Journal: PLoS ONE

Article Title: Checkpoint Signaling, Base Excision Repair, and PARP Promote Survival of Colon Cancer Cells Treated with 5-Fluorodeoxyuridine but Not 5-Fluorouracil

doi: 10.1371/journal.pone.0028862

Figure Lengend Snippet: (A) Metabolism of 5-FU and FdUrd. (B, C) HT29 (B) and HCT-8 (C) cells were treated with 5-FU (80 µM, HT29 cells; 200 µM HCT-8 cells), FdUrd (40 µM for both cell lines), or 10 mM hydroxyurea (HU) for the indicated times. Cell extracts were blotted for phospho-Ser317-Chk1 (P-Chk1), phospho-Thr68-Chk2 (P-Chk2), Chk1, or Chk2. TS, thymidylate synthase; TP, thymidine phosphorylase; UP, uridine phosphorylase; UK, uridine kinase; OPRT, orotate phosphoribosyltransferase; RR, ribonucleotide reductase; FUR, 5-fluorouridine; FUMP, 5-fluorouridine monophosphate; FUDP, 5-fluorouridine diphosphate; FUTP, 5-fluorouridine triphosphate; FdUMP, 5-fluorodeoxyuridine monophosphate; FdUDP, 5-fluorodeoxyuridine diphosphate; FdUTP, 5-fluorodeoxyuridine triphosphate.

Article Snippet: Antibodies were as follows: phospho-Ser317-Chk1 (R&D Systems); phospho-Thr68-Chk2, ATR, horseradish peroxidase-linked rabbit IgG, and horseradish peroxidase-linked mouse IgG (Cell Signaling); Chk1 (Santa Cruz Biotechnology); Chk2 and ATM (Epitomics); APE1 (Abcam); XRCC1 (Bethyl Laboratories); beta-actin (Sigma-Aldrich); and HSP90, D. Toft (Mayo Clinic, Rochester, MN).

Techniques:

Journal: NAR Molecular Medicine

Article Title: ATM-mediated co-chaperone DNAJB11 phosphorylation facilitates α-synuclein folding upon DNA double-stranded breaks

doi: 10.1093/narmme/ugae007

Figure Lengend Snippet: Etoposide induces ATM-dependent DNAJB11 T188 phosphorylation. ( A ) Etoposide-induced DNAJB11 phosphorylation is reduced in ATM inhibitor-treated cells. 293T and SH-SY5Y cells were pretreated with the ATM-specific inhibitor Ku55933, ATR-specific inhibitor AZD6738, or solvent DMSO for 24-h, followed by treatment with 10 μM of etoposide for 24-h. IPs were performed with an anti-DNAJB11 antibody. Immunoprecipitates were sequentially probed with anti-pSQ/TQ and anti-DNAJB11 antibodies. Five percent of lysates used for IP were loaded as the inputs and probed with anti-pSQ/TQ, anti-pCHK2, anti-CHK2, anti-pCHK1, anti-CHK1 and anti-DNAJB11 antibodies. β-actin is a loading control. ( B ) The amounts of pSQ/TQ and DNAJB11 were quantified, using the ImageJ software. The results of the quantitative analysis are shown as the relative values to the solvent controls (Student's t -test, *, P < 0.05). ( C ) ATM phosphorylates DNAJB11 T188 in vitro . 293T and SH-SY5Y cells were pretreated with or without the ATM-specific inhibitor Ku55933 and followed by treatment with or without 10 μM of etoposide. In vitro ATM kinase assay was conducted using immunoprecipitants of ATM on recombinant His6-DNAJB11, His6-DNAJB11 T188A, and His6-DNAJB11 T188E substrates. Samples were loaded onto a 10% SDS-PAGE, and the phosphorylated proteins were detected by anti-pSQ/TQ antibodies (upper panel, n = 3). The same samples were stained with Coomassie blue to confirm that all proteins were equally loaded (left panel). The precipitated ATM kinases were resolved by a 6% SDS-PAGE and detected with anti-ATM antibodies (right panel, n = 3).

Article Snippet: The antibodies used for immunoblotting were Myc (Roche, Switzerland), GFP (Santa Cruz, USA), FLAG (Sigma, USA), Phospho-CHK2 (Thr68) (Cell Signaling, USA), CHK2 (Cell Signaling, USA), His (Cytiva, USA), pSQ/TQ (Cell Signaling, USA), DNAJB11 (Proteintech, USA), Phospho-CHK1 (Ser317) (Cell Signaling, USA), CHK1 (Santa Cruz, USA), ATM (Cell Signaling, USA), HSPA8 (Novus Biologicals, USA), BIP (Abcam, UK), GST (Genetex, USA), p-AKT (S473) (Cell Signaling, USA), AKT (Cell Signaling, USA), γH2AX (upstate USA) and β-actin (Sigma, USA).

