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

Journal: iScience

Article Title: Frequent loss of FAM126A expression in colorectal cancer results in selective FAM126B dependency

doi: 10.1016/j.isci.2024.109646

Figure Lengend Snippet: Loss of FAM126A expression causes FAM126B dependency in CRC cell lines (A) Scatterplot depicting the correlation between FAM126A expression and FAM126B gene effect. TPM stands for transcripts per million clean reads. Pearson correlation coefficient (r) and p value were indicated on the plot. Linear regression was represented by the red line. (B) Detection of FAM126A and FAM126A-V5 in indicated cell lines by western blotting. (C) Competitive cell growth assay after inactivation of FAM126B or POLD3 in RKO-Cas9 and SW48-Cas9 cells expressing vector or FAM126A-V5. Data are the mean ± SD. from three technical replicates and normalized to control (sgChr2-4). (D) Verification of FAM126A knock out clones from DLD1 and HCT116. (E) Competitive cell growth assay after inactivation of FAM126B or POLD3 in FAM126A knock out clones relative to control cells expressing non-targeting control (NTC) sgRNA. Data are the mean ± SD from three technical replicates and normalized to control (sgChr2-4).

Article Snippet: DLD1 , Dr. Deepak Nijhawan’s lab at University of Texas Southwestern Medical Center , N/A.

Techniques: Expressing, Western Blot, Growth Assay, Plasmid Preparation, Control, Knock-Out, Clone Assay

Journal: iScience

Article Title: Frequent loss of FAM126A expression in colorectal cancer results in selective FAM126B dependency

doi: 10.1016/j.isci.2024.109646

Figure Lengend Snippet:

Article Snippet: DLD1 , Dr. Deepak Nijhawan’s lab at University of Texas Southwestern Medical Center , N/A.

Techniques: Recombinant, CRISPR, Software

Journal: Molecular cell

Article Title: Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

doi: 10.1016/j.molcel.2018.12.008

Figure Lengend Snippet: (A) Immunofluorescence was performed on ovarian B2MUT cells transfected with the indicated siRNAs, using an antibody to γH2AX, to evaluate foci formation. (B) Quantification of the number γH2AX foci formed in (G) in 30 representative cells transfected with siRNA to the indicated genes. (C) Examination of DDR signaling in extracts of WT and BRCA2 mutant cells transfected with the indicated siRNAs and immunoblotted with the indicated antibodies. (D) Quantitation of phospho-Chk1, phospho-H2AX and phospho-RPA32 from the BRCA2 mutant cell extracts in experiment (C).

Article Snippet: Generation of isogenic cell lines We obtained a BRCA2 mutant cell line ( BRCA2 −/− DLD-1) from Horizon Pharma.

Techniques: Immunofluorescence, Transfection, Mutagenesis, Quantitation Assay

Journal: Molecular cell

Article Title: Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

doi: 10.1016/j.molcel.2018.12.008

Figure Lengend Snippet: (A) Extracts from B2MUT ovarian cells expressing Cas9 and the indicated gRNAs were immunoblotted with the indicated antibodies. (B) Quantification of signal detected Western blotting in (A), normalized to vinculin. (C-D) MCA assays in which the indicated GFP-labeled B2MUT cells and E2-Crimson-labeled B2WT cells were mixed and co-infected with 3 individual gRNAs to FEN1. The cell mixture was cultured for 7 d, and the change in percent GFP+ cells was quantified by FACS and normalized to the average of the negative control Grna-expressing cells shown in Supplemental Figure 3A. Error bars reflect the variability of biological triplicates, and the number of asterisks indicates the statistical significance for the corresponding experiment (*=p<0.05,**=p<0.01,***=p<0.001). (E) MCA assay in which colonic GFP-labeled BRCA2 MUT cells and E2-Crimson-labeled BRCA2 WT cells were mixed and co-treated with the indicated doses of a FEN1 inhibitor. The cell mixture was cultured for 7 d, and the change in percent GFP+ cells was quantified by FACS and normalized to negative control gRNA-expressing cells. Error bars reflect the variability of biological triplicates, and asterisks indicate statistical significance as described above. (F) Dependency of BRCA1/2 MUT or BRCA1/2 WT cell lines on FEN1, determined from genome-scale CRISPR-Cas9 essentiality screens across 324 cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) (Meyers et al., 2017). (G) List of gRNA-resistant ORFs tested for complementation in BRCA2 MUT cells expressing Cas9 and a FEN1 gRNA. (H) Examination of the ability of the ORFs listed in (G) to rescue the growth defect caused by expression of Cas9 and a FEN1 gRNA. BRCA2 MUT ovarian cells were co-infected with lentivirus expressing a FEN1 gRNA and the indicated gRNA-resistant FEN1 ORF or negative control peptide. After selection and growth for 8 d, survival was quantified with a FACS-based cell counting method. (I) Extracts from BRCA2 WT cells expressing Cas9, a validated FEN1 gRNA, and the indicated gRNA-resistant ORF were immunoblotted with the indicated antibodies; all panels are derived from the same blot.

