Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: The stem cell factor-c-kit system and mast cells in human pancreatic cancer.

doi: 10.1097/01.lab.0000036875.21209.f9

Figure Lengend Snippet: Figure 1. A and B, Stem cell factor (SCF) and c-kit immunostaining in normal pancreatic tissues. A duct showing hyperplastic features exhibits strong c-kit-immunoreactivity (A) and it is surrounded by c-kit–positive (A) and SCF-positive (B) inflammatory cells. C, Immunostaining performed with the highly specific anti-tryptase antibody reveals that most of the inflammatory cells described around large and hyperplastic ducts are mast cells (C). Scale bar 25 m.

Article Snippet: The following materials were purchased: FCS, RPMI and DMEM, trypsin-EDTA solution, and penicillinstreptomycin solution from Biochrom KG (Berlin, Germany); human recombinant SCF from R&D Systems (Abingdon, United Kingdom) and from Amgen GmbH (Munich, Germany); anti-SCF antibody from Santa Cruz Biotechnology (Santa Cruz, California); anti-c-kit polyclonal antibodies from Santa Cruz Biotechnology and from NeoMarkers (Fremont, California); antitryptase and anti-chymase monoclonal antibodies from Chemicon International (Temecula, California); biotinylated secondary antibodies and streptavidin/ peroxidase complex from Kierkegaard and Perry Laboratories (Gaithersburg, Maryland); FITC-conjugated anti-rabbit antibody from Zymed (San Francisco, California); OCT compound from Miles Inc. (Elkhart, Indiana); horseradish peroxidase-conjugated secondary antibodies and ECL Western blotting detection reagents from Amersham Life Science (Amersham, United Kingdom); and Protease Inhibitor Cocktail Tablets from Roche (Basel, Switzerland).

Techniques: Immunostaining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: The stem cell factor-c-kit system and mast cells in human pancreatic cancer.

doi: 10.1097/01.lab.0000036875.21209.f9

Figure Lengend Snippet: Figure 4. Western blot analysis of SCF, c-kit, and tryptase in normal pancreas and pancreatic cancer samples, as described in “Materials and Methods.” The highly specific anti-SCF antibody detected a single band of approximately 45 kDa in the cancer samples. The anti-c-kit antibody detected one band of approximately 80 kDa in two of the cancer samples, and a band of about 125 kDa in a third cancer sample. The anti-tryptase antibody detected two bands of approximately 30 and 35 kDa in two pancreatic cancer samples, and a single, intermediate-sized band in one cancer and one normal pancreas sample.

Article Snippet: The following materials were purchased: FCS, RPMI and DMEM, trypsin-EDTA solution, and penicillinstreptomycin solution from Biochrom KG (Berlin, Germany); human recombinant SCF from R&D Systems (Abingdon, United Kingdom) and from Amgen GmbH (Munich, Germany); anti-SCF antibody from Santa Cruz Biotechnology (Santa Cruz, California); anti-c-kit polyclonal antibodies from Santa Cruz Biotechnology and from NeoMarkers (Fremont, California); antitryptase and anti-chymase monoclonal antibodies from Chemicon International (Temecula, California); biotinylated secondary antibodies and streptavidin/ peroxidase complex from Kierkegaard and Perry Laboratories (Gaithersburg, Maryland); FITC-conjugated anti-rabbit antibody from Zymed (San Francisco, California); OCT compound from Miles Inc. (Elkhart, Indiana); horseradish peroxidase-conjugated secondary antibodies and ECL Western blotting detection reagents from Amersham Life Science (Amersham, United Kingdom); and Protease Inhibitor Cocktail Tablets from Roche (Basel, Switzerland).

Techniques: Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: The stem cell factor-c-kit system and mast cells in human pancreatic cancer.

doi: 10.1097/01.lab.0000036875.21209.f9

Figure Lengend Snippet: Figure 5. Western blot analysis of SCF and c-kit in TAKA-1 cells and in six pancreatic cancer cell lines. TAKA cells did not express SCF, whereas SCF was expressed by Bx-PC-3, MiaPaCa-2, and T3M4 cell lines, the most intense band being present in MiaPaCa-2 cells. The anti-c-kit antibody detected two bands of approximately 80 kDa and one band of 125 kDa in TAKA cells and two bands of approximately 80 kDa in all the cancer cell lines; Capan-1 showed the highest c-kit expression levels.

Article Snippet: The following materials were purchased: FCS, RPMI and DMEM, trypsin-EDTA solution, and penicillinstreptomycin solution from Biochrom KG (Berlin, Germany); human recombinant SCF from R&D Systems (Abingdon, United Kingdom) and from Amgen GmbH (Munich, Germany); anti-SCF antibody from Santa Cruz Biotechnology (Santa Cruz, California); anti-c-kit polyclonal antibodies from Santa Cruz Biotechnology and from NeoMarkers (Fremont, California); antitryptase and anti-chymase monoclonal antibodies from Chemicon International (Temecula, California); biotinylated secondary antibodies and streptavidin/ peroxidase complex from Kierkegaard and Perry Laboratories (Gaithersburg, Maryland); FITC-conjugated anti-rabbit antibody from Zymed (San Francisco, California); OCT compound from Miles Inc. (Elkhart, Indiana); horseradish peroxidase-conjugated secondary antibodies and ECL Western blotting detection reagents from Amersham Life Science (Amersham, United Kingdom); and Protease Inhibitor Cocktail Tablets from Roche (Basel, Switzerland).

Techniques: Western Blot, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: The stem cell factor-c-kit system and mast cells in human pancreatic cancer.

doi: 10.1097/01.lab.0000036875.21209.f9

Figure Lengend Snippet: Figure 8. Specificity of the effect of SCF on TAKA-1 cells. TAKA-1 cells were incubated in the absence or presence of SCF (100 ng/ml), anti-c-kit antibody (5 g/ml), or the combination of both reagents. MTT assay was performed after 72 hours. Percent growth inhibition was determined by comparison with control cell growth. * p 0.05.

Article Snippet: The following materials were purchased: FCS, RPMI and DMEM, trypsin-EDTA solution, and penicillinstreptomycin solution from Biochrom KG (Berlin, Germany); human recombinant SCF from R&D Systems (Abingdon, United Kingdom) and from Amgen GmbH (Munich, Germany); anti-SCF antibody from Santa Cruz Biotechnology (Santa Cruz, California); anti-c-kit polyclonal antibodies from Santa Cruz Biotechnology and from NeoMarkers (Fremont, California); antitryptase and anti-chymase monoclonal antibodies from Chemicon International (Temecula, California); biotinylated secondary antibodies and streptavidin/ peroxidase complex from Kierkegaard and Perry Laboratories (Gaithersburg, Maryland); FITC-conjugated anti-rabbit antibody from Zymed (San Francisco, California); OCT compound from Miles Inc. (Elkhart, Indiana); horseradish peroxidase-conjugated secondary antibodies and ECL Western blotting detection reagents from Amersham Life Science (Amersham, United Kingdom); and Protease Inhibitor Cocktail Tablets from Roche (Basel, Switzerland).

Techniques: Incubation, MTT Assay, Inhibition, Comparison, 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 , 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