Journal: Frontiers in Genetics

Article Title: DOT1L inhibition does not modify the sensitivity of cutaneous T cell lymphoma to pan-HDAC inhibitors in vitro

doi: 10.3389/fgene.2022.1032958

Figure Lengend Snippet: Histone modification levels after treatment of CTCL lines with HDAC inhibitors and DOT1L inhibitors. (A–C) Western blots showing H3K79me1/2, pan H4ac, H3K9ac and H2BK120ub1 levels in the indicated CTCL cell lines after treatment with 25 µM Pinometostat (Pino), and 0.25 µM or 1 µM Vorinostat (Vor) for 2 h (A) , 24 h (B) or 72 h (C) . (D) Western blot on unsonicated samples showing DOT1L protein levels in the indicated CTCL cell lines after treatment with 25 µM Pinometostat (Pino), 0.25 µM or 1 µM Vorinostat for 72 h. (E–F) Cell viability of the indicated CTCL cell lines after treatment with 25 µM Pinometostat (Pino), and 0.25 µM or 1 µM Vorinostat for 72 h determined by Annexin V-DAPI staining. Bars indicate average value of two independent biological replicates; individual data points are shown.

Article Snippet: Subsequently, membranes were incubated overnight at 4°C with the primary antibodies diluted in Tris-buffered saline containing 0.05% Tween-20 (TBST) and 2% Nutrilon: anti-H3K79me1 (RRID: AB_2631105) (1:1000), anti-H3K79me2 (04-835, Millipore) (1:1000), anti-H3K9ac (ab4441, Abcam) (1:2500), anti-H3K9ac (13-0020, Epicypher) (1:1000), anti-H4Ac pan (06-866, Millipore) (1:2000), anti-H2BK120ub1 (5546, Cell Signaling Technology) (1:1000) DOT1L (90878, Cell Signaling Technology) (1:500), anti-α-Tubulin (9023948, Santa Cruz Biotechnology) (1:10000) or anti-H3 (ab1791; Abcam) (1:5000).

Techniques: Modification, Western Blot, Staining

Journal: bioRxiv

Article Title: AURKB/Ipl1 restarts replication forks to recover from replication stress

doi: 10.1101/2021.05.27.446025

Figure Lengend Snippet: AURKB has a role in the response to DNA damage. A . Yeast cells limited for Ipl1 are sensitive to DNA damage. Ten-fold serial dilution of cells spotted onto YPD plates and grown continually with 0.01%MMS, 50mM HU, or after 50 uJ/cm 2 of ultraviolet light. WT (W303), ipl1 (4145-1-3) and mec1 (JRY7274) B . Replication stressed HeLa cells are sensitive to two distinct AURKB inhibitors but not an AURKA inhibitor. HeLa cells were treated with the indicated drugs (100nM AZD1152 (AZD), ZM-0.4uM ZM447439, 50nM MLN8237 (Alisertib, MLN), 2mM hydroxyurea (HU)) for 6 hours and ∼350 cells were plated in 10cm plates and grown for 15 days. The number of colonies formed were counted after staining with crystal violet. P-values calculated by Anova with Tukey post-test. C . Proteins at stalled replication forks. Quantitative iPond SILAC analysis of the indicated proteins Log 2 Abundance Ratio of HU treated cells over time compared with a non-HU treated control (Data mined from Dungrawala et al ., 2015 ). D . aniPOND of HeLa cells synchronized into S phase. Cells were pulsed with EdU for 10 minutes, and then chased with 10 μM Thymidine for 20 minutes. Negative controls for the EdU labeling (No EdU) and the conjugation to biotin (No Click) were also included. Samples were subjected to western blotting of the respective proteins after normalization to the total amount of input protein. E . aniPOND of HeLa cells synchronized into S phase and incubated with 3 mM HU to induce replication stress. Top, schematic of experiment, with red arrows indicating where EdU is incorporated in the various samples. Bottom, samples are subjected to western blotting of the respective proteins after normalization to the total amount of input protein in each sample. F . Quantification of CPC proteins from aniPOND samples, normalized to H2B. P-values calculated by one-way ANOVA with Tukey post-test with multiple comparison correction.

