Journal: The Journal of Cell Biology

Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

doi: 10.1083/jcb.201707160

Figure Lengend Snippet: Aurora A kinase binds and localizes to INCENP. (A) Volcano plot of quantitative liquid chromatography–tandem mass spectrometry experiments identifying interactors of INCENP. Plotted are the differences in label-free quantification intensity between the INCENP-GFP (right) and control group against the transformed (−log 10 ) p-value of a Student’s t test. The red line indicates the permutation-based false discovery rate (FDR) threshold (0.001) to correct for multiple testing. (B) Western blot analysis of an anti-GFP immunoprecipitation (IP) performed in mitotic cell lysates, using either nocodazole (N) or STLC (S) from HeLa cells expressing either LAP or INCENP-LAP. After SDS-PAGE, the Western blot was probed for GFP and Aurora A kinase (AAK). 10% of input was loaded. (C) Western blot analysis of an anti-GFP immunoprecipitation performed in cell lysates from HEK-293T cells transiently expressing either GFP, full-length (FL) INCENP-GFP, or various truncation mutants of INCENP-GFP (1–820, 1–878, and 1–897). The Western blot was probed for GFP and Aurora A kinase and reprobed for Aurora B kinase (ABK). 10% of input was loaded. (D) Immunofluorescence images of U2OS cells expressing either LacI-mCherry or LacI-mCherry-ΔCEN-INCENP (a version of INCENP lacking the N-terminal 57 residues; ). Cells were immunostained with an Aurora A kinase antibody. Enlarged images show recruitment of Aurora A kinase to the LacI-mCherry-ΔCEN-INCENP spot. Quantification of Aurora A kinase intensity is shown on the right. (E) Immunofluorescence images of U2OS cells expressing VSV-INCENP-GFP under a doxycycline-inducible promoter, either noninduced or induced. Cells were immunostained with GFP and Aurora A kinase antibodies. Enlarged images show colocalization of INCENP-GFP and Aurora A kinase upon overexpression of INCENP-GFP. (F) Line graphs of kinetochore pairs highlighted in E. (G) Immunofluorescence images showing kinetochore localization of pS69 in HeLa cells treated with siRNA to either luciferase (control) or INCENP (siINCENP). Quantification of pS69 and INCENP fluorescence intensity is shown on the right. For pS69 quantification, n = 3 experiments of 15–20 cells; for INCENP quantification, n = 1 experiment of 15–20 cells. Error bars indicate SD. ****, P < 0.0001; unpaired t test. Bars: (main images) 10 µm; (insets) 1 µm.

Article Snippet: Cells were transfected with a LacI-mCherry or LacI-mCherry–ΔCEN-INCENP vector (45236; Addgene; from M. Lampson; ) using Lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Liquid Chromatography, Mass Spectrometry, Quantitative Proteomics, Control, Transformation Assay, Western Blot, Immunoprecipitation, Expressing, SDS Page, Immunofluorescence, Over Expression, Luciferase, Fluorescence

Journal: Frontiers in Chemistry

Article Title: Microscopic distribution of taxanes in freeze-fixed stems of Taxus cuspidata

doi: 10.3389/fchem.2024.1437141

Figure Lengend Snippet: Cryo-TOF-SIMS spectra and chemical structures of (A) 10-DAB, (B) BAC, (C) 10-DAT, (D) EDT, (E) CE, (F) paclitaxel, (G) 7-epi-taxol, and (H) 7-xyl-10-DAT. Cryo-TOF-SIMS spectra obtained from the frozen, hydrated transverse surface of T. cuspidata stems from (I) late summer and (J) spring in the phloem region. Standard chemicals of taxanes were dissolved at ca. 50 mM in 80% EtOH and frozen for measurements.

Article Snippet: Standard chemicals including paclitaxel (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), 10-DAB (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), BAC and CE (Selleck Chemicals LLC., TX, United States), 7-xyl-10-DAT, 7-epi-taxol, EDT, and 10-DAT (MedChem Express LLC., NJ, United States) were purchased and used as received.

Techniques: