Journal: Neuro-Oncology

Article Title: SYK inhibition blocks proliferation and migration of glioma cells and modifies the tumor microenvironment

doi: 10.1093/neuonc/noy008

Figure Lengend Snippet: Immune signaling molecules are expressed by glioma cells in vivo. (A) Microarray analysis of SYK, LYN, PLCG2, and SLP76 in NHAs, normal brain (Brain), glioblastoma (GBM), secondary glioblastoma (2ary-GBM), lower-grade astrocytoma (Astro), and oligodendrogliomas (Oligo) using Genedata Expressionist analyst software. (B) Representative IHC staining of GBM samples for SYK, LYN, and PLCG2; n > 15. (C) Tumor score of GBM samples stained for SYK, LYN, and PLCG2. (D) SYK expression in glioma mouse models (GFAP tvaPDGF ARF−/−; GFAP Cre+ NF1−/+ p53−/f PTEN +/f; S100b-v-ErbB; p53−/−). (E) SYK expression in 2 orthotopic glioma mouse models (U87MG-luc in HSD nude; GL261-L2G in C57BL/6J). (F) SYK expression of pilocytic astrocytoma n = 16 and (G) tumor score.

Article Snippet: The following monoclonal antibodies were used: Ki67 (Thermo Scientific); PLCG2, GFAP (Sigma Aldrich); cyclin D1 (C-20), LYN (H-6), SYK (2D10 and C-20), Zap70 (M-20), SLP76 (F-7), ACTIN (I-19), GFAP (C-19 for FACS) (SCBT); goat anti-rabbit-Alexa488, goat anti-mouse-Alexa568, goat anti-rabbit-Alexa647 (Invitrogen); GFP-Alexa488, CD45APC-CY7, CD44-PE, CD4-BV785, CD8-BV711, CD19-v570, CD19-APC, CD11b-BV510, Dextran Texas Red, CD206-APC, CD25-PerCPCy5.5, CD11c-PECy5.5, CD3-Alexa700, GR-1-APC, F4/80-BV421, and MHCII IA/E-APCCy7 (Biolegend and E-Bioscience).

Techniques: In Vivo, Microarray, Software, Immunohistochemistry, Staining, Expressing

Journal: Neuro-Oncology

Article Title: SYK inhibition blocks proliferation and migration of glioma cells and modifies the tumor microenvironment

doi: 10.1093/neuonc/noy008

Figure Lengend Snippet: SYK is expressed by glioma cells. (A) SYK and beta actin western blot of GBM and normal brain samples. (B) Western blot of SYK, LYN, SLP76, and beta actin expression in 18 GBM cell lines. (C) IF of SYK (green) and nuclei (blue) in BS287. (D) Western blot of SYK and tubulin on BS287 cancer spheres and NHA. (E) IF of GBM sections co-stained for SYK (red) and glioma markers GFAP and oligodendrocyte transcription factor (green). (F) IF of GBM sections co-stained for SYK (red), leukocyte marker CD45 (green), and monocyte/macrophage/microglia marker CD68 (green).

Article Snippet: The following monoclonal antibodies were used: Ki67 (Thermo Scientific); PLCG2, GFAP (Sigma Aldrich); cyclin D1 (C-20), LYN (H-6), SYK (2D10 and C-20), Zap70 (M-20), SLP76 (F-7), ACTIN (I-19), GFAP (C-19 for FACS) (SCBT); goat anti-rabbit-Alexa488, goat anti-mouse-Alexa568, goat anti-rabbit-Alexa647 (Invitrogen); GFP-Alexa488, CD45APC-CY7, CD44-PE, CD4-BV785, CD8-BV711, CD19-v570, CD19-APC, CD11b-BV510, Dextran Texas Red, CD206-APC, CD25-PerCPCy5.5, CD11c-PECy5.5, CD3-Alexa700, GR-1-APC, F4/80-BV421, and MHCII IA/E-APCCy7 (Biolegend and E-Bioscience).

Techniques: Western Blot, Expressing, Staining, Marker

Journal: Cancer Research

Article Title: NPM-ALK Oncogenic Tyrosine Kinase Controls T-Cell Identity by Transcriptional Regulation and Epigenetic Silencing in Lymphoma Cells

doi: 10.1158/0008-5472.can-09-2655

Figure Lengend Snippet: Figure 1. ALK+ lymphoma cells do not express TCR complex–related molecules. A, immunohistochemical staining on primary ALCL. Lymphoma cells (arrows) are positive for ALK and negative for the expression of CD3ε, LAT, ZAP70, and SLP76. Admixed reactive T cells are positive for all T-cell markers. Bar, 50 μm. B, ALK+ (Karpas-299, SU-DHL1, TS, and JB6) and ALK−(CEM, Jurkat, MAC-1, ST-4, and PF382) T-cell lines and peripheral blood mononuclear cells from healthy donors were lysed and immunoblotted for the indicated antibodies. C, RT-PCR analysis. Differential expression of the indicated genes was analyzed by RT-PCR using 5-fold serial dilutions of cDNA prepared from in vitro cultured ALK+ (TS and SU-DHL1) and ALK−(MAC-1 and Jurkat) cell lines. β2-Microglubulin (B2M) expression was used to normalize the cDNA input. Results are from one experiment and representative of four independent experiments.

Article Snippet: For immunohistochemical stainings, sections from tissue microarray were incubated with the following primary antibodies: ALK (ALK-1; DAKO), CD3ε (NCLCD3-PS1; Novocastra), ZAP70 (clone 2F3.2; Upstate), LAT (FL-233; Santa Cruz Biotechnology), and SLP76 (Cell Signaling).

Techniques: Immunohistochemical staining, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative Proteomics, In Vitro, Cell Culture

Journal: Cancer Research

Article Title: NPM-ALK Oncogenic Tyrosine Kinase Controls T-Cell Identity by Transcriptional Regulation and Epigenetic Silencing in Lymphoma Cells

doi: 10.1158/0008-5472.can-09-2655

Figure Lengend Snippet: Figure 3. CD3ε, ZAP70, LAT, and SLP76 expression is controlled by NPM-ALK–mediated transcriptional repression or gene hypermethylation. A, TS, SU-DHL1, and Jurkat cells were treated with 300 nmol/L of the specific ALK kinase inhibitor CEP-14083 for 12 h and then lysed and immunoblotted for the indicated antibodies. Results are from one experiment and representative of five independent experiments. B and C, TS and SU-DHL1 cells were treated for the indicated time points with 1 μmol/L DAC and then analyzed either by RT-PCR on cDNAs (B) or by immunoblotting (C) for the expression of the indicated mRNA and proteins, respectively. Jurkat cells were used as controls. Results are from one experiment and representative of three independent experiments. D, DNAs purified from TS and SU-DHL1 cells and from MAC-1 cells untransduced or transduced with NPM-ALK or NPM-ALKK210R were treated with bisulfite. Sequences were obtained from the region that surrounds the intron 1-exon 2 boundary of the ZAP70 gene and contains 8 CpG islands. The locations of the analyzed CpG sites within the CpG island are shown. Arrow, transcription starting site (TSS); filled circles, methylated CpG sites; open circles, unmethylated sites.

Article Snippet: For immunohistochemical stainings, sections from tissue microarray were incubated with the following primary antibodies: ALK (ALK-1; DAKO), CD3ε (NCLCD3-PS1; Novocastra), ZAP70 (clone 2F3.2; Upstate), LAT (FL-233; Santa Cruz Biotechnology), and SLP76 (Cell Signaling).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Purification, Transduction, Methylation