Article Snippet: The antibodies used were: 12Ca5 (mouse monoclonal antiHA produced in UVA hybridoma facility), Borealin (Banerjee JCB 2014), AURKA (homemade), H3S10P (Mouse Millipore Cat#06570 multiple Lot#, Rabbit Cell Signaling Technologies Cat#D2C8 Lot# 3377S), H3T3 (EMD Millipore Cat#7-424 Lot#3012075), H2AP.T120 (Active Motif Cat#61195 Lot#31511001), BUB1 (GeneTex Cat#GTx30097 Lot#821701160, PCNA (Novus Biotechne-Brand NB500-106 Lot # A6), H2B (Upstate Cat#07371 Lot#30216) H4 (Cell Signaling Technologies), PLK1P.T210 (Cell Signaling Technologies Lot#A3730), Tubulin (DM1a, UVA hybridoma facility), P-CHK1-S345(Cell Signaling Cat#133D3, Lot#2348S), P-CHK1-S317 (Cell Signaling Cat#2344S) P-ATR90, antiBrdU(Mouse-BD Biosciences 347580 Lot# 7157935 AbCam Rat Cat#AB6326 multiple lots #GR3173537-7, #GR3269246-1), gamma-H2aX (Upstate Cell Signaling Solutions Cat# 07-164 Lot#23646), Pan Aurora T-loop (Cell Signaling Technologies), Claspin (A300267A-Bethyl), IdU (from Burke), CldU (from Burke).

Techniques: Serial Dilution, Staining, Labeling, Conjugation Assay, Western Blot, Incubation

Journal: bioRxiv

Article Title: AURKB/Ipl1 restarts replication forks to recover from replication stress

doi: 10.1101/2021.05.27.446025

Figure Lengend Snippet: AURKB/Ipl1 and PLK1/Cdc5 regulate ATR/Mec1. A . In situ kinase assay of cycling wild type, ipl1-321 and cdc5-1 cells tagged with 3xHA (labeled C) treated with MMS for 45 min at 36 degrees (labeled M), the MMS was removed and cells were grown in YPD for the indicated times (hours). Proteins were separated by PAGE and Mec1 kinase activity was detected by autophosphorylation after kinase renaturation and incubating with 32 P-labelled ATP followed by autoradiography. B . Quantification of PLK1 T-loop phosphorylation during S phase after Aurora kinase inhibition. C . Cells were treated as in , then immunoblotted for pan-Aurora T-loop phosphorylation (AURKA, yellow, AURKB, blue), PLK1 pT210, H3-pS10, and H2B as a loading control. D . Quantification of chromatin bound Claspin (CLSPN), CHK1, and PLK1. Claspin and CHK1 stay bound to chromatin across multiple experiments after AURKB inhibition, while chromatin bound PLK1 is unaffected by AURKB inhibition.

Article Snippet: The antibodies used were: 12Ca5 (mouse monoclonal antiHA produced in UVA hybridoma facility), Borealin (Banerjee JCB 2014), AURKA (homemade), H3S10P (Mouse Millipore Cat#06570 multiple Lot#, Rabbit Cell Signaling Technologies Cat#D2C8 Lot# 3377S), H3T3 (EMD Millipore Cat#7-424 Lot#3012075), H2AP.T120 (Active Motif Cat#61195 Lot#31511001), BUB1 (GeneTex Cat#GTx30097 Lot#821701160, PCNA (Novus Biotechne-Brand NB500-106 Lot # A6), H2B (Upstate Cat#07371 Lot#30216) H4 (Cell Signaling Technologies), PLK1P.T210 (Cell Signaling Technologies Lot#A3730), Tubulin (DM1a, UVA hybridoma facility), P-CHK1-S345(Cell Signaling Cat#133D3, Lot#2348S), P-CHK1-S317 (Cell Signaling Cat#2344S) P-ATR90, antiBrdU(Mouse-BD Biosciences 347580 Lot# 7157935 AbCam Rat Cat#AB6326 multiple lots #GR3173537-7, #GR3269246-1), gamma-H2aX (Upstate Cell Signaling Solutions Cat# 07-164 Lot#23646), Pan Aurora T-loop (Cell Signaling Technologies), Claspin (A300267A-Bethyl), IdU (from Burke), CldU (from Burke).

Techniques: In Situ, Kinase Assay, Labeling, Activity Assay, Autoradiography, Inhibition