tissues. h, i , FACS showed H3K79me2 and STAT5 levels in human tumor infiltrating CD8 + T cells. j-m , Effect of methionine on human tumor infiltrating CD8 + T cells. Human colorectal cancer infiltrating CD8 + T cells were cultured with or without methionine. T cell cytokine production ( j, k ), H3K79me2 ( l ), and STAT5 ( m ) were analyzed by FACS. One representative of four is shown. n , Effect of methionine supplementation on apoptosis of tumor infiltrating CD8 + T cells and ID8 tumor cells in vivo. ID8 tumor bearing mice were treated with methionine or PBS. T cell and tumor cell apoptosis was determined by FACS. o , Methionine levels in ID8 tumor after methionine or PBS treatment. p-r: Effect of anti-PD-L1 on methionine-affected CT26 tumor progression. Mice bearing CT26 tumor were treated with anti-PD-L1, methionine, and their combination. Tumor volume ( p ), T cell tumor infiltration ( q ) and apoptosis ( r ) were assessed. Data are mean ± s.e.m. Information on sample sizes, experimental number, times, biological replicates, statistical tests, and P values is available in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Cell Culture, In Vivo

Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a-e , Methionine supplementation restored T cell immunity in B16F10-bearing mice. Tumor growth ( a ), tumor infiltrating CD8 + T cell H3K79me2 ( b ) and STAT5 ( c ) were monitored. FACS showed intratumor CD8 + T cell apoptosis ( d ) and cytokines ( e ) . f-h , Methionine supplementation restored T cell immunity in ID8-bearing mice. Tumor growth was monitored by bioluminescence imaging ( f ). FACS showed ascites CD8 + T cell ( g ) and intratumor CD8 + T cell cytokines ( h ) . i-l , Studies on colorectal cancer patients treated with methionine. Western blot showed p-STAT5 and H3K79me2 in peripheral CD8 + T cells prior and post methionine treatment ( i ). FACS showed IL-2 + T cells ( j ), CD8 + T cell effector cytokines ( k ) and apoptosis ( l ) in patients prior and post methionine treatment. Data are mean ± s.e.m. Sample sizes (n), P values, statistical tests and number of times experiments were replicated are listed in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Imaging, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Suppression of Inflammatory and Fibrotic Signals by Cinnamon ( Cinnamomum cassia ) and Cinnamaldehyde in Cyclophosphamide-Induced Overactive Bladder in Mice

doi: 10.1155/2021/5205759

Figure Lengend Snippet: Effects of CNP and CNA on changes in selected protein expression levels of cyclophosphamide- (CYP-, 300 mg/kg) induced OAB mice 24 hours after CYP induction. (a) Upper, typical Western blotting analysis using whole bladder tissue from different groups was conducted to show changes in CD11b, pp65NF κ B, TLR4, SCF, and M3; β -actin was included for normalization. (b) Statistical results from the densitometry measurements after normalization to β -actin. Animal groups are as described in . Data are presented as mean ± SEM ( n = 5 for each group). † ∗ p < 0.05, compared with the corresponding vehicle- (Veh-) treated OAB group (CYP + Veh) or sham control group (C), respectively, by one-way ANOVA followed by S-N-K t -test.

Article Snippet: After fixation, permeabilization, and blocking, the bladder slices were randomly selected for incubation with proper primary antibodies against muscarinic M2 and M3 (1 : 200) (US Biological, CO, USA), CD11b and β -catenin (1 : 50, Abcam, Cambridge, UK), pp65 NF κ B (1 : 50, BD PharMingen, San Diego, CA, USA), MIF and TLR4 (1 : 100, GeneTex, Irvine, CA, USA), SCF (1 : 50, Santa Cruz Biotechnology, Inc., Santa.

Techniques: Expressing, Western Blot, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Suppression of Inflammatory and Fibrotic Signals by Cinnamon ( Cinnamomum cassia ) and Cinnamaldehyde in Cyclophosphamide-Induced Overactive Bladder in Mice

doi: 10.1155/2021/5205759

Figure Lengend Snippet: Graphical illustration showing the key pathways regulated by CNP and CNA. CNP and CNA have a profound effect on the inhibition of cyclophosphamide- (CYP-) induced OAB bladder injury in inflammation and fibrosis pathways via inhibiting MIF/TLR4- and SCF/c-Kit-mediated signal transduction, which in turn suppresses pp65NF κ B/CD11b and β -catenin/ α -SMA upregulation, respectively.

Article Snippet: After fixation, permeabilization, and blocking, the bladder slices were randomly selected for incubation with proper primary antibodies against muscarinic M2 and M3 (1 : 200) (US Biological, CO, USA), CD11b and β -catenin (1 : 50, Abcam, Cambridge, UK), pp65 NF κ B (1 : 50, BD PharMingen, San Diego, CA, USA), MIF and TLR4 (1 : 100, GeneTex, Irvine, CA, USA), SCF (1 : 50, Santa Cruz Biotechnology, Inc., Santa.

Techniques: Inhibition, Transduction

Journal: Cell death & disease

Article Title: SCF and IL-33 regulate mouse mast cell phenotypic and functional plasticity supporting a pro-inflammatory microenvironment.

doi: 10.1038/s41419-023-06139-7

Figure Lengend Snippet: Fig. 7 SCF controls connective tissue-like MC accumulation in tumor lesions. A Schematic representation of anti-SCF neutralizing Ab administration in vivo. AOM/DSS-treated mice were i.p. injected three times with anti-SCF or control Ab (100 μg/mouse) starting from the fourth DSS cycle. Treated and control mice were sacrificed at 13 weeks from AOM administration. B Colon paraffin-embedded sections from AOM/DSS mice treated with Ctrl-Ig or anti-SCF neutralizing antibodies as described in (A) were stained with anti-MCP4 Ab followed by Alexa Fluor 488 secondary Abs (green). Nuclei were counterstained with DAPI (blue) and images were acquired with a Zeiss LSM980 confocal microscopy using a 20× objective. The frequencies of MCs positive for mMCP4 protease were analyzed in 20 fields randomly acquired from tumor lesions and shown as mean ± SD cells/field. Paired Student’s t test: *p < 0.05. Number of adenomas and colon lengths are shown in (C). Paired Student’s t test: *p < 0.05. Graphs are representative of two independent experiments with a total of 5 mice/group.

Article Snippet: For blocking experiments, mice were i.p. injected with anti-SCF neutralizing antibody (AB-455-NA R&D Systems) or normal goat control IgG (AB-108-C R&D Systems) (100μg/mouse) three times every 10 days starting one day before the fourth DSS cycle (see Fig. 7A).

Techniques: In Vivo, Injection, Control, Staining, Confocal Microscopy

Journal: PLoS ONE

Article Title: Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

doi: 10.1371/journal.pone.0076822

Figure Lengend Snippet: A , KIT Y721 phosphorylation was activated by exogenous rhKITLG (100 ng/mL for 10 min following 2 h serum deprivation [ , ]) in GIST-T1 cells containing a heterozygous activating KIT mutation (n=4/group) and in GIST-T1-5R cells containing an additional, imatinib-resistant KIT mutation (n=6/group), but not in GIST882 cells lacking a WT KIT allele (n=5/group). B , Culturing with anti-human KITLG neutralizing antibody for 4 days inhibited baseline proliferation of GIST-T1 cells ( P <0.001; n=3; regression and 95% confidence band are shown as solid and dashed lines, respectively) and GIST-T1-5R cells ( P <0.001; n=6). The effect of KITLG immunoneutralization on GIST-T1 cells was blocked by preabsorbing the antibody with 10-fold molar excess of rhKITLG (see open symbols in the left panel, second row; n=3/group). KITLG immunoneutralization did not inhibit the proliferation of GIST882 or GIST48B cells lacking a WT KIT allele (note that GIST48B cells also express very low to undetectable levels of KIT protein, see ) and of LX-2 cells lacking KIT protein (n=3/cell line). C , Inhibition of the proliferation of GIST-T1 and GIST-T1-5R cells by siRNA-mediated knock-down of KITLG (n=4/cell line/group; P GIST-T1 : day 2: 0.008, days 4, 6 and 8: <0.001; P GIST-T1-5R : day 2: <0.02, day 4: 0.004, days 6 and 8: <0.001).

Article Snippet: The role of endogenous KITLG in the proliferation of GIST-T1, GIST-T1-5R, GIST882, GIST48B and LX-2 cells was tested by culturing in the above media in the presence of purified, azide-free goat polyclonal anti-human KITLG antibody (AB-255-NA, R&D Systems, Minneapolis, MN; applied for 4 days at concentrations indicated in the Results), which has been shown to neutralize KITLG-induced proliferation in the TF1 human erythroleukemic cell line [ ].

Techniques: Phospho-proteomics, Mutagenesis, Inhibition, Knockdown

Journal: PLoS ONE

Article Title: Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

doi: 10.1371/journal.pone.0076822

Figure Lengend Snippet: Results obtained in murine and human smooth muscle cells ( A - E ), human LX-2 stellate cells ( F - G ) and human GIST-T1 cells ( H - I ) are shown. A , Hoffman modulation contrast image of primary human gastric smooth muscle cells. B , KITLG mRNA (total: soluble+membrane-bound) was readily detectable in primary human smooth muscle cells (passage 3) maintained with Smooth Muscle Growth Medium-2 containing insulin, hFGF-B, hEGF and 5% FBS (Lonza) but not in 24-h growth factor- and serum-deficient basal medium. C , Both IGF1 (100 ng/mL) and the GSK3α/β inhibitor SB415286 (30 µM) stimulated Kitl expression in murine gastric tunica muscularis organotypic cultures (n=3/group). D - E , SB415286 stimulated endogenous Kitl expression in murine primary gastric smooth muscle cells ( D ; n=3/group) and KITLG transcriptional activity in the same cell type transfected with a KITLG promoter (-2120 bp to +407 bp)-pGL3 luciferase construct ( E ; n=3/group). IGF1 (100 ng/mL; n=3/group) and SB415286 (30 µM; n=6/group) also increased endogenous KITLG mRNA expression in LX-2 ( F ) and GIST-T1 cells ( H ) and stimulated KITLG promoter activity in a time-dependent fashion (LX-2: n=6-9/group; G ; GIST-T1: n=3-11/group; I ). Groups marked by asterisk are different from the control group, and groups not sharing the same superscript letter are different from each other ( P <0.05 by post-hoc multiple comparisons).

Article Snippet: The role of endogenous KITLG in the proliferation of GIST-T1, GIST-T1-5R, GIST882, GIST48B and LX-2 cells was tested by culturing in the above media in the presence of purified, azide-free goat polyclonal anti-human KITLG antibody (AB-255-NA, R&D Systems, Minneapolis, MN; applied for 4 days at concentrations indicated in the Results), which has been shown to neutralize KITLG-induced proliferation in the TF1 human erythroleukemic cell line [ ].

Techniques: Membrane, Expressing, Activity Assay, Transfection, Luciferase, Construct, Control

Journal: PLoS ONE

Article Title: Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

doi: 10.1371/journal.pone.0076822

Figure Lengend Snippet: A , Increased occupancy of the Kitl core promoter region by H4ac in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. Only SB415286 increased occupancy by H3K9ac ( B ). Representatives of two independent experiments, each performed in triplicates, are shown. C , Dose-dependent stimulation of KITLG expression in LX-2 cells by 24-h treatment with the class I-II HDAC inhibitor SAHA (n=3/group). D , Dose-dependent inhibition of the SB415286-induced stimulation of KITLG expression by the p300/PCAF HAT inhibitor garcinol (24 h) in LX-2 cells (n=3/group). Drugs were applied following 24-h serum deprivation. Groups marked by asterisk are different from the control group, and groups not sharing the same superscript letter are different from each other ( P <0.05 by post-hoc multiple comparisons).

Article Snippet: The role of endogenous KITLG in the proliferation of GIST-T1, GIST-T1-5R, GIST882, GIST48B and LX-2 cells was tested by culturing in the above media in the presence of purified, azide-free goat polyclonal anti-human KITLG antibody (AB-255-NA, R&D Systems, Minneapolis, MN; applied for 4 days at concentrations indicated in the Results), which has been shown to neutralize KITLG-induced proliferation in the TF1 human erythroleukemic cell line [ ].

Techniques: Muscles, Expressing, Inhibition, Control

Journal: PLoS ONE

Article Title: Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

doi: 10.1371/journal.pone.0076822

Figure Lengend Snippet: A , Increased occupancy of the Kitl core promoter by the trithorax group-mediated, activating H3K4me2 histone modification in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. B - C , Reduced occupancy of the Kitl core promoter by the PRC2-mediated, repressive H3K27me3 histone modification ( B ) and by the PRC2 histone methyltransferase EZH2 ( C ) in response to rhIGF1 and SB415286 in the same tissues. D - F , Stimulation of KITLG expression by the indirect histone methyltransferase inhibitor Adox in murine gastric smooth muscles ( D ), LX-2 cells ( E ) and GIST-T1 cells ( F ) (n=3/group). Adox was applied for 72 h following 24-h serum deprivation. See further details in the legend to .

Article Snippet: The role of endogenous KITLG in the proliferation of GIST-T1, GIST-T1-5R, GIST882, GIST48B and LX-2 cells was tested by culturing in the above media in the presence of purified, azide-free goat polyclonal anti-human KITLG antibody (AB-255-NA, R&D Systems, Minneapolis, MN; applied for 4 days at concentrations indicated in the Results), which has been shown to neutralize KITLG-induced proliferation in the TF1 human erythroleukemic cell line [ ].

