Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: Functional siRNA screen reveals candidate mitotic phosphatases. (A) Screen schematic. 801 siRNAs targeting 267 phosphatases were used in the screen. (B) Nuclear morphology of cells transfected with indicated siRNAs and treated with 100 nm taxol for 24 h 2 d after siRNA transfection. Negative control cells arrest in mitosis when exposed to taxol. Multinucleation resulting from SAC failure occurs in cells transfected with siRNAs targeting CDKN3, ANP32A, INPP5E, 5NT, SAC1, and PP1M as well as MAD2 (positive control). CDC25A knockdown results in premitotic arrest. (C) Quantification of screen results. Broken lines: 95% confidence interval. P-values were calculated using one-way ANOVA. n = 3 counts for each siRNA (a single representative experiment out of two repeats). Error bars represent mean values ± SEM. (D) Subquantification of phenotypes into premitotic arrest versus multinucleation. Knockout of all screen hits except for CDC25A results in SAC failure. Error bars represent mean values of three independent counts ( n = 3). (E) Generation of cell lines expressing tetracycline-inducible shRNAs and GFP. Western blots show target knockdown as a function of time in response to shRNA induction. The SAC failure in MAD2 and CDKN3 shRNA cells is indicated by a decreased phospho-H3 fraction in cells exposed to 100 nm taxol for 24 h after 72 h of tetracycline induction (P < 0.0001 for MAD2 and CDKN3 shRNA cells compared with LACZ shRNA cells in one-way ANOVA; n = 10). Error bars show mean values ± SEM. (F) CDKN3 knockout cells fail to activate the spindle checkpoint in response to three mitotic poisons. (G) CDKN3 knockdown disrupts the SAC response to three spindle poisons. HeLa GFP-H2B/mCherry–α-tubulin cells were transfected with control and CDKN3 siRNAs, and 48 h later treated with inhibitors for 24 h. Cells were fixed and imaged to count nuclear fractions. n = 3 counts per siRNA per condition. P < 0.0001 ( t test). Error bars represent mean values ± SEM. (H) CDKN3 is essential for the spindle checkpoint in human primary brain stem cells. The SC-23 cells were challenged with 200 nm taxol 72 h after siRNA transfection. Mitotic arrest occurs in control cells (green arrows), and CDKN3 knockdown led to multinucleation (red arrows).

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Functional Assay, Transfection, Negative Control, Positive Control, Knock-Out, Expressing, Western Blot, shRNA

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: Spindle checkpoint failure in CDKN3 siRNA-treated cells is not caused by off-target RNAi effects. (A) Overexpression of siRNA-resistant GFP-CDKN3 rescues the SAC failure phenotype in cells transfected with CDKN3 siRNA. Phenotype rescue in CDKN3 knockdown cells transfected with siRNA-resistant GFP-CDKN3 is indicated by mitotic arrest in taxol (red). No phenotype rescue and minimal GFP fluorescence were observed in CDKN3 siRNA cells transfected with siRNA-sensitive GFP-CDKN3 construct (top). Transfection with either GFP-CDKN3 construct resulted in a similar level of GFP-CDKN3 expression in cells pretransfected with control siRNA (bottom two rows). (B) Quantification of the rescue experiment. Error bars indicate SEM. n = 3 experiments; P < 0.0001 in t test. (C) Validated siRNAs against CDKN3 recapitulate the SAC failure phenotype. (D) Quantification of the SAC failure phenotype induced by three CDKN3 siRNAs shown in C. n = 9, P < 0.0001 in one-way ANOVA. (E) Validation of CDKN3 knockdown by four separate siRNAs in quantitative Western blots. n = 6, P < 0.0001 in one-way ANOVA. Note the normal CDKN3 signal in cells transfected with MAD2 siRNA. (F) Cells transfected with four separate CDKN3 siRNAs express normal MAD2 level. The MAD2/actin ratio was quantified by Western blotting. n = 6, (not significant) n.s. in one-way ANOVA for each CDKN3 siRNA. Note the MAD2 knockdown (P = 0.0025) in cells transfected with positive control MAD2 siRNA. (G) Normal localization of endogenous MAD2 on prometaphase kinetochores in cells transfected with CDKN3 siRNAs. Note the lack of MAD2 signal in cells transfected with validated MAD2 siRNA. (H) Quantification of MAD2-positive prometaphase kinetochores in cells transfected with CDKN3 siRNAs and MAD2 siRNAs. P < 0.0001 in ANOVA, n = 50 200-µm nuclear cross sections per siRNA. (I) MAD2 overexpression does not rescue the SAC failure phenotype induced by CDKN3 siRNA. Note the similar nuclear morphology of cells transfected with CDKN3 siRNA alone compared with CDKN3 siRNA followed by MAD2 overexpression. (J) The SAC failure in CDKN3 siRNA cells is not rescued by ectopic overexpression of MAD2. n = 10; n.s. in t test. (K) Expression of Flag-MAD2 in cells transfected with the construct used in rescue experiments (I and J).

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Over Expression, Transfection, Fluorescence, Construct, Expressing, Western Blot, Positive Control

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: CDKN3 is essential for normal mitosis. (A) Inducible knockdown of CDKN3 and MAD2 leads to multinucleation (arrows, 200× magnification). (B) Quantification of multinucleation in CDKN3 and MAD2 knockout cells. P < 0.0001 for both CDKN3 and MAD2 siRNA (one-way ANOVA; n = 5). Error bars represent mean values ± SEM. (C) Knockdown of CDKN3 and MAD2 decreases mitotic index in unsynchronized cells as shown by flow cytometry. P < 0.001 in one-way ANOVA; n = 6. Error bars represent mean values ± SEM. (D) Representative time-lapse frames of cells dividing 72 h after transfection with negative control siRNA (A) and CDKN3 siRNA (B). Note the shortened time between NEB (black arrows) and anaphase (red arrows) upon CDKN3 knockdown. Time-lapse images were taken on an automated imaging system (Pathway 855; BD) in a controlled environment (5% CO 2 , 37°C) every 192 s (3.2 min) using laser autofocus and a 20× NA 0.75 objective lens (Olympus); only every other frame from relevant sequences is shown for simplicity. (E) Frequency distributions of anaphase times. Movies of individual cells in unsynchronized populations were followed manually frame-by-frame to detect NEB. 76 control cells and 137 siCDKN3-transfected cells were measured in three independent experiments. P < 0.0001 in t test. (F) Gallery of mitotic cells transfected with CDKN3 siRNA. Note the multipolar spindles, unattached chromosomes, multinucleation, and cleavage furrows cutting through partially decondensed chromosomes. (G) Quantification of abnormal mitoses in unsynchronized CDKN3 and MAD2 knockout cells. P < 0.0001 (one-way ANOVA; n = 5). Error bars represent mean values ± SEM.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Knock-Out, Flow Cytometry, Transfection, Negative Control, Imaging

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: CDKN3 is a nucleolar protein during interphase and controls G1/S transition. (A) Endogenous CDKN3 localizes to interphase nuclei. Detergent extraction before fixation helps visualize the nucleolar fraction of endogenous CDKN3 (bottom). The signal is lost from cells transfected with CDKN3 siRNA (right), demonstrating antibody specificity. (B) Endogenous CDKN3 colocalizes with nucleolin. CDKN3 foci (green) are surrounded by diffuse nucleolin staining (red) in each nucleolus. (C) CDKN3 phosphatase activity is not required for subcellular targeting of GFP-CDKN3. HeLa cells were transfected with wild-type GFP-CDKN3 (top) and phosphatase-dead GFP-CDKN3 C79S point mutant (bottom). Nucleolar GFP-CDKN3 is visible in detergent-extracted nuclei. At least 100 nuclei were visualized per construct and condition. (D) Representative cell cycle profiles of cells transfected with control siRNA and CDKN3 siRNA. (E) Mildly increased S-phase fraction in HeLa cells transfected with CDKN3 siRNA. P < 0.01 in t test; n = 4. (F) CDKN3 is required for serum starvation–induced G1 arrest. Serum-starved HCT cells transfected with control and CDKN3 siRNAs were pulsed with EdU in serum-free medium to visualize cells that enter S phase (see micrographs on the right; red arrows indicate EdU-positive nuclei). P < 0.0001 in t test; n = 4.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Transfection, Staining, Activity Assay, Mutagenesis, Construct

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: CDKN3 localizes to centrosomes. (A) Endogenous CDKN3 colocalizes with pericentrin during mitosis. (B) Stably overexpressed DDK-CDKN3 associates with centrosomes. (C) A small fraction of endogenous CDKN3 localizes to the midzone in anaphase. (D) Stably overexpressed DDK-CDKN3 colocalizes with KIF20A on midbodies in telophase. (E) A fraction of endogenous CDKN3 remains at centrosomes in interphase. (F) Localization of stably overexpressed DDK-CDKN3 to interphase centrosomes.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Stable Transfection

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: CDKN3 is essential for centrosome maintenance. (A) Abnormal centrosome clusters in HeLa GFP-CENPA/GFP–γ-tub cells transfected with CDKN3 siRNA. Endogenous pericentrin was visualized by immunofluorescence. Arrows indicate multinucleated cells with supernumerary centrosomes. (B) Loss of CDKN3 causes generation of supernumerary centrosomes. P = 0.0073 in t test; n = 5.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Transfection, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: CDKN3 modulates progression through mitosis via regulation of CDC2 phosphorylation at Thr-161. (A) The CDKN3 interface is conserved in human CDK2 (Protein Database [PDB] accession no. 1B39 ) and CDC2 (PDB accession no. 3LFQ ) kinases (GDSEID/DYK motifs, blue). Activation loops (magenta) include putative CDKN3 target residue (pThr160/161; yellow). CDC25 target sites are green. Regions of full conservation are red; other regions are gray. (B) Endogenous CDKN3 colocalizes with endogenous CDC2 on centrosomes during mitosis. (C) Endogenous CDC2 pThr161 localizes to centrosomes and the mitotic spindle during cell division. Dephosphorylation of CDC2 pThr161 occurs in anaphase. HeLa cells were stained with antibody recognizing CDC2 pThr-161 , anti–α-tubulin antibody, and Hoechst 33342. (D) CDC pThr161 localizes to kinetochores in early mitosis. CDC2 pThr161 (red) colocalizes with the kinetochore marker GFP-CENPA (green) in prometaphase but not in anaphase. (E) CDC2 pThr161 is dephosphorylated at exit from mitosis. Cells were arrested in G2 through 24 h of exposure to RO3306 and washed to trigger mitotic entry. Decreasing cyclin B1 levels indicate cell cycle progression toward the mitotic exit. (F) Hyperphosphorylation of CDC2 pThr161 in HeLa cells transfected with CDKN3 siRNA. Total CDC2 and CDC2 pTyr15 levels are unaffected by CDKN3 siRNA. (G) Recombinant CDKN3 inactivates recombinant CDC2/cyclin B in an in vitro kinase assay in a dose-dependent manner. Increasing amounts of recombinant CDKN3 (0.5–5 µg) were incubated for 30 min in kinase buffer with a constant amount of active CDC2–cyclin B kinase complex, CDC2 substrate (histone H1), and radioactive [P 32 ]γ-ATP. CDC2-dependent H1 phosphorylation was detected by autoradiography. 150 mM olomoucine (a CDK kinase inhibitor) was used as a control. (H) Cells transfected with CDKN3 siRNA fail to dephosphorylate CDC2 pThr161 in early anaphase. Note the normal CDC2 pThr-161 metaphase signal in control and CDKN3 siRNA cells. The CDC2 pThr-161 signal persists in CDKN3 siRNA cells during anaphase.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Activation Assay, De-Phosphorylation Assay, Staining, Marker, Transfection, Recombinant, In Vitro, Kinase Assay, Incubation, Autoradiography

