Journal: Journal of Neuroscience

Article Title: Specific Acetylation of p53 by HDAC Inhibition Prevents DNA Damage-Induced Apoptosis in Neurons

doi: 10.1523/jneurosci.5214-12.2013

Figure Lengend Snippet: Figure 8. Acetylation of lysines 381 and 382 promotes the pro-apoptotic functions of p53 in proliferating tumor cell lines. A, HCT116, DLD1, and HT22 cells were transfected with an equal amount of plasmid DNA encoding wild-type human p53 (WT), DNA-bindingmutantp53(R175H),orthep53acetylationmutantsK373QandK381/382Q.Immunoblotanalysiswasperformedon total cell lysates 24 h after electroporation using antibodies specific for human p53. Plasmid DNA encoding eGFP was used as a transfection control. B, Luciferase reporter assay of plasmid DNA encoding a fragment of the human PUMA proximal promoter (PUMAFrag1-Luc)inhumancolorectalcarcinomacelllinesHCT116andDLD-1andinmousehippocampalcelllineHT22.Promoter reporterplasmidwascotransfectedwithDNA-bindingmutantp53(R175H),wild-typep53(WT),K373Q,orK381/382Qacetylation mutants of p53. Error bars indicate SD of mean from three independent experiments. ***p 0.001, significant difference compared with R175H; ###p 0.001, ##p 0.01, significant difference compared with WT (one-way ANOVA followed by Bonferroni’stest).C,CellviabilitymeasuredbyMTTassayofHCT116,DLD-1,andHT22cultures24haftertransfectionwithR175H, WT, K373Q, or K381/382Q acetylation mutants of p53. Error bars indicate SD of mean from three independent experiments. ***p0.001,**p0.01,*p0.05,significantdifferencecomparedwithR175H; ##p0.01,significantdifferencecompared withWT(one-wayANOVAfollowedbyBonferroni’stest).D,HCT116celldeathassessedbyethidiumhomodimerstaining(EthD-1; red) 24 h after transfection with R175H, WT, K373Q, or K381/382Q acetylation mutants of p53.

Article Snippet: DLD-1 and HCT116 human cell lines were purchased from American Type Culture Collection.

Techniques: Transfection, Plasmid Preparation, Electroporation, Control, Luciferase, Reporter Assay

Journal: Journal of Neuroscience

Article Title: Specific Acetylation of p53 by HDAC Inhibition Prevents DNA Damage-Induced Apoptosis in Neurons

doi: 10.1523/jneurosci.5214-12.2013

Figure Lengend Snippet: Figure 9. HDAC inhibition does not prevent PUMA induction and DNA damage-induced death in HCT116 cells.A, Immunoblot analysisofpan-p53,phosphorylatedp53(pS15-p53),andacetylatedp53(AcK381-p53andAcK382-p53)inextractsfromHCT116 cells treated with CPT (20 M) with or without TSA (1.3 M) for 8 h. B, Quantitative RT-PCR of PUMA expression in HCT116 cells treatedwithCPT(20M)withorwithoutTSA(1.3M)for8h.ErrorbarsindicateSDofmeanfromthreeindependentexperiments. ***p 0.001, significant difference compared with untreated control; ##p 0.01, significant difference compared with CPT (one-wayANOVAfollowedbyBonferroni’stest).C,HCT116cellviabilitymeasuredbyMTTassayaftertreatmentwithCPT(20M) with or without TSA (1.3 M). Error bars indicate SD of mean from three independent experiments. ***p 0.001, **p 0.01, significant difference compared with untreated control; #p 0.05, significant difference compared with CPT (one-way ANOVA followedbyBonferroni’stest).D,HCT116celldeathassessedbyethidiumhomodimerstaining(EthD-1;red)aftertreatmentwith CPT (20 M) with or without TSA (1.3 M).

Article Snippet: DLD-1 and HCT116 human cell lines were purchased from American Type Culture Collection.

Techniques: Inhibition, Western Blot, Quantitative RT-PCR, Expressing, Control

Journal: Developmental cell

Article Title: Inheritance of CENP-A nucleosomes during DNA replication requires HJURP

doi: 10.1016/j.devcel.2018.09.003

Figure Lengend Snippet: (A) Schematic representation of the experimental approach used in B. Cells were blocked at the G1/S boundary or early S phase by addition of thymidine, and allowed to under S phase following thymidine removal. (B) Cells expressing BirA*-fused proteins under doxycycline inducible promoter were treated as shown in A. Biotinylated proteins were isolated by streptavidin purification following by immunoblot analysis with an HJURP antibody. The experiment was conducted twice, each experiment was conducted using 0.9*107 cells. Input and pull down fractions in the experiment were run on independent gels. (C) Schematic representation of the DLD1-Tir1 cell line where HJURP was endogenously tagged at both alleles with AID-YFP. ChiP from HJURP-AID-YFP cells (left) or YFP-CENP-A cells (right) at indicated time points. ChIP was performed using anti-GFP antibody or normal rabbit IgG. RT-PCR was performed using primers specific for α-satellite DNA of chromosome 7. The graph represents an average of two independent experiments, +/− SEM. (D) FACS profiles of cells used as an input for ChIP.

Article Snippet: DLD-1 cell lines (male) expressing Tir1 were obtained from A. Holland (Johns Hopkins University).

Techniques: Expressing, Isolation, Purification, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Developmental cell

Article Title: Inheritance of CENP-A nucleosomes during DNA replication requires HJURP

doi: 10.1016/j.devcel.2018.09.003

Figure Lengend Snippet: (A) Schematic representation DLD1-Tir1 cell line where HJURP was endogenously tagged with AID-YFP at both alleles. (B) Immunoblot analysis showing the efficiency of IAA dependent HJURP degradation. Ponceau was used as a loading control. The blots in B were separated into two sections due to removal of extraneous samples present on the same blot. (C) Clonogenic assay using the parental DLD1-Tir1 and HJURP-AID-YFP cells plus or minus IAA treatment for 10 days. (D) Representative images of DLD1-Tir1 cells in G1/S arrest and G2 phase. Insets are showing single centromeres and sister centromeres in G1/S and G2 phase cells, respectively. DNA was visualized by DAPI, immunofluorescence for CENP-T is shown in green and CENP-A is shown in red. Scale bar is 2μm. (E) Schematic representation of the experiment in F and H. (F)(H) Representative images of cells in G2 phase and mitosis, respectively, and treated as shown in E. DNA was visualized by DAPI, immunofluorescence for CENP-T is shown in green and CENP-A is shown in red. Scale bar is indicated. (G)(I) Quantification of F and H, respectively. The data normalized to G1/S phase condition (G) and untreated condition (I) within cell lines. Normalized data from four (G) or three (I) independent experiments was plotted using box-and-whisker plot: 5–95 percentile, n > 2946 (G) and 8414 centromeres (I). The statistical significance was calculated using unpaired t-test and the p values are indicated. For reference, red line indicates level of CENP-A retention in (G) untreated parental control in G2 phase or (I) untreated parental control in mitosis. The percent loss of CENP-A was calculated to be 31.53% and 36.68% in G and I, respectively.

Article Snippet: DLD-1 cell lines (male) expressing Tir1 were obtained from A. Holland (Johns Hopkins University).

Techniques: Western Blot, Control, Clonogenic Assay, Immunofluorescence, Whisker Assay

Journal: Developmental cell

Article Title: Inheritance of CENP-A nucleosomes during DNA replication requires HJURP

doi: 10.1016/j.devcel.2018.09.003

Figure Lengend Snippet: (A) Schematic representation of the DLD1-Tir1 cell line where HJURP was endogenously tagged with AID-YFP and CENP-A was endogenously tagged with the SNAP tag. (B) The immunofluorescence images of the localization profile of HJURP-AID-YFP in cell line shown in A. (C) Immunoblot analysis of the efficiency of IAA dependent HJURP degradation during mitosis demonstrated by staining with HJURP antibody. Ponceau staining was used as a loading control. The blots were separated in two sections due to removal of empty lanes from the original blot. (D) (F) Schematic representation of the experiment (top). Representative images of cells at indicated time points and treated as shown in the top panel. DNA was visualized by DAPI, immunofluorescence for CENP-T is shown in green and CENP-A is shown in red (bottom). Scale bar is 2μm. (E)(G) Quantification of D and F, respectively. The data was normalized to initial G1/S condition (E) or initial signal (G) within each individual experiment. Normalized data from two (E) or three (G) independent experiments was plotted using box-and-whisker plot: 5–95 percentile, n at least 4674 (E) and 2295 (G). The statistical significance was calculated using unpaired t-test and the p values are indicated. For reference, red line indicates the median level of CENP-A retention in untreated control. The percent loss of CENP-A was calculated to be 38.41% and 27.91% in E and G, respectively.

Article Snippet: DLD-1 cell lines (male) expressing Tir1 were obtained from A. Holland (Johns Hopkins University).