Techniques: Phospho-proteomics, Solvent, Control, Software, In Vitro, Kinase Assay, Recombinant, SDS Page, Staining

Journal: Cancer Cell

Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

doi: 10.1016/j.ccell.2017.08.010

Figure Lengend Snippet:

Article Snippet: Chk1 [p Ser317] Rabbit polyclonal Antibody , Novus Biologicals , Cat# NB100-92499; RRID: AB_1216466.

Techniques: Purification, Imaging, Virus, Control, Mutagenesis, Recombinant, Staining, Reverse Transcription, SYBR Green Assay, Microarray, RNA Expression, RNA Sequencing, Methylation, Labeling, Software, Light Microscopy

Journal:

Article Title: Cryptotanshinone has diverse effects on cell cycle events in melanoma cell lines with different metastatic capacity

doi: 10.1007/s00280-010-1440-8

Figure Lengend Snippet: Effect of Cryptotanshinone on the expression of cell cycle-associated proteins in B16 and B16BL6 melanoma cells. Cells were treated with Cryptotanshinone (0, 1, 10 and 25 µM) for 24 h. Whole-cell extracts were analyzed by western blotting using the indicated antibody. β-actin or GAPDH was used as an internal control to monitor equal protein loading. a The effect of Cryptotanshinone on the expression of p53, Chk1 and Chk2 in B16 and B16BL6 melanoma cells. b The effect of Cryptotanshinone on the expression of p21 and Cdc25c in B16 and B16BL6 melanoma cells. c The effect of Cryptotanshinone on the expression of Cyclin A1, Cyclin B1 and Cdk1/cdc2 in B16 and B16BL6 melanoma cells

Article Snippet: Protein samples were resolved by SDS–PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA). p53, checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), p21, Cyclin A1, Cyclin B1, cyclin-dependent kinase 1/cell division cycle2 (Cdk1/Cdc2) and cell division cycle 25 homolog C (Cdc25c) proteins were detected by immunoblot with anti-p53 (1:500, NCL-p53-CM5p, NovoCastra, Newcastle, UK), anti-Chk1 (1:200, BS1053, Bioworld Technology, Bioworld, USA), anti-Chk2 (1:200, BS1391, Bioworld Technology, Bioworld, USA), anti-p21 (1:200, sc-6246, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cyclin A1 (1:200, BS1804, Bioworld Technology, Bioworld, USA), anti-Cyclin B1 (1:200, sc-752, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cdk1/Cdc2 (1:200, BS1820, Bioworld Technology, Bioworld, USA), anti-Cdc25c (1:200, sc-327, Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal antibody and β-actin (1:15000; Sigma St Louis, MO, USA) or GAPDH (1:5000, sc-32233, Santa Cruz Biotechnology, Santa Cruz, CA), then followed by incubation with peroxidase-coupled secondary antibodies.

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1

doi: 10.1101/2020.02.10.942573

Figure Lengend Snippet: A.) (From left to right) A549 HDAC6 KO cells generated with the CRISPR-Cas9 system. H157 and H1975 HDAC6 KO cells generated with the CRISPR-Cas9 system. H1299 and A549 inducible HDAC6 knockdown cells (termed H1299i and A549i, respectively) pre-treated with doxycycline for two weeks. Mouse embryonic fibroblasts (MEFs) harvested from age-matched wild-type and transgenic HDAC6 KO mice (both from a C57Bl/6 background). Liver, kidney, lung, heart, spleen, and brain tissue harvested from age-matched wild type and transgenic HDAC6 KO mice (both from a C57Bl/6 background). All cell lines and tissues were lysed and analyzed via Western Blot for Chk1, HDAC6, acetylated tubulin, and GAPDH expression. B.) RT-PCR was used to determine whether HDAC6 knockdown influences Chk1 mRNA levels in A549 control and HDAC6 stable knockdown cells, as well as WT and HDAC6 knockout murine lung tissue. C.) (Above) A549 stable knockdown cells were treated with 10μg/mL cycloheximide (CHX), harvested at the indicated timepoints, and analyzed via Western blot. Representative Western blot of Chk1 and GAPDH from the trials used to determine Chk1 half-life. (Below) The average intensity of Chk1 relative to GAPDH expression from three independent experiments was obtained (via ImageJ) and graphed. D.) 293T HDAC6 knockout cells were plated, and 24 hours later were transfected with 2.4μg HA-tagged HDAC6. Control cells were treated with transfection reagent PEI for 24 hours. HA-HDAC6-transfected cells were harvested at the indicated timepoints and probed for the indicated proteins. Fold-change in Chk1 expression was evaluated via ImageJ. E.) Mammalian expression vectors containing Myc-Chk1, Flag-HDAC6, and His-Ub were transfected into HEK-293T cells. Cells were incubated for 48 hours, harvested, and passed through a Ni-NTA column to pull down for His-Ub. Bound proteins were subsequently eluted from the columns, run on an SDS-PAGE gel, and probed for Chk1.

