porcine tubulin  (Cytoskeleton Inc)

Journal: Nature Communications

Article Title: CPT1A-mediated IDO1 succinylation shapes EGFRvIII-driven resistance to tumor electric field therapy in glioblastoma

doi: 10.1038/s41467-026-77927-w

Figure Lengend Snippet: A Western blot analysis of AhR in cytoplasmic and nuclear fractions of TBD0220 and LN229-EGFRvIII cells. TEFT treatment promotes AhR nuclear translocation, B Immunofluorescence staining of AhR (red) and DAPI (blue). TEFT induces AhR nuclear accumulation, which is reversed by IDO1 knockdown or osimertinib. Scale bars, 10 μm, C ATAC-seq footprinting showing increased chromatin accessibility at the AhR binding motif in EGFRvIII cells versus wild-type, D GO enrichment analysis of AhR target genes (identified by intersecting ATAC-seq and ChIP-Atlas), highlighting “Mitotic Cell Cycle” and “Microtubule Cytoskeleton” as top pathways, E Representative immunofluorescence images of mitotic spindles (α-tubulin, green) and chromosomes (DAPI, blue) in LN229 cells. TEFT causes severe spindle disruption and multipolar mitosis in wild-type cells, whereas EGFRvIII cells maintain bipolar spindles. This protection is lost upon IDO1 knockdown or osimertinib treatment, F Quantification of cells with abnormal nuclei following TEFT treatment ( n = 3 biological replicates per group), G Venn diagram intersecting RNA-seq (upregulated), ATAC-seq (accessible peaks), and ChIP-Atlas (AhR binding targets) data, identifying DCLK1 as a key downstream target, H Western blot confirming the upregulation of DCLK1 protein in EGFRvIII cells upon TEFT, I Immunofluorescence images of mitotic spindles in DCLK1 -knockdown cells. Silencing DCLK1 sensitizes EGFRvIII cells to TEFT-induced spindle defects, J Quantification of abnormal nuclear morphology in DCLK1-depleted cells ( n = 3 biological replicates per group), K Venn diagram integrating multi-omics data with microtubule/GTPase-related genes, identifying ARHGEF2 (GEF-H1) as another target, L In vitro tubulin polymerization assay in TBD0220 cells under the indicated conditions. IDO1 knockdown, ARHGEF2 knockdown, osimertinib, or epacadostat reduced the microtubule-polymerizing activity associated with EGFRvIII-mediated TEFT resistance. Data are presented as mean ± SD. The exact n values and definitions of biological replicates are specified for the relevant panels. Statistical tests were as follows: two-tailed unpaired Student’s t test for panel J ; ordinary one-way ANOVA followed by two-sided Šídák-adjusted multiple-comparisons tests for panel F . Exact P values, adjusted for multiple comparisons where applicable, are shown in the corresponding panels. Source data are provided as a Source Data file.

Article Snippet: Tubulin polymerization kinetics were monitored using the Tubulin Polymerization Assay Kit (BK006P, Cytoskeleton, Inc.) according to the manufacturer’s protocol.

Techniques: Western Blot, Analysis, Translocation Assay, Immunofluorescence, Staining, Knockdown, Footprinting, Binding Assay, Disruption, RNA Sequencing, Morphology, Biomarker Discovery, In Vitro, Polymerization Assay, Activity Assay, Two Tailed Test

Journal: Journal of Medicinal Chemistry

Article Title: Dissecting Structural and Functional Determinants of Microtubules Stabilization through Guided Chemical Modulation

doi: 10.1021/acs.jmedchem.6c01666

Figure Lengend Snippet: Cellular and biochemical characterization of compounds 1a and 1b . (A) Effects of compounds on A549 cells. Each row shows α-tubulin immunostaining (DM1A antibody), DNA labeling (DAPI staining), and the merge of the previous ones (tubulin in green and DNA in magenta) of interphase cells (first three columns) and mitotic cells (following three). Top row panels are control cells treated with 0.5% DMSO (vehicle), with the usual and evenly distributed microtubular network in interphase and normal bipolar spindle with chromosomes aligned at the metaphase plate during mitosis. The following rows display cells treated at selected concentrations showing equivalent effect (PTX 100 nM, 1a 2 μM and 1b 1 μM), where the microtubular network is denser and with the presence of bundles during interphase, and a diversity of abnormal mitotic spindle containing misaligned chromosomes. All images are shown at the same magnification; scale bar 10 μm. (B) Time course assembly of 25 μM tubulin in PEDTA buffer in the presence of the vehicle (DMSO, black line) or 35 μM of compound; PTX (brown), FTX2 (yellow), 1a (orange), 1b (purple). (C) Van’t Hoff Plot of the equilibrium binding constants of compounds 1a and 1b at the assayed temperatures. Error bars represent the standard error of the measurement.

Article Snippet: Porcine brain tubulin was isolated using the high-molarity PIPES procedure then mixed with Biotin labeled tubulin (Cytoskeleton Inc., T333P) in 1:49 ratio.

Techniques: Immunostaining, DNA Labeling, Staining, Control, Binding Assay