Article Snippet: Generation of isogenic cell lines We obtained a BRCA2 mutant cell line ( BRCA2 −/− DLD-1) from Horizon Pharma.

Techniques: Expressing, Western Blot, Labeling, Infection, Cell Culture, Negative Control, MCA Assay, CRISPR, Selection, Cell Counting, Derivative Assay

Journal: Molecular cell

Article Title: Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

doi: 10.1016/j.molcel.2018.12.008

Figure Lengend Snippet: (A) Extracts from the indicated cell lines, untreated or treated with the indicated siRNAs, were immunoblotted with antibodies to BRCA2 and GAPDH. Left and right panels were run as separate gels. (B) Immunofluorescence was performed on cells of the indicated genotypes, with antibodies to γH2AX and Rad51 protein, to evaluate foci formation after 10 Gy IR. (C) The indicated cell lines were passaged in the presence of the indicated dose of olaparib or DMSO. After 3 d, cell survival was quantified using CellTiter-Blue. Error bars reflect the variability of biological triplicates. (D) Schematic diagram depicting the experimental procedure for BRCA2 SL screening in isogenic BRCA2 cell lines. (E) Schematic of wild-type BRCA2 structure, depicted with its functional domains and sites of interaction with key binding partners. Brca2 truncation mutant proteins possessed by the colonic and ovarian BRCA2 MUT cell lines used in this study are shown for comparison.

Article Snippet: Generation of isogenic cell lines We obtained a BRCA2 mutant cell line ( BRCA2 −/− DLD-1) from Horizon Pharma.

Techniques: Immunofluorescence, Functional Assay, Binding Assay, Mutagenesis, Comparison

Journal: Molecular cell

Article Title: Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

doi: 10.1016/j.molcel.2018.12.008

Figure Lengend Snippet: (A-C) Volcano plots for the shRNA colonic, CRISPR colonic, and CRISPR ovarian B2SL screens. Significance (-log10FDR) is plotted against the genetic interaction (GI) score (average log2 fold-change for each gene in BRCA2 MUT versus BRCA2 WT cells). Genes that met a significance threshold of -log10FDR>1 are color-coded as green for relative dropout or red for relative enrichment in BRCA2 MUT vs BRCA2 WT cells. (D-F) Results from the colonic shRNA, colonic CRISPR, and ovarian CRISPR B2SL screens, plotted as the log2 fold-change in BRCA2 MUT cells against the log2 fold-change in BRCA2 WT cells. (G-H) Comparison of GI score (average log2 fold-change for each gene in BRCA2 MUT versus BRCA2 WT cells) in the ovarian CRISPR screen versus the colonic CRISPR screen (G), and the ovarian CRISPR screen versus primary shRNA screen (H).

Article Snippet: Generation of isogenic cell lines We obtained a BRCA2 mutant cell line ( BRCA2 −/− DLD-1) from Horizon Pharma.

Techniques: shRNA, CRISPR, Comparison

Journal: Molecular cell

Article Title: Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

doi: 10.1016/j.molcel.2018.12.008

Figure Lengend Snippet: (A) Performance of genes in several key pathways, plotted on a color scale for genetic interaction (GI) score: the normalized average log2 fold-change across both colonic and ovarian CRISPR screens. The asterisk indicates reporting of GI score from the shRNA screen instead of combined CRISPR screens. (B) Schematic of the base excision repair (BER) pathway, showing the strength of the GI score for each gene in the pathway, plotted on the same color scale as in (A). (C-F) MCA assays in which colonic GFP-labeled BRCA2 MUT and E2-Crimson-labeled BRCA2 WT cells were mixed and co-treated with the indicated drugs. The change in percent GFP+ cells was measured by FACS after 12 d and normalized to vehicle control. Error bars reflect the variability of biological triplicates.

Article Snippet: Generation of isogenic cell lines We obtained a BRCA2 mutant cell line ( BRCA2 −/− DLD-1) from Horizon Pharma.