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: A proteome-wide phospho-mass spectrometry screen identifies downstream mitotic effectors of CDKN3. (A) Strategy to identify the CDKN3-CDC2 targets. (B) Mass spectrometry reveals hyperphosphorylation of CKβ pSer-209 upon CDKN3 knockdown. Two samples per siRNA were analyzed in duplicate runs (with a total of four LC-MS/MS experiments); representative images are shown. (C) Western blot verification of proteomic screen. Note the increased phosphorylation of endogenous CKβ upon CDKN3 knockdown, whereas the total endogenous CKβ protein level remains the same. (D) Endogenous CKβ pSer-209 localizes to centrosomes. (E) Endogenous CKβ pSer-209 localizes to centrosomes throughout mitosis and disappears from centrosomes in telophase. (F) Western blot verification of CKβ knockdown. (G) CKβ is essential for mitotic spindle checkpoint. HeLa cells were transfected with CK2β siRNAs and treated with 100 nm taxol (24 h) 2 d after transfection. (H) Quantification of SAC failure resulting from CKβ knockdown. P < 0.0001 in one-way ANOVA; n = at least 5 counts per siRNA. Error bars represent mean values ± SEM.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transfection

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: Loss of CDKN3 expression and elevated CDK activity in glioblastoma. (A) CDKN3 is ubiquitously expressed in human brain. (B) The CDKN3 protein is expressed in healthy brain (red arrows). CDKN3 expression is diminished (green arrows) in human GBM tumors. Images are shown at 100× magnification. (C) Immunohistochemistry quantification confirms loss of the CDKN3 protein in GBM. Percentages of CDKN3-positive cells in healthy brain cores and tumor specimens were compared by t test (P = 0.0003). Error bars represent mean values ± SEM. (D) Activation of CDKs in GBMs. Immunohistochemistry reveals increased CDK substrate phosphorylation in brain tumors compared with normal brain. (E) Quantification of increased CDK activity in GBMs. Percentages of phospho-CDK substrate–positive cells in healthy brain cores and tumors were compared by paired t test (P = 0.0133). Error bars represent mean values ± SEM. (F) Decreased expression of CDKN3 accompanied by CDK activation in an independent cohort of human GBMs. Note the increased phosphorylation of CDK substrates in all tumors. (G) Quantification of CDKN3 expression in healthy brain and GBM. CDKN3 and actin were quantified in at least three independent Westerns per specimen, and the ratios were compared with healthy brain (one-way ANOVA; n = 3 experiments for each tumor and 13 experiments for healthy brain). Yellow columns indicate tumors with significantly decreased total CDKN3 expression. Broken lines: 95% confidence interval. Error bars represent mean values ± SEM. (H) Quantification of the CDKN3 expression in 26 GBMs. 23% of tumors (6/26) showed significant loss of CDKN3 expression (one-way ANOVA; n = 3 experiments for each GBM and 13 experiments for healthy brain).

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Expressing, Activity Assay, Immunohistochemistry, Activation Assay

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: Model of sequential CDC2 dephosphorylation during mitosis. Phosphatases identified in this work are marked with asterisks. Dephosphorylation of Tyr-15 by CDC25 activates CDC2 at mitosis entry, whereas dephosphorylation of Thr-161 by CDKN3, PP2A, and other phosphatases guides CDC2 through late mitosis.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: De-Phosphorylation Assay