Techniques: Modification, Muscles, Expressing

Journal: PLoS ONE

Article Title: Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

doi: 10.1371/journal.pone.0076822

Figure Lengend Snippet: A - B , Reduced occupancy of the Kitl core promoter by the repressive H3K9me2 ( A ) and H3K9me3 ( B ) histone modifications in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. C - D , Probing the role of HP1 isoforms in transcriptional repression of KITLG in LX-2 cells by siRNA- (CBX5: HP1α; 25 nM, 72 h; C ) or shRNA-mediated knock-down (CBX1: HP1β; CBX3: HP1γ; 30 µg plasmid, 72 h; D ). Note activation of KITLG expression by shRNA-mediated knock-down of HP1γ (n=3/group). E - F , Stimulation of KITLG expression by the G9A/GLP H3K9me1/2 HKMT inhibitor BIX-01294 in LX-2 cells ( E ) and GIST-T1 cells ( F ) (n=3/group). G , Stimulation of KITLG expression by the H3K9 HKMT inhibitor chaetocin in LX-2 cells (n=3/group). Drugs were applied for 24 h following 24-h serum deprivation. See further details in the legend to .

Article Snippet: The role of endogenous KITLG in the proliferation of GIST-T1, GIST-T1-5R, GIST882, GIST48B and LX-2 cells was tested by culturing in the above media in the presence of purified, azide-free goat polyclonal anti-human KITLG antibody (AB-255-NA, R&D Systems, Minneapolis, MN; applied for 4 days at concentrations indicated in the Results), which has been shown to neutralize KITLG-induced proliferation in the TF1 human erythroleukemic cell line [ ].

Techniques: Muscles, shRNA, Knockdown, Plasmid Preparation, Activation Assay, Expressing

Journal: Molecular Therapy Oncology

Article Title: Development of multivalent CAR T cells as dual immunotherapy and conditioning agents

doi: 10.1016/j.omton.2025.200944

Figure Lengend Snippet: Overview of ELECTRIC CAR (A) Schematic of ELECTRIC CARs. SCF is N terminus, followed by a (G 4 S) 3 linker, TPO, a second (G 4 S) 3 linker, FLT3LG, a CD28 hinge, transmembrane, and intracellular (IC) signaling domain, and CD3ζ. (B) ColabFold-predicted structure of extracellular domain of ELECTRIC CAR, with representative binding modes to the KIT, MPL, and FLT3. (C) Comparison of in vitro T cell expansion after viral transduction to Mock (non-transduced) T cells (two-way ANOVA; ∗∗∗∗ p < 0.0001). (D) Transduction efficiency (left) and ELECTRIC CAR MFI (right) of transduced CAR T cells 8 days after viral transduction. (E) Representative flow cytometry plot (left) of anti-hSCF detection by murine anti-IGG1-PE fluorescent antibody (right). (F–H) Nalm-6 (N6) B-ALL cells were transduced with a virus encoding the extracellular and transmembrane domains of KIT (F), MPL (G), and FLT3 (H). (I–K) Monovalent natural ligand CARs expressing SCF, TPO, or FLT3LG and ELECTRIC CARs were co-cultured with GFP+ N6 cells overexpressing (OE) KIT (I), MPL (J), or FLT3 (K) and N6 GFP expression was measured over time via Incucyte Live Imaging. (L–N) ELECTRIC CAR T cells were co-cultured across a range of E:T ratios with N6 KIT OE (L), N6 MPL OE (M), and N6 FLT3 OE (N) and monitored via Incucyte Live Imaging. (O) Monovalent natural ligand CARs expressing SCF, TPO, or FLT3LG and ELECTRIC CARs were co-cultured with a heterogeneous mixture of N6 KIT OE, N6 MPL OE, and N6 FLT3 OE (1:1:1 ratio of KIT:MPL:FLT3) and monitored via Incucyte Live Imaging. (P) Monovalent natural ligand CARs expressing SCF, TPO, or FLT3LG and ELECTRIC CARs were co-cultured with N6 KIT OE, N6 MPL OE, and N6 FLT3 OE at 1:1 E:T ratios for 24 h, and supernatants were harvested for cytokine production measurement by ELISA. Cytokine production was compared between Mock and CAR T cells for each type of target cell (two-way ANOVA, Tukey’s multiple comparison test). For I–P, data are mean ± s.e.m. of triplicate wells.

Article Snippet: Triplekine CARs were detected with an anti-human SCF antibody (Catalog #MAB655, R&D Systems, Minneapolis, MN) followed by an anti-IGG1 PE antibody (Clone: RMG1-1, BioLegend, San Diego, CA) or an anti-G 4 S Linker antibody (Clone: E702V, Cell Signaling Technology, Danvers, MA).

Techniques: Binding Assay, Comparison, In Vitro, Transduction, Flow Cytometry, Virus, Expressing, Cell Culture, Imaging, Enzyme-linked Immunosorbent Assay