Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: ( a ) Cell proliferation and ( b ) cell viability alterations of HT-29 and SW480 colorectal cancer cell lines following different folic acid (FA) supplies. Sulforhodamine B (SRB) was used for cell proliferation detection (* p ≤ 0.05, *** p ≤ 0.001), while cell viability data were obtained by alamarBlue assay (** p ≤ 0.01). FA-depleted cells were kept in media containing 0 ng/mL FA, whereas treated cells were exposed to 100 and 10,000 ng/mL FA for 72 h. The percentages of cell proliferation and viability were given relative to samples kept in the normal growth media. FA: folic acid.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Alamar Blue Assay

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: Genomic stability detection of HT-29 and SW480 cells exposed to different folic acid (FA) concentrations (0, 100, 10,000 ng/mL). ( a ) Micronucleus (MN) scoring was performed on DAPI- and anti-γ-H2AX-stained slides. Left: We obtained the results by proportioning the cells with MN with all cells counted (** p ≤ 0.01, *** p ≤ 0.001). Right: Representative γ-H2AX-positive micronuclei are indicated with arrows. ( b ) DNA integrity was evaluated with comet assay, additionally. Left: Graphs show the changes in genomic stability in consideration of comet tail DNA percentage (* p ≤ 0.05). Right: Characteristic comets of both cell lines were captured following different treatments. FA: folic acid.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Staining, Single Cell Gel Electrophoresis

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: DNA methylation analysis of HT-29 and SW480 cell lines exposed to different folic acid (FA) concentrations. The methylation levels of long interspersed nuclear element 1 (LINE-1) CpG positions (pos 1, pos 2, pos 3) were ( a ) summarized and also ( b ) visualized individually to detect global DNA methylation changes. With the use of Reduced Representation Bisulfite Sequencing (RRBS) method, a genome-wide methylome profile of 10,000 ng/mL FA-treated cells was established in the comparison of cells kept in FA-free (0 ng/mL FA) media. ( c ) Firstly, the number of genes with altered methylation in the investigated CpG sites was assessed. “Hyper” and “hypo” sections indicate the number of genes with methylated and unmethylated CpG sites, respectively. The intersection of these two categories refers to the genes that possess both methylated and unmethylated CpG dinucleotides. ( d ) Heatmap shows the top 10 significantly ( p ≤ 0.05) enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with the number of differentially methylated genes. ( e ) Pie charts represent the localization of differentially methylated sites (DMS) in distinct chromatin states. FA: folic acid; pos: CpG position; hyper: hypermethylation; hypo: hypomethylation; DMSs: differentially methylated sites; heterochrom/lo: heterochromatin or low signal region; txn: transcription; CNV: copy number variation; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: DNA Methylation Assay, Methylation, Methylation Sequencing, Genome Wide, Comparison

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: Genome-wide transcriptome alterations of HT-29 and SW480 cells following 10,000 ng/mL folic acid (FA) supplementation detected by Human Transcriptome Array 2.0 (HTA 2.0). ( a ) Pie charts represent the proportion of up- and downregulated genes. ( b ) Visual networks of protein–protein interactions were generated by the StringApp of Cytoscape software based on the list of genes with significant ( p ≤ 0.05) expression alterations and ≥|1.5| fold change (FC). Colors refer to the expression level of protein-coding genes (dark blue: FC ≤ −2, light blue: FC ≥ −2 and ≤−1.5, light red: FC ≥ 1.5 and ≤2, dark red: FC ≥ 2). ( c ) Top 10 genes showing significant ( p ≤ 0.05) up- and downregulation visualized with volcano plots. Gray points represent all the transcripts detected by HTA 2.0 microarray, while significantly ( p ≤ 0.05) altering genes with FC ≥ |1.5| value were marked with red and blue. P-val: p -value.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Genome Wide, Protein-Protein interactions, Generated, Software, Expressing, Microarray