Article Snippet: Tissue arrays were then stained with 1:25 Chk1 S317 anti-rabbit (Novus Biologicals, Catalog#: NB100-92499) and pan-cytokeratin AE1/AE3 anti-mouse (1:200, DAKO Cytomation, Catalog#: M3515) using the following diluent: 1% NGS+ 0.1% T20+PBS.

Techniques: Generated, CRISPR, Knockdown, Transgenic Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Knock-Out, Transfection, Incubation, SDS Page

Journal: bioRxiv

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1

doi: 10.1101/2020.02.10.942573

Figure Lengend Snippet: A , B.) Mammalian expression vectors containing Flag-Chk1 and HA-HDAC6 were transfected into HEK-293T cells with PEI. 48 hours after overexpression, cells were harvested in lysis buffer, incubated with either HA-coated (A) or Flag-coated (B) agarose beads, and the resultant immunoprecipitated protein was run on an SDS-page gel and probed for the reciprocal tag. C.) 293T lysates were probed with anti-Chk1 antibody complexed with protein A/G beads, the beads were washed, and the resulting milieu probed for HDAC6 to detect an endogenous interaction between Chk1 and HDAC6. D.) His-Chk1 was overexpressed in E. coli . His-Chk1 was purified with Ni-NTA agarose beads. Then, GST and GST-HDAC6 were overexpressed in E. coli , and GST-tagged protein was pulled-down and purified by glutathione-agarose. Purified His-Chk1 was incubated with either glutathione agarose-bound GST or GST-HDAC6, and then bound proteins were eluted. The samples were subjected to SDS-PAGE and Western blot analysis. E.) The indicated Flag-tagged HDAC6 deletion mutant constructs were transfected into 293T cells along with Myc-Chk1. 48 hours later, cells were lysed, and lysates were pulled down for Flag. F.) Schematic of the Flag-tagged HDAC6 deletion mutant constructs used for the coimmunoprecipitation in (C). G.) The indicated Myc-tagged Chk1 deletion mutant constructs were transfected into 293T cells along with Flag-HDAC6. 48 hours later, cells were lysed, and lysates pulled down for Flag. H.) Schematic of the Myc-tagged Chk1 deletion mutant constructs used for the coimmunoprecipitation in (E).

Article Snippet: Tissue arrays were then stained with 1:25 Chk1 S317 anti-rabbit (Novus Biologicals, Catalog#: NB100-92499) and pan-cytokeratin AE1/AE3 anti-mouse (1:200, DAKO Cytomation, Catalog#: M3515) using the following diluent: 1% NGS+ 0.1% T20+PBS.

Techniques: Expressing, Transfection, Over Expression, Lysis, Incubation, Immunoprecipitation, SDS Page, Purification, Western Blot, Mutagenesis, Construct

Journal: bioRxiv

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1

doi: 10.1101/2020.02.10.942573

Figure Lengend Snippet: A.) A549 HDAC6 knockdown cells were transfected with a TRIPZ inducible lentiviral shRNA-expressing plasmid against Chk1, creating the Chk1Tripz line that is HDAC6 and Chk1 knocked-down. B.) Chk1Tripz and A549 HDAC6 knockdown cells were plated in triplicate at a concentration of 150 cells/well and treated with the indicated dose of radiation. Cells were incubated for 12 days, fixed with crystal violet, and quantified. Single sample t test, *p<0.05, **p<0.0008. C.) Representative images of A549 HDAC6 stable knockdown and Chk1Tripz colony formation assays described in (C). D.) A549 control and HDAC6 stable knockdown cells were pre-treated with 0.25μM of potent Chk1 inhibitor CHIR-124 prior to 10Gy irradiation. At the indicated timepoints, cells were harvested and probed for the indicated proteins via western blot.

Article Snippet: Tissue arrays were then stained with 1:25 Chk1 S317 anti-rabbit (Novus Biologicals, Catalog#: NB100-92499) and pan-cytokeratin AE1/AE3 anti-mouse (1:200, DAKO Cytomation, Catalog#: M3515) using the following diluent: 1% NGS+ 0.1% T20+PBS.

Techniques: Knockdown, Transfection, shRNA, Expressing, Plasmid Preparation, Concentration Assay, Incubation, Control, Irradiation, Western Blot

Journal: bioRxiv

Article Title: HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1

doi: 10.1101/2020.02.10.942573

Figure Lengend Snippet: Kaplan-Meier curve, univariate analysis of the overall survival of 187 NSLCL patients stratified by p S317 Chk1 status, with this status determined via AQUA staining.

Article Snippet: Tissue arrays were then stained with 1:25 Chk1 S317 anti-rabbit (Novus Biologicals, Catalog#: NB100-92499) and pan-cytokeratin AE1/AE3 anti-mouse (1:200, DAKO Cytomation, Catalog#: M3515) using the following diluent: 1% NGS+ 0.1% T20+PBS.

Techniques: Staining