Journal: Cell Reports Medicine

Article Title: Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling

doi: 10.1016/j.xcrm.2024.101658

Figure Lengend Snippet:

Article Snippet: Dapi4′,6-Diamidin-2-phenylindole (DAPI) , Sigma-Aldrich , Cat# D 9564.

Techniques: Plasmid Preparation, Recombinant, Transfection, Fluorescence, Staining, Reverse Transcription, Expressing, Liposomes, Mutagenesis, shRNA, Control, Construct, Software, Imaging, Functional Assay, Dissection, Sequencing, Real-time Polymerase Chain Reaction

Journal: Molecular Oncology

Article Title: DAPK3 participates in the mRNA processing of immediate early genes in chronic lymphocytic leukaemia

doi: 10.1002/1878-0261.12692

Figure Lengend Snippet: H3T6 and H3T11 phosphorylation at IEGs is catalysed by DAPK3 and is similarly abrogated by both BTK and DAPK3 inhibition. (A) qPCR data analysis of EGR1 and DUSP2 gene expression at 30 ( n = 6 patients) and 60 min ( n = 4 patients) post‐anti‐IgM stimulation in U‐CLL cells. CLL cells were pretreated with 1 µ m ibrutinib or 25 µ m DAPKi for 1 h as indicated below the graphs before anti‐IgM stimulation. Expression changes were quantified using the ∆ C t method with TBP and PPP6C as control genes. Bars represent the grand mean of the samples. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with anti‐IgM as control. EGR1 (30 m) P values = 0.0009, 0.0015 and 0.0009 for anti‐IgM vs Unstim, Ibr and DAPKi respectively. DUSP2 (30 m) P values = 0.0013, 0.0023 and 0.0011 for the same comparisons. EGR1 (60 m) P values = 0.0039, 0.0052 and 0.0052 for the same comparisons. DUSP2 (60 m) P values = 0.0204, 0.0269 and 0.0205 for the same comparisons. (B) ChIP‐qPCR data assessing levels of H3T6‐P and H3T11‐P at the EGR1 and DUSP2 gene loci. CLL cells pretreated with either 1 µ m ibrutinib or 25 µ m DAPKi for 1 h before stimulation with anti‐IgM for 30 min. Values on the x axis refer to specific gene regions relative to the TSS in kilobases (kb) as indicated on the gene schematics below (not to scale). Error bars represent the SD of three independent experiments. CTCF1/3 (C1/C3) were used as negative control regions which are not indicated on the gene schematics. (C) co‐IP of CLL cells stimulated with anti‐IgM for 1 h as indicated. Immunoprecipitates from histone H3 pulldown were analysed by SDS/PAGE followed by western blot probing for DAPK3‐T265P. Untreated, crude cell lysate was used as positive control (input), and IgG beads were used for negative control (Ctrl IgG). Blot is representative of three independent co‐IP experiments. Western blot is cropped due to different exposure times for input and co‐IP lysates. (D) Kinase assay quantified by dot‐blot with recombinant histone H3 and recombinant DAPK3 to measure DAPK3 kinase activity over time (5–60 min) with or without (negative control) ATP and in the presence of 1 µ m ibrutinib and 25 µ m DAPK inhibitor. (E) Kinase assay displaying histone H3 phosphorylation quantified relative to untreated assay with error bars representing the SD of three independent experiments. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with the untreated sample as control for each time point. H3T6‐P P values = 0.3075 and 0.0139 (5 m), 0.4157 and 0.0021 (20 m), 0.1583 and 0.0001 (60 m) for untreated vs. Ibr and DAPKi, respectively. H3T11‐P P values = 0.6679 and 0.6844 (5 m), 0.0672 and 0.0095 (20 m), 0.0043 and 0.0001 (60 m) for the same comparisons. Ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Cells were pretreated with ibrutinib (Pharmacyclics, Sunnyvale, CA, USA) at 1 μ m or a DAPK inhibitor (DAPKi) (Calbiochem, San Diego, CA, USA; 324788‐10MG) at 10–120 μ m as required and where indicated.

Techniques: Inhibition, Expressing, Negative Control, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Positive Control, Kinase Assay, Dot Blot, Recombinant, Activity Assay