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: The intersection of genome-wide DNA methylation and gene expression data obtained by Reduced Representation Bisulfite Sequencing (RRBS) and Human Transcriptome Array (HTA) 2.0 analyses. Values represent the methylome and transcriptome pattern changes of 10,000 ng/mL folic acid (FA)-treated HT-29 and SW480 cells compared to non-treated samples (0 ng/mL FA). Only genes with promoter methylation status alteration in accordance with their expression level ( p ≤ 0.05 and fold change ≥|1.5|) were listed (left) and also visualized in volcano plots (right). Gray points represent all the transcripts detected by the microarray, while blue ones highlight down- and red ones show upregulating genes from the list. met. status: DNA methylation status; met. diff.: DNA methylation difference; expr. status: gene expression status; P-val: p -value.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Genome Wide, DNA Methylation Assay, Gene Expression, Methylation Sequencing, Methylation, Expressing, Microarray

Journal: Cell reports

Article Title: Single-cell secretion analysis reveals a dual role for IL-10 in restraining and resolving the TLR4-induced inflammatory response

doi: 10.1016/j.celrep.2021.109728

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were then stained in 50 uL for 1 hour at room temperature with anti-Phospho-p38 MAPK (Thr180/Tyr182, 3D7, Alexa Fluor 488 Conjugate) Rabbit mAb (Cell Signaling Technology #41768, 1:50 dilution) or anti-Phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204, D13.14.4E) Rabbit mAb (Cell Signaling Technology #4370) as a primary followed by staining in 50 uL using Goat anti-Rabbit IgG (H+L, Alexa Fluor 488) as a secondary antibody.

Techniques: Blocking Assay, Recombinant, Software, Single-cell Analysis, Flow Cytometry

Journal: Cell reports

Article Title: Single-cell secretion analysis reveals a dual role for IL-10 in restraining and resolving the TLR4-induced inflammatory response

doi: 10.1016/j.celrep.2021.109728

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were then stained in 50 uL for 1 hour at room temperature with anti-Phospho-p38 MAPK (Thr180/Tyr182, 3D7, Alexa Fluor 488 Conjugate) Rabbit mAb (Cell Signaling Technology #41768, 1:50 dilution) or anti-Phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204, D13.14.4E) Rabbit mAb (Cell Signaling Technology #4370) as a primary followed by staining in 50 uL using Goat anti-Rabbit IgG (H+L, Alexa Fluor 488) as a secondary antibody.

Techniques: Blocking Assay, Recombinant, Software, Single-cell Analysis, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: Impact of the modulation of TYRO3 expression on the radiosensitivity of bladder cancer cell lines. ( a – c ) Representative clonogenic survival curves of RT112 and 5637 cell lines ( upper panel). 48 h after transfection with siLUC (control, red), siTYRO3#4 (blue), siTYRO3#801 (green) or diluted concentration of siRNA#1 (black) cells were exposed to increased doses of gamma-rays. In each case, downregulation was confirmed by western blot at 48 h after transfection. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( d ) Representative survival curves of UM-UC-3 control (empty plasmid, red) and TYRO3 over-expressed cell lines (TYRO3 encoding- plasmid, purple) ( upper panel). The upregulation was confirmed by western blot. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( e ) Phosphorylated TYRO3 shown in western blot ( lower panel) after immunoprecipitation of the cell extracts with phospho-Tyrosine antibodies and its relative expression in 5637 cells quantified using Image-J software and normalized to that of total TYRO3 ( upper panel). Data represents mean ± SD of 3 independent experiments. Unpaired t-test analysis: * p < 0.05; ** p < 0.005, ns: not significant.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Expressing, Transfection, Control, Concentration Assay, Western Blot, Plasmid Preparation, Immunoprecipitation, Software