Techniques: CRISPR, shRNA, Labeling, Control

Journal: Journal of Cancer

Article Title: Integrated Multi-omics Analyses Identify CDCA5 as a Novel Biomarker Associated with Alternative Splicing, Tumor Microenvironment, and Cell Proliferation in Colon Cancer Via Pan-cancer Analysis

doi: 10.7150/jca.91082

Figure Lengend Snippet: Correlation between CDCA5 and TME of colorectal cancer with TISCH database. (A) Correlation of CDCA5 with TME in CRC. (B, C) The annotation and distribution of the immune cell types in CRC GSE136394 and CRC GSE13955. The proportion of CDCA5 in different immune cell types in CRC GSE136394 dataset (D, E) and CRC GSE13955 dataset (F, G).

Article Snippet: The human colorectal cancer cell line DLD1 was purchased from Procell Life Science& Technology Co., Ltd (Wuhan,China), which was cultured in DMEM supplemented with 10% heat‐inactivated fetal bovine serum (FBS) (Sijiqing Biologic, Hangzhou, China) and was incubated at 37 ̊C in a humid incubator with air containing 5% CO2.

Techniques:

Journal: Journal of Cancer

Article Title: Integrated Multi-omics Analyses Identify CDCA5 as a Novel Biomarker Associated with Alternative Splicing, Tumor Microenvironment, and Cell Proliferation in Colon Cancer Via Pan-cancer Analysis

doi: 10.7150/jca.91082

Figure Lengend Snippet: Effect of CDCA5 silencing on DLD1 cell proliferation, apoptosis, migration and clone formation abilities. (A) mRNA level of CDCA5 after Si-CDCA5 transfection. (B) The viability of DLD1 cells after Si-CDCA5 transfection. (C) Cell clone formation after CDCA5 knockdown. (D) Cell proliferation after CDCA5 knockdown. (E) CDCA5-siRNA reduced the migration of colon cancer cells. (F) CDCA5-siRNA reduced the invasion of colon cancer cells. (G) Cell apoptosis after CDCA5 knockdown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001.

Article Snippet: The human colorectal cancer cell line DLD1 was purchased from Procell Life Science& Technology Co., Ltd (Wuhan,China), which was cultured in DMEM supplemented with 10% heat‐inactivated fetal bovine serum (FBS) (Sijiqing Biologic, Hangzhou, China) and was incubated at 37 ̊C in a humid incubator with air containing 5% CO2.

Techniques: Migration, Transfection, Knockdown

Journal: Genes & Nutrition

Article Title: Effect of genistein on cholesterol metabolism-related genes in a colon cancer cell line

doi: 10.1007/s12263-008-0082-5

Figure Lengend Snippet: Effects of increasing genistein concentrations on 3H-thymidine incorporation in DNA of DLD-1 cells after 24 h of hormonal treatment. Data displayed as mean ± SE. *P < 0.05 Dunnett’s multiple comparisons test versus control (CTR)

Article Snippet: Cell culture condition Human colon adenocarcinoma-derived DLD-1 cells were obtained from the Interlab Cell Line Collection (IST, Genoa, Italy).

Techniques:

Journal: Genes & Nutrition

Article Title: Effect of genistein on cholesterol metabolism-related genes in a colon cancer cell line

doi: 10.1007/s12263-008-0082-5

Figure Lengend Snippet: Genistein effects on HMG-CoA reductase gene expression in DLD-1 cells

Article Snippet: Cell culture condition Human colon adenocarcinoma-derived DLD-1 cells were obtained from the Interlab Cell Line Collection (IST, Genoa, Italy).

Techniques: Expressing

Journal: Genes & Nutrition

Article Title: Effect of genistein on cholesterol metabolism-related genes in a colon cancer cell line

doi: 10.1007/s12263-008-0082-5

Figure Lengend Snippet: LDL-R mRNA levels in DLD-1 cells exposed for 5 h to genistein (0.01 μM) alone and genistein (0.01 μM) plus ICI 182,780 (0.1 μM). Data displayed as mean ± SE of number molecules mRNA LDL receptor/number molecules mRNA β-actin. P value was determined by one-way analysis with Dunnett’ post-test. *P < 0.05 versus control (CTR)

Article Snippet: Cell culture condition Human colon adenocarcinoma-derived DLD-1 cells were obtained from the Interlab Cell Line Collection (IST, Genoa, Italy).

Techniques:

Journal: Genes & Nutrition

Article Title: Effect of genistein on cholesterol metabolism-related genes in a colon cancer cell line

doi: 10.1007/s12263-008-0082-5

Figure Lengend Snippet: Genistein effects on LDL receptor gene expression in DLD-1 cells

Article Snippet: Cell culture condition Human colon adenocarcinoma-derived DLD-1 cells were obtained from the Interlab Cell Line Collection (IST, Genoa, Italy).

Techniques: Expressing