Journal: Cancer Research

Article Title: NPM-ALK Oncogenic Tyrosine Kinase Controls T-Cell Identity by Transcriptional Regulation and Epigenetic Silencing in Lymphoma Cells

doi: 10.1158/0008-5472.can-09-2655

Figure Lengend Snippet: Figure 4. CD3ε, ZAP70, LAT, SLP76, and DNMT1 expression in ALCL depends on STAT3. A to C, TS and SU-DHL1 cells were transduced with a control shRNA lentivirus or the shSTAT3 lentivirus. Jurkat cells were used as controls. After 96 h, the percentages of transduced cells were >95% by enhanced green fluorescent protein expression as assessed by flow cytometry. Cells (TS and SU-DHL1 in A and SU-DHL1 in B and C) were harvested and analyzed by RT-PCR (A) and quantitative PCR (B) on cDNAs for the indicated genes or by immunoblotting with the indicated antibodies (C). Results are from one experiment and representative of two independent experiments. D, DNA was purified from TS and SU-DHL1 cells transduced with a control shRNA lentivirus or the shSTAT3 lentivirus. Sequences of clones from bisulfite-treated DNA were obtained from the region that surrounds the intron 1-exon 2 boundary of the ZAP70 gene as indicated above. Filled circles, methylated CpG sites; open circles, unmethylated sites. Statistical analysis was calculated by χ2 P value comparing the ratios of unmethylated CpG sites of TS and SU-DHL1 cell lines treated with shSTAT3 versus control (P < 0.0001).

Article Snippet: For immunohistochemical stainings, sections from tissue microarray were incubated with the following primary antibodies: ALK (ALK-1; DAKO), CD3ε (NCLCD3-PS1; Novocastra), ZAP70 (clone 2F3.2; Upstate), LAT (FL-233; Santa Cruz Biotechnology), and SLP76 (Cell Signaling).

Techniques: Expressing, Transduction, Control, shRNA, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Purification, Clone Assay, Methylation

Journal: Cancer Research

Article Title: NPM-ALK Oncogenic Tyrosine Kinase Controls T-Cell Identity by Transcriptional Regulation and Epigenetic Silencing in Lymphoma Cells

doi: 10.1158/0008-5472.can-09-2655

Figure Lengend Snippet: Figure 6. Schematic representation of the effects of NPM-ALK on TCR signaling molecules. In ALCL cells, the TCR signaling pathway is nonfunctional, because NPM-ALK suppresses through STAT3 the expression of CD3ε, ZAP70, SLP76, and LAT by transcriptional repression and epigenetic silencing. In the absence of TCR signaling, the activated phenotype of ALCL cells, as well as their cytoskeletal rearrangements, survival, and proliferation, could be sustained by the activation of the Rho family GTPases. Rac1 and Cdc42 GTPases are activated by an ALK-dependent VAV1 and VAV3 phosphorylation (10, 15).

Article Snippet: For immunohistochemical stainings, sections from tissue microarray were incubated with the following primary antibodies: ALK (ALK-1; DAKO), CD3ε (NCLCD3-PS1; Novocastra), ZAP70 (clone 2F3.2; Upstate), LAT (FL-233; Santa Cruz Biotechnology), and SLP76 (Cell Signaling).

Techniques: Expressing, Activation Assay, Phospho-proteomics

Journal: Cell reports

Article Title: DNA-PKcs controls the cytotoxic T cell response to cancer and transplant allograft through regulating LAT-dependent signaling

doi: 10.1016/j.celrep.2025.116796

Figure Lengend Snippet: (A) JLAT transductants were stimulated with 5 μg/mL αCD3/CD28 for 15 min. mCherry coIP western blot shows that phosphorylation of LAT by DNA-PKcs at S241 is required for signalosome formation, given altered pull-down of GADS and SLP-76 in JLAT cells expressing S241A LAT and S2A LAT. Representative blots from n = 3 independent experiments. (B) CoIP coupled mass spectrometry was completed in JLAT transductants expressing WT, S224A, or S241A LAT stimulated with 5 μg/mL αCD3/CD28 for 15 min. Pull-down was normalized to mCherry-expressing JLAT cells, and protein pull-down was further normalized to relative LAT pull-down with log-fold differences shown between the groups, n = 5 replicates/transductant. The top statistically different proteins pulled down with mCherry-tagged LAT are represented in WT, S224A, and S241A LAT transductants.

Article Snippet: SLP-76 (D1R1A) Rabbit Monoclonal Antibody #70896 , Cell Signaling Technologies , Cat#70896; RRID: AB_2799792.

Techniques: Western Blot, Phospho-proteomics, Expressing, Mass Spectrometry