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on Ionizing Radiation-Induced Foci and DNA damage. γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 downregulated RT112 ( a ) or 5637 ( c ) cells (scale bar 5 microns); Quantification of cells containing more than 10 γH2AX foci at 24 h after 2 Gy of irradiation in the downregulated RT112 ( b ) and 5637 ( d ) cell lines; ( e ) γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 overexpressing UM-UC-3 cell line (Scale bar 5 microns); ( f ) Quantification of cells containing more than 10 γH2AX foci at 30 min and 24 h after 2 Gy of irradiation in TYRO3 overexpressing cell lines. The data shown above is from three different experiments and error bars represent the SD. Unpaired t -test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0005, ns: non-significant. Representative images of the alkaline Comet assay performed on TYRO3 downregulated RT112 ( g ) and 5637 ( h ) cells and the resulting Olive tail moments analysis in TYRO3 downregulated RT112 ( i ) and 5637 ( j ) cells irradiated at 6 Gy. The data shown here is from three independent experiments analyzing 200 nucleus per condition per experiment, horizontal bars represent the median values. Kruskal-Wallis nonparametric tests with Multiple comparisons were used: * p < 0.05; ** p < 0.005, ns: non-significant; ( k ) western blot of DNA damage repair proteins; TYRO3 was downregulated, irradiated at 6 Gy and the lysates were prepared and analyzed 0.5–24 h after.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Alkaline Single Cell Gel Electrophoresis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on DNA damage response pathways. ( a ) Volcano plot showing the fold change (Log2 Ratio) versus negative log of the p-value of differentially expressed genes after Nanostring analysis between BCa (RT112 and 5637) 6 Gy irradiated cells versus BCa 6 Gy irradiated cells after complete TYRO3 knock-down (siTYRO3#4 and siTYRO3#801). Significant: p -value < 0.05 ( b ) Log2 ratio of the significantly expressed genes ( p < 0.05) after Nanostring analysis between the two groups. The name of the up- or downregulated genes are listed on the left. On the right are the corresponding Nanostring gene annotations. ( c ) Protein-protein association network of the up and downregulated genes assessed using the STRING database.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 downregulation affects cell cycle following irradiation. Analysis of the cell cycle distribution 24 h after 6 Gy irradiation on TYRO3-downregulated RT112 ( a ) and 5637 ( b ) cells. Comparison of percentage (%) of cells in G2/M in RT112 ( c ) and 5637 ( d ) cells. Data shown is from three different experiments and error bars represent the SD. Unpaired t-test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005, ns: non-significant. ( e ) western blot of cell cycle proteins 30 min and up to 24 h after irradiation.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Comparison, Western Blot

Journal: eLife

Article Title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

doi: 10.7554/eLife.74342

Figure Lengend Snippet:

Article Snippet: Antibody , Anti- PERK (Rabbit monoclonal) , Cell Signaling Technology , 3192 , WB (1:1000).

Techniques: Cloning, CRISPR, Knock-Out, Expressing, Recombinant, Plasmid Preparation, Transfection, Sequencing, Software, Diffusion-based Assay, Single Particle

Journal: Cell Reports Medicine

Article Title: Leucine restriction ameliorates Fusobacterium nucleatum- driven malignant progression and radioresistance in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2024.101753

Figure Lengend Snippet: Intracellular F. nucleatum promotes radioresistance in NPC cells by suppressing host apoptosis and DNA damage (A–G) Fn-infected and uninfected NPC cells were exposed to 2, 4, and 8 Gy irradiation, respectively. (A) Representative images of NPC cells. Fn (MOI = 10:1) or E. coli -infected NPC cells (MOI = 1:100). Scale bar: 150 μm. (B) Cellular viability with live/dead assay. Statistical results are presented in the below panels. Data are mean values of three biology repeats. Scale bar: 100 μm. (C) Representative photographs of colony formation assays. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (D) The apoptosis rates were determined by flow cytometry. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (E) LDH activity in supernatant was assessed by LDH Cytotoxicity Assay Kit; optical density (OD) values of 490 nm were present with histogram. (F) Representative images of the comet assay. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (G) Western blot analysis of γH2AX was performed. Statistical results are presented in the right panels. Data are mean values of three biology repeats. Data are shown as mean ± SD. p values were determined by independent sample t tests (C–E and G), ∗ p < 0.05, ∗∗ p < 0.001, and ∗∗∗ p < 0.001.

Article Snippet: E. coli DH5α , TIANGEN , Cat# CB101-02.

Techniques: Infection, Irradiation, Live Dead Assay, Flow Cytometry, Activity Assay, LDH Cytotoxicity Assay, Single Cell Gel Electrophoresis, Western Blot

Journal: Cell Reports Medicine

Article Title: Leucine restriction ameliorates Fusobacterium nucleatum- driven malignant progression and radioresistance in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2024.101753

Figure Lengend Snippet:

Article Snippet: E. coli DH5α , TIANGEN , Cat# CB101-02.

Techniques: Recombinant, Reverse Transcription, SYBR Green Assay, Viability Assay, ROS Assay, LDH Cytotoxicity Assay, ATP Assay, Sequencing, Virus, Synthesized, Software

Journal: Developmental cell

Article Title: Mechanosensing by the lamina protects against nuclear rupture, DNA damage, and cell cycle arrest

doi: 10.1016/j.devcel.2019.04.020

Figure Lengend Snippet: Key resources table

Article Snippet: Rabbit polyclonal anti-lamin B1 , Abcam , ab16048.

Techniques: Recombinant, Single Cell Gel Electrophoresis, Expressing, Control, Software

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: ATR Target Activation by the CHK1 Inhibitor AZD7762 in U2OS Cancer Cells (A) Western blot showing activation of ATR targets. U2OS cells were treated with the indicated concentrations for 30 and 60 min, lysed, and probed with anti-phospho (Serine 345) CHK1 and β-actin antibodies. (B) Induction of pre-apoptotic pan-nuclear γ-H2AX by ATR and CHK1 inhibitor in combination in cancer cells. U2OS cells were treated with the indicated drug concentrations for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody. Scale bar, 20 μm. (C) Quantitative data of γH2AX- (nine or more foci per cells) positive cells or pan-nuclear γH2AX signal after indicated treatments are shown (n = 3, mean ± SEM). (D) Western blot showing increased phosphorylation of H2AX after combination treatment. U2OS cells were treated with the indicated concentrations for 24 hr. At the end of incubation time, western blotting was performed using anti-phospho (Serine 139) H2AX, anti-phospho (Serine 345) CHK1, cleaved PARP, anti-phospho (Serine 10) H3, and β-actin antibodies. (E) Comet assay showing DNA damage induction by ATR and CHK1 inhibitor in combination. U2OS cells were treated with the indicated drug concentrations for 24 hr. At the end of incubation, cells were harvested and alkaline comet assay was performed. (F) Quantitative data of the tail moment are shown (n = 3, mean ± SEM, in each experiment ≥100 comets were measured). (G) Cancer-specific ssDNA formation by VE-821 and AZD7762, either alone or in combination. U2OS cells were treated with the indicated drug concentrations for 24 hr and pre-extracted using CSK buffer before fixation. Cells were stained with anti-RPA32 antibody; images were taken using a confocal microscope and were analyzed using ImageJ software. A mean intensity of ≥70 a.u. per cell was considered as positive. Quantitative data are presented as mean ± SEM from three independent experiments. (H) ssDNA formation in normal fibroblast VH-10 cells is shown. (I) Pre-apoptotic pan-nuclear γH2AX induction by combination treatment of ATR and CHK1 inhibitors in U2OS is mediated through the JNK pathway. U2OS cells were treated with the indicated drug concentrations for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody, and high-throughput microscopy was used to determine the percentage of γH2AX-positive cells (nine or more γH2AX foci per cell) or an average intensity of ≥2,000 a.u. for pan-nuclear γH2AX-positive cells (n = 2 with multiple wells, mean ± SEM). Statistical significance was determined using one-way ANOVA ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Incubation, Single Cell Gel Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Staining, Microscopy, Software, High Throughput Screening Assay

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: Combination of the ATR Inhibitor VE-821 and the CHK1 Inhibitor AZD7762 Synergistically Kills Cancer Cells (A) Clonogenic survival of U2OS, VH-10, and MCF-7 cells. The 500 (U2OS and MCF-7) or 1,000 (VH-10) cells were seeded in 10-cm 2 dishes, and, after 5-hr incubation, the inhibitors were added directly to the media. After 72-hr incubation, drug-containing media were replaced with fresh media and cells were kept for another 5–8 days before colonies were stained with methylene blue. Quantitative data: n = 3, mean ± SEM. (B) Parental and cMYC-transformed cells were treated with the indicated doses for 72 hr. At the end of the incubation period, resazurin was added and cell viability was measured. Quantitative data: n = 3, mean ± SEM. (C) BJ-hTERT, BJ-hTERT SV40, and BJ SV40 RAS cells were treated with the indicated doses for 72 hr. At the end of the incubation period, resazurin was added and cell viability was measured. Quantitative data: n = 3, mean ± SEM. (D) CHK1 functionally compromised cells are sensitive to ATR inhibitor. Clonogenic survival of DLD-1, DLD-1 CHK1 S317A/− , DLD-1 CHK1 +/− , and DLD-1 ATR S/S after ATR inhibitor VE-821 treatment is shown. A similar protocol was used as for U2OS and VH-10 cells. Quantitative data: n = 3, mean ± SEM. (E) Therapeutic efficacy of combined inhibition of ATR and CHK1 in mouse tumor models. Therapeutic efficacy of VX-970 and AZD7762 in H460 lung cancer xenografted mice is shown. BALB/c nude mice bearing H460 xenograft were divided in four groups (five animals in each group) with a tumor volume of ∼130 mm 3 in each group. The first control group of animals was treated with vehicle (orally and intraperitoneally). The second group of animals was treated with 25 mg/kg body weight of CHK1 inhibitor AZD7762 (intraperitoneal route). The third group of animals was treated with 60 mg/kg body weight of ATR inhibitor VE-822 (oral administration), and the fourth group received a combination of both CHK1 and ATR inhibitors. Vehicle and drugs were administered on days 0–3, 10–12, and 18–20 irrespective of no mice survival in each group. Tumor volume was measured with calipers and is shown here as mean ± SEM. Statistical significance was determined using two-way ANOVA with repeated measurement ( ∗ p < 0.05 and ∗∗ p < 0.01). (F) Kaplan-Meier survival curve of H460-xenografted mice. When tumor size reached 1,000 mm 3 , the animal was sacrificed.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Incubation, Staining, Transformation Assay, Drug discovery, Inhibition, Control

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: ATR and CHK1 Inhibitors, Alone or in Combination, Decrease Replication Fork Speed Only in Cancer Cells (A) Treatment regimen is shown. U2OS and VH-10 cells were treated for 60 min with the indicated drug concentrations and sequentially labeled with 5-chlorodeoxyuridine (CldU) and 5-iododeoxyuridine (IdU) for 30/20 min each in the presence of the inhibitors. DNA fibers were stained and replication speed was measured by IdU labeling. (B and C) Representative images show stained replication fork tracts for each treatment group. (D) Quantitative data of replication fork speed (kb/min), mean ± SEM, and p values were analyzed with one-way ANOVA for each condition and cell line. (E and F) Average distribution of replication fork rates. A minimum of 450 forks per condition were analyzed from at least three independent repeats.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Labeling, Staining

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: Combination Treatment of VE-821 and AZD7762 Results in S Phase Arrest in U2OS Cells (A) U2OS cells were treated with the indicated drug concentrations for 24 hr and propidium iodide (PI) staining was carried out to measure cell-cycle profile using flow cytometry. (B) Quantitative data were obtained using Modfit software. (C) ATR and CHK1 inhibitors in combination decrease EdU incorporation in U2OS cells. U2OS cells were treated for 24 hr with the indicated doses. Images were taken with a confocal microscope and analyzed using ImageJ software. A mean intensity of ≥80 a.u. per cell was considered as EdU-positive cells. Quantitative data: n = 3, mean ± SEM. (D) No significant decrease in EdU incorporation in normal fibroblast VH-10 cells treated with the ATR and CHK1 inhibitors either alone or in combination. VH-10 cells were treated for 24 hr with the indicated doses. Quantitative data: n = 3, mean ± SEM. Statistical significance was determined using one-way ANOVA ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Staining, Flow Cytometry, Software, Microscopy

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: Synergistic Cytotoxic Effect in U2OS Cancer Cells by Combination Treatment of AZD7762/VE-821 Is Mainly Due to CDK-Mediated Excess Origin Firing (A) U2OS cells were pretreated with the indicated concentrations of the CDK inhibitor Roscovitine for 1 hr prior to the addition of VE-821 and AZD7762 for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody and anti-53BP1, and DNA was counterstained with ToPro. (B) Quantitative data of pan-nuclear γH2AX are shown (mean ± SEM from two independent experiments). (C) Treatment regimen for DNA fiber assay in U2OS cells is shown. (D) CDK inhibitors Roscovitine and PHA-767491 enhance the replication fork speed of U2OS cells treated with VE-821 and AZD7762 in combination. U2OS cells were pre-treated with Roscovitine or PHA-767491 for 1 hr prior to the addition of VE-821 and AZD7762. Representative images show stained replication fork tracts for each treatment group. (E) Average distribution of replication fork rates. A minimum of 450 forks per condition were analyzed from at least three independent repeats. (F) Roscovitine abolishes the synergistic cytotoxic effect of combination treatment of AZD7762 and VE-821 in U2OS cancer cells. U2OS cells were individually treated with VE-821, AZD7762, or the combination with our without Roscovitine for 24 hr, followed by recovery for another 48 hr. Cell viability was measured by using resazurin at 72 hr. (G) Model for ATR/CHK1 synthetic lethality. CHK1 is activated by replication stress both by ATR-dependent and -independent pathways ( Yang et al., 2008 ) to suppress replication stress in cancer, promoting restart and survival. CHK1 inhibitors increase oncogene-activated CDK activity and origin firing, leading to replication stress and accumulation of stalled replication forks, requiring ATR activity to prevent replication collapse. Red arrows indicate primary route in the presence of both ATR and CHK1 inhibitors.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Staining, Activity Assay

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: HU-Induced Replication Stress in Combination with VE-821 and AZD7762 Causes Fragmented Nuclei and the Early Onset of Apoptosis Only in U2OS Cells (A) U2OS cells were treated for 24 hr with the indicated drug concentrations and stained with anti-cleaved caspase 3 and β-actin antibodies. Representative confocal images are shown. (B) Quantitative data of fragmented nuclei and cleaved caspase-3 positive cells presented as mean ± SEM from three independent experiments. (C) Western blot showing apoptosis in U2OS cells treated with ATR and CHK1 inhibitors alone or in combination. U2OS cells were treated with the indicated concentrations for 24 hr; lysed; protein extracted; and western blotting was performed with anti-Cleaved PARP, anti-phospho (Serine 10) Histone H3, and anti-β-actin antibodies. (D) HU-induced replication stress does not cause fragmentation of nuclei or apoptosis in combination with VE-821 and AZD7762 in VH-10 normal fibroblast cells in 24 hr. Etoposide treatment (4 μM) was used to induce apoptosis as a positive control. Scale bar represents 20 μM.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Staining, Western Blot, Positive Control