Techniques: Immunofluorescence, Western Blot, Staining, Control, Whisker Assay

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: The role of eEF2K in cancer cell survival under chronic extracellular acidosis. A549 (A) or HCT116 (B) cells were cultured in pH 6.7 (6.7EXT) or pH 7.4 (7.4EXT) buffered medium for about 3 months. Cells were then cultured in pH 6.7 or 7.4-buffered media for 48 h with/without 1 mM IPTG and/or 30 μM A484954, before lysis and Western blot analysis. A549 (C) or HCT116 (D, G) cells were also subjected to flow cytometry to determine subG1 population. (E) HCT116 cells were cultured at pH 7.4 in the presence or absence of 1 mM IPTG and 30 μM A484954 for 48 h, and G1 cell size was then determined by flow cytometry. Results are shown as forward scatter-height (FSC-H). (F) HCT116 cells were cultured as described for panel E and then subjected to flow cytometry analysis; the percentage of sub-G1 population is shown. (H) HCT116 cells cultured at pH 6.7 (6.7EXT) or 7.4 (7.4EXT) for approximately 3 months were transfected with GFP-spectrin alone or GFP-spectrin plus FLAG-tagged eEF2K as indicated. Cells were lysed 8 h after transfection, and eEF2K levels were analyzed by Western blotting. Data are expressed as mean ± SE from 3 independent experiments. P values were obtained by two-way ANOVA. *, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001. For panels A and B, data shown are representative of three independent experiments.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Cell Culture, Lysis, Western Blot, Flow Cytometry, Transfection

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: Acidosis activates eEF2K in cancer cells. (A) Validation of the P-eEF2 Thr56 antibody for immunohistochemistry. eEF2K WT and knockout (KO) MEFs were stained with P-eEF2 Thr56. Scale bar, 50 μm. (B) Representative sequential sections of human lung cancer to support expression of NHE-1, GLUT-1, and P-eEF2 Thr56. Nuclei were counterstained with hematoxylin. Ten lung cancer carcinoma samples were stained in total, 7 of which showed strong areas of GLUT-1 expression; of these, 6 codisplayed NHE-1 and P-eEF2 Thr56. Scale bar, 100 μm. A549 (C) or HCT116 (D) cells were cultured in medium buffered at pH 6.4, 6.8, or 7.4 for the indicated times. IPTG (1 μM) was added to A549 (E) or HCT116 (F) cells 5 days before the experiment. Cells were then incubated in medium buffered at different pH values for 48 h with/without 1 mM IPTG, 30 μM A484954, or 25 μM etoposide, followed by lysis and Western blot analysis. (G) A549 cells were treated as described for panel E, and Western blot analysis was then performed to study the phosphorylation of p70S6K1 at Thr389 and S6 at Ser240/Ser244. For panels C to G, data shown are representative of three independent experiments.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Biomarker Discovery, Immunohistochemistry, Knock-Out, Staining, Expressing, Cell Culture, Incubation, Lysis, Western Blot, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: Activation of eEF2K is important in cancer cell survival under acute acidic conditions. A549 (A) or HCT116 (B) cells were treated as described for Fig. 9E and ​andF,F, respectively, and then subjected to cell ATP assay using the CellTitre-Glo kit. A549 (C) or HCT116 (E) cells were treated as described for panels A and B, respectively, and then subjected to cytotoxicity assay using CellTox Green kit. A549 (D) or HCT116 (F) cells were cultured as described for panels A and B, respectively, and then subjected to flow cytometry analysis. The percentage of sub-G1 population is presented. For panels A, B, C, and E, results are expressed as means ± SE from 3 independent experiments in duplicate. For panel D, results are means ± SE from 3 independent experiments. For panel E, results are means ± SE from 2 independent experiments and a third one in duplicate. P values were obtained either by two-way ANOVA (*, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001) or by one-way ANOVA followed by Dunnett's test (@, 0.01 ≤ P < 0.05; #, 0.01 < P ≤ 0.001; $, P < 0.001).

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Activation Assay, ATP Assay, Cytotoxicity Assay, CellTox Assay, Cell Culture, Flow Cytometry

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: The role of eEF2K in protein synthesis under acidosis. (A) IPTG (1 μM) was added to A549 cells 5 days before experiment. A549 cells were then transferred to media at different pH values for 1 h with/without 1 μM IPTG. Rates of protein synthesis were determined, and results are presented as counts per minute (cpm) per microgram protein and expressed as means ± SE from 4 independent experiments. (B) Quantification of IPTG-treated cells as described for panel A expressed as a percentage of control (no IPTG treatment). For panels A and B, P values were obtained either by two-way ANOVA (*, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001) or by one-way ANOVA followed by Dunnett's test (@, 0.01 ≤ P < 0.05; #, 0.01 < P ≤ 0.001; $, P < 0.001). (C) A549 cells were incubated at pH 6.7 or 7.4 for 1 h. Lysates were fractionated on sucrose density gradients. Positions of the 40S, 60S, and 80S ribosomal particles and polysome fractions are shown. Absorbance values (254 nm) are in arbitrary units and on the same scale for each panel. Representative data from 4 independent experiments are shown.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Control, Incubation