Journal: bioRxiv

Article Title: An ATM/Wip1-dependent timer controls the minimal duration of a DNA-damage mediated cell cycle arrest

doi: 10.1101/042119

Figure Lengend Snippet: ( A ) U2OS cells transfected with GAPDH or Wip1 siRNA were treated with NCS (5 nM) and collected after 2, 6 and 20 h. Chromatin fractions were probed with indicated antibodies. ( B ) RPE cells transfected with Wip1 siRNA were microirradiated, fixed 1 or 24 h later, and stained with the indicated antibodies. ( C) HA-KAP1-WT or-S824A were immunopurified from cells exposed to NCS, incubated with His-Wip1 and probed with pS824-KAP1 or KAP1 antibody. (D) U2OS cells were fixed 1 h after treatment with NCS, incubated with His-Wip1 (0-5ng/ul) and probed for gH2AX and pS824-KAP1. Plot shows mean nuclear fluorescence intensity, error bars indicate SD. (E ) Kap1 is dephosphorylated before Plk1 activation. RPE cells transfected with TP53 siRNA and U2OS cells were followed as in and stained for pS824-Kap1. For RPE cells, the times were modified as indicated. (F) RPE cells transfected with GAPDH or TP53 siRNA were synchronized by HU, released to fresh media for 5 h (R5) and treated with NCS for indicated times. Nocodazole (NZ) was added 1 h after NCS. Where indicated, cells were incubated in the presence of BI2536. Whole cell lysates were probed with indicated antibodies. ( G ) Overexpression of Kap1-S824A phenocopies ATM inhibition. Cumulative mitotic entry of ≥300 U2OS cells expressing inducible HA-tagged Kap1-wt (red) or Kap1-S824A (green) after treatment with NCS (4 nM) and subsequent treatment after 1h with ATRi or ATRi + ATMi.

Article Snippet: Antibodies against pSMC3-S1083 (#IHC00070), pKap1-S824 (#A300-767A and GTX63711), Kap1 (#A300-274A and GTX62973), pS966-SMC1 (A300-050A), and PP4C (GTX114659) were from Bethyl Lab and GeneTex.

Techniques: Transfection, Staining, Incubation, Fluorescence, Activation Assay, Modification, Over Expression, Inhibition, Expressing

Journal: bioRxiv

Article Title: An ATM/Wip1-dependent timer controls the minimal duration of a DNA-damage mediated cell cycle arrest

doi: 10.1101/042119

Figure Lengend Snippet: (A) U2OS or RPE cells were transfected with Wip1 siRNA, microirradiated, and stained after 1 or 24 h with the indicated antibodies. (B) U2OS and RPE cells were microirradiated in the presence of indicated inhibitors. After 1 h, cells were stained with the indicated antibodies. (C) EGFP or EGFP-Wip1 was immunoprecipitated from HEK293 cells using GFP-Trap. Endogenous KAP1 and p53 were probed with antibodies. (D) U2OS cells transfected with GAPDH or Wip1 siRNA were treated with NCS in combination with DMSO or ATMi for indicated times. Whole cell lysates were probed with indicated antibodies. (E) U2OS cells transfected with GAPDH, Wip1 or PP4C siRNA were treated with NCS for indicated times. Soluble and chromatin fractions were probed with indicated antibodies. (F) RPE cells transfected with GAPDH, Wip1 or PP4C siRNA were treated with NCS for indicated times. Whole cell lysates were probed with indicated antibodies.

Article Snippet: Antibodies against pSMC3-S1083 (#IHC00070), pKap1-S824 (#A300-767A and GTX63711), Kap1 (#A300-274A and GTX62973), pS966-SMC1 (A300-050A), and PP4C (GTX114659) were from Bethyl Lab and GeneTex.

Techniques: Transfection, Staining, Immunoprecipitation

Journal: Journal of Virology

Article Title: A Mechanism-Based Targeted Screen To Identify Epstein-Barr Virus-Directed Antiviral Agents

doi: 10.1128/jvi.01179-20

Figure Lengend Snippet: Figure 2. Validating 30 candidate compounds by measuring their effects on KAP1 432

Article Snippet: 348 349 Antibodies 350 Antibodies included mouse anti-FLAG Ab (Sigma F3165), goat anti-KAP1 Ab (Bethyl 351 Laboratories A303-838A), rabbit anti-KAP1 Ab (Bethyl Laboratories A300-274A), rabbit anti-p-352 S824-KAP1 Ab (Bethyl Laboratories A300-767A), mouse anti-EA-D Ab (EMD MAB8186), 353 IRDye 800CW-conjugated donkey anti-rabbit IgG (LI-COR Bioscience 926-32213), and 354 IRDye 680RD-conjugated donkey anti-goat IgG (LI-COR Bioscience 926-68047).

Techniques:

Journal: Journal of Virology

Article Title: A Mechanism-Based Targeted Screen To Identify Epstein-Barr Virus-Directed Antiviral Agents

doi: 10.1128/jvi.01179-20

Figure Lengend Snippet: Figure 9. Selected candidate compounds inhibit phosphorylation of KAP1 in an independent 487

Article Snippet: 348 349 Antibodies 350 Antibodies included mouse anti-FLAG Ab (Sigma F3165), goat anti-KAP1 Ab (Bethyl 351 Laboratories A303-838A), rabbit anti-KAP1 Ab (Bethyl Laboratories A300-274A), rabbit anti-p-352 S824-KAP1 Ab (Bethyl Laboratories A300-767A), mouse anti-EA-D Ab (EMD MAB8186), 353 IRDye 800CW-conjugated donkey anti-rabbit IgG (LI-COR Bioscience 926-32213), and 354 IRDye 680RD-conjugated donkey anti-goat IgG (LI-COR Bioscience 926-68047).

Techniques: Phospho-proteomics

Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in immortalized MEFs. (A) Cells (WT, MJI-53; KO, MJI-1) were treated for 24 h using 0 or 3 μM aphidicolin. (B) Cells (lane 1–3, WT, MJI-91; lanes 4–6, KO, MJI-186; lanes 7–9, WT, MJI-105; lanes 1–12, KO, MJI-84) were treated for 24 h using 0, 1 or 3 μM aphidicolin. Actin and 53BP1 serve as loading controls. DYNLL1 is used as a surrogate marker for loss of ASCIZ. (C) Quantification of western blot band intensities. An arbitrary unit of 100 represents the average band intensity for the respective phospho-protein in the 3 μM aphidicolin-treated wildtype samples on each membrane. Graphs indicate the mean ± standard error, n = 3. Additional loading controls for total KAP1, p53 and H2AX are shown in Supplementary Fig. S1A and B.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Marker, Western Blot, Membrane

Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in primary MEFs. Freshly prepared primary MEFs from a wildtype and an Asciz −/− embryo were treated for 24 h with 0, 1 or 3 μM aphidicolin. For comparison, cultures from the same embryos were also irradiated with 2-Gy and allowed to recover for 30 min. Similar results for another independent pair of wildtype and Asciz −/− primary MEFs are shown in Supplementary Fig. S1C, and additional loading controls for total KAP1, p53 and H2AX are shown in Supplementary Fig. S1D.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Comparison, Irradiation

Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in human ASCIZ/ATMIN knockout cells. (A, B) BL30 cells were cultured as indicated in the presence or absence of doxycycline to induce a non-targeting control guide RNA or the guide RNA against ASCIZ , and aphidicolin to elicit prolonged DNA replication stress as indicated. For comparison, control guide RNA transduced cells were also irradiated for canonical ATM activation without doxycycline or aphidicolin treatment in panel A. Panel B shows a blot of the same samples except the irradiated control. Additional loading controls for total ATM, KAP1 and p53 are shown in Supplementary Fig. S1E.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Knock-Out, Cell Culture, Control, Comparison, Irradiation, Activation Assay