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, Transgenic Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, 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: Transfection, shRNA, Expressing, Plasmid Preparation, Concentration Assay, Incubation, 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

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: PLoS Genetics

Article Title: Protein Phosphatase 6 Protects Prophase I-Arrested Oocytes by Safeguarding Genomic Integrity

doi: 10.1371/journal.pgen.1006513

Figure Lengend Snippet: (A) Western blots showing up-regulated level of γH2AX and down-regulated CHK1/2-p53 pathway. Level of GAPDH was used as internal controls. Molecular mass is given in kilodaltons. Oocytes were isolated from ovaries of PD35 mice and used for western blot. For each lane, 200 GV oocytes were used. For each experiment, at least 5 mice of each genotype were used. (B) Immunofluorescent staining of 2-month-old ovarian sections showing increased γH2AX in Ppp6c F/F ;GCre+ oocytes. Green: γH2AX; Red: MVH; Blue, DAPI. White arrows point to nucleus of control oocytes; yellow arrows point to nucleus of mutant oocytes. Bar = 20 μm. At least 3 mice of each genotype were used for analysis, and representative images are shown. (C) Decreased incidence of GVBD and PBE of Ppp6c F/F ;GCre+ oocytes. PD35 GV oocytes were isolated and matured in vitro , oocytes that resumed meiosis I (GVBD) and extruded the first polar body (PBE) were counted at 4 h and 13 h, respectively. Data are shown as mean ± SEM. *P< 0.05; **P< 0.01. Representative images of immunostaining for DNA (red) and α-tubulin (green) showing abnormal spindle assembly and aberrant chromosome alignment in Ppp6c F/F ;GCre+ oocytes at 8 h and 13 h, respectively. Bar = 10 μm. In vitro maturation experiments were repeated at least three times.

Article Snippet: Commercial antibodies were used to detect PPP6C (rabbit, A300-844A; Bethyl Laboratories, Inc.), α-tubulin (mouse, DM1A; Sigma-Aldrich), MVH (rabbit, ab13840; Abcam), γH2AX (rabbit, 9718; Cell Signaling Technology, Inc.), p-CHK1 (S345) (rabbit, BS4041; Bioworld Technology, Inc.), p-CHK2 (T68) (rabbit, BS4043; Bioworld Technology, Inc.), p-p53 (S15) (rabbit, 12571; Cell Signaling Technology, Inc.), CHK1 (rabbit, BS1052; Bioworld Technology, Inc.), p-AKT (S473) (rabbit, 4060; Cell Signaling Technology, Inc.), p-AMPK (T172) (rabbit, 2535; Cell Signaling Technology, Inc.), p-mTOR (S2448) (rabbit, 5536; Cell Signaling Technology, Inc.), p-S6K (T389) (rabbit, 9234; Cell Signaling Technology, Inc.), p-rpS6 (S240/244) (Rabbit, 5364; Cell Signaling Technology, Inc.), GAPDH (rabbit, 5174; Cell Signaling Technology, Inc.) and β-actin (mouse, sc-47778, Santa Cruz).

Techniques: Western Blot, Isolation, Staining, Mutagenesis, In Vitro, Immunostaining

Journal: PLoS Genetics

Article Title: Protein Phosphatase 6 Protects Prophase I-Arrested Oocytes by Safeguarding Genomic Integrity

doi: 10.1371/journal.pgen.1006513

Figure Lengend Snippet: (A) Western blots showing up-regulated CHK1/2-p53 pathway activity in zeocin-treated Ppp6c F/F ;GCre+ oocytes. Level of β-actin was used as internal controls. Molecular mass is given in kilodaltons. GV oocytes were isolated from ovaries of PD35 mice and treated with zeocin in vitro . For each lane, 200 GV oocytes were used. For each experiment, at least 5 mice of each genotype were used. (B) Western blots showing up-regulated CHK2-p53 pathway activity in zeocin-treated Ppp6c F/F ;GCre+ ovaries. Level of β-actin was used as internal controls. Molecular mass is given in kilodaltons. Ovary lysates were prepared from ovaries of PD35 mice after zeocin treatment in vivo . For each lane, 30 μg proteins were loaded. For each experiment, at least 3 mice of each genotype were used.

Article Snippet: Commercial antibodies were used to detect PPP6C (rabbit, A300-844A; Bethyl Laboratories, Inc.), α-tubulin (mouse, DM1A; Sigma-Aldrich), MVH (rabbit, ab13840; Abcam), γH2AX (rabbit, 9718; Cell Signaling Technology, Inc.), p-CHK1 (S345) (rabbit, BS4041; Bioworld Technology, Inc.), p-CHK2 (T68) (rabbit, BS4043; Bioworld Technology, Inc.), p-p53 (S15) (rabbit, 12571; Cell Signaling Technology, Inc.), CHK1 (rabbit, BS1052; Bioworld Technology, Inc.), p-AKT (S473) (rabbit, 4060; Cell Signaling Technology, Inc.), p-AMPK (T172) (rabbit, 2535; Cell Signaling Technology, Inc.), p-mTOR (S2448) (rabbit, 5536; Cell Signaling Technology, Inc.), p-S6K (T389) (rabbit, 9234; Cell Signaling Technology, Inc.), p-rpS6 (S240/244) (Rabbit, 5364; Cell Signaling Technology, Inc.), GAPDH (rabbit, 5174; Cell Signaling Technology, Inc.) and β-actin (mouse, sc-47778, Santa Cruz).

Techniques: Western Blot, Activity Assay, Isolation, In Vitro, In Vivo

Journal: The EMBO Journal

Article Title: E2F1 proteolysis via SCF ‐cyclin F underlies synthetic lethality between cyclin F loss and Chk1 inhibition

doi: 10.15252/embj.2018101443

Figure Lengend Snippet:

Article Snippet: pCHK1 S317 , GeneTex , GTX22834.

Techniques: