Journal: Photochemistry and photobiology

Article Title: The REV-ERB antagonist SR8278 modulates keratinocyte viability in response to UVA and UVB radiation.

doi: 10.1111/php.13930

Figure Lengend Snippet: FIGURE 3 KS15 and SR8278 absorb UV wavelengths of light to limit DNA photoproduct formation and signaling responses. (A, B) Initial (6-4)PP and CPD levels in genomic DNA were quantified from experiments performed in Figure 2. (C) The absorbance spectrum of KS15 and SR8278 was determined by spectrophotometry. (D) Representative western blots of phosphorylated KAP1 (Ser824) and CHK1 (Ser345) in HaCaT keratinocytes pre-treated for 24 h with vehicle (DMSO) or KS15 and SR8278. (E, F) Quantitation of phospho-KAP1 and phospho-CHK1 from HaCaT cells from three experiments performed as in (C).

Article Snippet: The blots were blocked in 5% non- fat milk in TBST (Tris- buffered saline containing 0.1% Tween- 20) and then probed overnight with primary antibodies from Cell Signaling Technology recognizing XPA (#14607), BMAL1 (#14020), or phosphorylated CHK1 (Ser345; #2348) or from Bethyl Laboratories against phosphorylated KAP1 (Ser824; A300- 767A).

Techniques: Spectrophotometry, Western Blot, Quantitation Assay

Journal: Journal of Clinical Investigation

Article Title: CHK1 targets spleen tyrosine kinase (L) for proteolysis in hepatocellular carcinoma

doi: 10.1172/jci61380

Figure Lengend Snippet: Figure 1 SYK(L) is regulated by CHK1 in HCC cells. (A) Sequence alignment of the putative CHK1 phos- phorylation motif in SYK(L) from different species. The domain structure of SYK(L) protein and its alternative splicing variant SYK(S) are shown. SYK(S) has 23 amino acid residues (DEL) miss- ing in the IDB region, and the sequence homology of the 23-residue DEL across species is shown. (B) The indicated cells were transfected with the indicated plasmids for 24 hours (left) or the indi- cated siRNAs for 48 hours (right) and then were analyzed by Western blot. (C) SMMC7721 cells without detectable SYK(L) expression were trans- fected with the indicated plasmids for 24 hours, and the whole cell lysates (WCLs) were resolved directly by SDS-PAGE or were first incubated with Flag agarose and then analyzed by Western blot. (D) Cell lysates from Huh7 cells were immuno- precipitated with anti-CHK1, anti-SYK(L), or pre- immune IgG as indicated and were subsequently immunoblotted with anti-SYK(L) or anti-CHK1 antibody. Note: CHK1 associates with endog- enous SYK(L).

Article Snippet: Antibodies against SYK (N-19), Ki67, actin, and GAPDH were from Santa Cruz Biotechnology Inc. Antibodies against phosphorylated-CHK1 (Ser 345), phosphorylated-cdc2 (Tyr 15), CHK2, His, and HA were obtained from Cell Signaling Technology.

Techniques: Sequencing, Alternative Splicing, Variant Assay, Residue, Transfection, Western Blot, Expressing, SDS Page, Incubation

Journal: Journal of Clinical Investigation

Article Title: CHK1 targets spleen tyrosine kinase (L) for proteolysis in hepatocellular carcinoma

doi: 10.1172/jci61380

Figure Lengend Snippet: Figure 2 SYK(L) is regulated by CHK1 through phosphorylation on Ser295. (A) His-tagged SYK(L) WT or the S295A mutant that was translated in vitro were incubated with samples immunoprecipitated from cells with Flag-CHK1 in the presence or the absence of ATP (as indicated) for 2 hours. The samples were then analyzed by Western blot. (B) His-tagged SYK(L) WT protein translated in vitro was incubated with Flag-CHK1 WT or its kinase dead (KD) form, which was immunoprecipitated from cells as indicated for 2 hours. The samples were then analyzed by Western blot. (C) SMMC7721 cells transfected with the indicated plasmids for 24 hours were analyzed by Western blot. (D) SMMC7721 cells were stably transfected with the WT SYK(L) or the mutant SYK(L)-S295A as indicated, and the cells were treated with GÖ6976 (100 nM) or HU (2.5 mM) for 10 hours as indicated and subjected to Western blot. (E) Huh7 cells were transfected with scramble or 3 different CHK1 siRNA duplexes as indicated for 48 hours. The cell lysates were resolved directly by SDS-PAGE (WCL) or were first incubated with anti-SYK(L) antibodies (IP: SYK) and then analyzed by Western blot.

Article Snippet: Antibodies against SYK (N-19), Ki67, actin, and GAPDH were from Santa Cruz Biotechnology Inc. Antibodies against phosphorylated-CHK1 (Ser 345), phosphorylated-cdc2 (Tyr 15), CHK2, His, and HA were obtained from Cell Signaling Technology.

Techniques: Phospho-proteomics, Mutagenesis, In Vitro, Incubation, Immunoprecipitation, Western Blot, Transfection, Stable Transfection, SDS Page

Journal: Journal of Clinical Investigation

Article Title: CHK1 targets spleen tyrosine kinase (L) for proteolysis in hepatocellular carcinoma

doi: 10.1172/jci61380

Figure Lengend Snippet: Figure 3 Regulation of SYK(L) by CHK1 occurs via the ubiquitin/proteasome pathway. (A and B) Huh7 cells were cotransfected with the indicated plasmids for 20 hours and were treated with or without MG132 (10 μM) for 4 hours. The cell lysates were resolved directly by SDS-PAGE or were first incubated with Flag agarose and then analyzed by Western blot. (C) Huh7 cells stably expressing SYK(L) WT or the S295A mutant were incubated with 20 μg/ml of CHX for the indicated times. Cell lysates were then analyzed by Western blot (left panel), and the densities of the SYK(L) protein bands at each time point were normalized to GAPDH and were calculated into percentages using 100% as the value of the zero time point (right panel). (D) Huh7 cells trans- fected with scrambled siRNA or CHK1 siRNA were incubated with 20 μg/ml of CHX for the indi- cated times. Cell lysates were then analyzed as described in C.

Article Snippet: Antibodies against SYK (N-19), Ki67, actin, and GAPDH were from Santa Cruz Biotechnology Inc. Antibodies against phosphorylated-CHK1 (Ser 345), phosphorylated-cdc2 (Tyr 15), CHK2, His, and HA were obtained from Cell Signaling Technology.

Techniques: Ubiquitin Proteomics, SDS Page, Incubation, Western Blot, Stable Transfection, Expressing, Mutagenesis

Journal: Journal of Clinical Investigation

Article Title: CHK1 targets spleen tyrosine kinase (L) for proteolysis in hepatocellular carcinoma

doi: 10.1172/jci61380

Figure Lengend Snippet: Figure 4 SYK(L) function is negatively regulated by CHK1-mediated phosphorylation of Ser295 in HCC in vitro and in vivo. (A) SMMC7721 cells were stably transfected with the indicated plasmids, as shown by Western blot (insertion panel), and the proliferative capacity of these cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at the indicated times (n = 3). The dots represent the mean, while the bars indicate the SEM. *P < 0.05; **P < 0.001, using Student’s t test. (B) The stable cell lines used in A were cultured for 12 days, and the number of colonies was counted and graphed (n = 3). Bars indicate the SEM. P values were obtained by Student’s t test. (C) The stable cell lines used in A were subjected to migration or invasion assays as described in Methods, and the representative photographs are shown in the left panel. Scale bar, 80 μm. The quantification of the left panel was graphed (right panel; n = 3). Bars indicate the SEM. P values were obtained by Student’s t test. (D) The stable cell lines used in A were injected into nude mice (2 × 106 cells per mouse), and after 4 weeks, the xenografts were excised from the mice and weighed. Each dot represents a tumor weight, and the mean tumor weights in each group are indicated by solid lines (n = 8). P values were obtained by Student’s t test. (E) The stable cell lines made from both Huh7 and SMMC7721 cells were subjected to RT-PCR using primers specific for the indicated genes.V, empty vector as control.

Article Snippet: Antibodies against SYK (N-19), Ki67, actin, and GAPDH were from Santa Cruz Biotechnology Inc. Antibodies against phosphorylated-CHK1 (Ser 345), phosphorylated-cdc2 (Tyr 15), CHK2, His, and HA were obtained from Cell Signaling Technology.

Techniques: Phospho-proteomics, In Vitro, In Vivo, Stable Transfection, Transfection, Western Blot, MTT Assay, Cell Culture, Migration, Injection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Control

Journal: Journal of Clinical Investigation

Article Title: CHK1 targets spleen tyrosine kinase (L) for proteolysis in hepatocellular carcinoma

doi: 10.1172/jci61380

Figure Lengend Snippet: Figure 5 Inhibition of CHK1 suppresses tumorigenicity through the CHK1/ SYK(L) pathway in HCC in vitro and in vivo. (A) SMMC7721 stable cell lines used in Figure 3A were treated with GÖ6976 (100 nM) or DMSO (Con) as indicated for different amounts of time and were subjected to an MTT assay (n = 3). The dots represent the mean, while the bars indicate the SEM. (B) SMMC7721 stable cell lines were injected into the flanks of nude mice and incubated for 6 days, and then the mice were treated with GÖ6976 (100 nM) or DMSO. The tumor volumes were measured and recorded every 3 days, and tumor growth curves were created for each group (n = 14). Dots represent the mean, while bars indicate the SEM. *P < 0.05, using Student’s t test. (C) On day 27, the xenografts were excised from mice and weighed, as shown in Supplemental Figure 9. Each dot represents a tumor weight; the mean tumor weights of each group were indicated by solid lines (left panel; n = 14), and P values were obtained using the Student’s t test.

Article Snippet: Antibodies against SYK (N-19), Ki67, actin, and GAPDH were from Santa Cruz Biotechnology Inc. Antibodies against phosphorylated-CHK1 (Ser 345), phosphorylated-cdc2 (Tyr 15), CHK2, His, and HA were obtained from Cell Signaling Technology.

Techniques: Inhibition, In Vitro, In Vivo, Stable Transfection, MTT Assay, Injection, Incubation

Journal: Journal of Clinical Investigation

Article Title: CHK1 targets spleen tyrosine kinase (L) for proteolysis in hepatocellular carcinoma

doi: 10.1172/jci61380

Figure Lengend Snippet: Figure 6 Overexpression of CHK1 indicates a poor prognosis, and there is an inverse correlation between the protein levels of SYK(L) and CHK1 in HCC. (A and B) RFS (A) and OS (B) curves were generated based on the CHK1 protein expression statuses from 162 HCC samples. Actuarial probabilities were calculated using the Kaplan-Meier method and were compared using the log-rank test. After resection of primary tumors, patients with low CHK1 expression in their primary tumors had better RFS and OS rates than those with high CHK1 expression (P = 0.011 and P = 0.013, respectively). (C) Immunohistochemical stain- ing of CHK1 and SYK(L) was performed in tumor tissues of patients with HCC. Representative examples of CHK1 and SYK(L) staining in the serial sections from the same tumor tissues are shown. Scale bars: 50 μm. Data represent mean ± SEM.

Article Snippet: Antibodies against SYK (N-19), Ki67, actin, and GAPDH were from Santa Cruz Biotechnology Inc. Antibodies against phosphorylated-CHK1 (Ser 345), phosphorylated-cdc2 (Tyr 15), CHK2, His, and HA were obtained from Cell Signaling Technology.

Techniques: Over Expression, Generated, Expressing, Immunohistochemical staining, Staining

Journal: Journal of Clinical Investigation

Article Title: CHK1 targets spleen tyrosine kinase (L) for proteolysis in hepatocellular carcinoma

doi: 10.1172/jci61380

Figure Lengend Snippet: Figure 7 A proposed model for the regulation of SYK(L) by CHK1 in HCC. (A) High levels of CHK1 protein may phosphorylate SYK(L) on Ser295, which triggers the degradation of SYK(L) by the ubiquitin/proteasome pathway and consequently favors the tumorigenesis of HCC. (B) When CHK1 is inhibited using small molecule inhibitors, such as GÖ6976, the phosphorylation of SYK(L) is diminished to stabilize the SYK(L) protein, which in turn suppresses tumor progression in HCC.

Article Snippet: Antibodies against SYK (N-19), Ki67, actin, and GAPDH were from Santa Cruz Biotechnology Inc. Antibodies against phosphorylated-CHK1 (Ser 345), phosphorylated-cdc2 (Tyr 15), CHK2, His, and HA were obtained from Cell Signaling Technology.

Techniques: Ubiquitin Proteomics, Phospho-proteomics

Journal: PLoS ONE

Article Title: Mutation of Serine 1333 in the ATR HEAT Repeats Creates a Hyperactive Kinase

doi: 10.1371/journal.pone.0099397

Figure Lengend Snippet: (A–C) The indicated ATR mutant or wild type proteins complexed with ATRIP were purified from HEK293T cells and incubated with GST-MCM2 substrate, [γ- 32 P]-ATP, and GST-TOPBP1-AAD (GST-AAD) or GST. Kinase reactions were separated by SDS-PAGE and [ 32 P]-MCM2 and [ 32 P]-GST-TOPBP1 detected by autoradiography. Quantitation of the fold activation compared to the wild type protein incubated with GST was measured by a phosphorimager and is indicated below each lane. The amount of ATR, TOPBP1, MCM2, and GST proteins in each reaction was detected with Coomassie blue (CB) or immunoblotting (IB) as indicated. Each mutant was tested at least three times and representative experiments are shown.

Article Snippet: Antibodies used include ATR-N19 (Santa Cruz Biotechnology), HA (Covance), CHK1-G4 (Santa Cruz Biotechnology), FLAG-M2 (Sigma), ATRIP403 , MCM2 (BD Transduction Labs), phosphorylated Ser-317 CHK1 (Cell Signaling Technology), phosphorylated Ser-345 CHK1 (Cell Signaling Technology), and phosphorylated Ser-10 Histone H3 (Cell Signaling Technology).

Techniques: Mutagenesis, Purification, Incubation, SDS Page, Autoradiography, Quantitation Assay, Activation Assay, Western Blot

Journal: PLoS ONE

Article Title: Mutation of Serine 1333 in the ATR HEAT Repeats Creates a Hyperactive Kinase

doi: 10.1371/journal.pone.0099397

Figure Lengend Snippet: The indicated ATR mutant or wild type proteins complexed with ATRIP were purified and incubated with GST-MCM2 substrate, [γ- 32 P]-ATP, and GST-TOPBP1-AAD (GST-AAD) or GST. Kinase reactions were separated by SDS-PAGE and [ 32 P]-MCM2 detected by autoradiography. Quantitation of the fold activation compared to wild type protein incubated with GST measured by a phosphorimager is indicated below each lane. The amount of ATR and MCM2 present was detected by Coomassie blue (CB) or immunoblotting (IB) as indicated. Each mutant was tested at least three times and a representative experiment is shown.

Article Snippet: Antibodies used include ATR-N19 (Santa Cruz Biotechnology), HA (Covance), CHK1-G4 (Santa Cruz Biotechnology), FLAG-M2 (Sigma), ATRIP403 , MCM2 (BD Transduction Labs), phosphorylated Ser-317 CHK1 (Cell Signaling Technology), phosphorylated Ser-345 CHK1 (Cell Signaling Technology), and phosphorylated Ser-10 Histone H3 (Cell Signaling Technology).

Techniques: Mutagenesis, Purification, Incubation, SDS Page, Autoradiography, Quantitation Assay, Activation Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: PARP Inhibition Increases the Reliance on ATR/CHK1 Checkpoint Signaling Leading to Synthetic Lethality—An Alternative Treatment Strategy for Epithelial Ovarian Cancer Cells Independent from HR Effectiveness

doi: 10.3390/ijms21249715

Figure Lengend Snippet: ATRi/CHK1i combined with PARPi increases apoptosis. Caspase 3/7 expression was used as an indicator of apoptosis in EOC cell lines exposed to 0.5 µmol/L PARPi, 0.5 µmol/L CHK1i, and 0.5 µmol/L ATRi for 24 and 48 h. Untreated cells were used as controls and considered 100%. In SKOV-3 cells, apoptosis was increased by both ATRi in combination with PARPi (30%) and CHK1 combination with PARPi (22%) after 48 h of treatment. In OV-90 (p53MUT) cells, apoptosis was increased after 24 and 48 h of incubation with ATRi in combination with PARPi (16% and 17%, respectively) and CHK1 in combination with PARPi (13% and 9% respectively). In PEO-1 (BRCA2 MUT ) cells, apoptosis was increased by ATRi in combination with PARPi (35%) and CHK1 in combination with PARPi (24%) after 48 h of treatment. Error bars denote standard deviation, * indicates statistically significant differences between the samples incubated with the drugs compared with control cells ( p < 0.05). + Statistically significant differences between the samples incubated with PARPi and combination treatment (PARPi/ATRi; PARPi/CHK1i) ( p < 0.05). # Statistically significant differences between samples incubated with ATRi or CHKi and combination treatment (PARPi/ATRi; PARPi/CHK1i). Statistical analysis was performed using the ANOVA test with the Tukey’s post-hoc test for multiple comparisons.

Article Snippet: After blocking nonspecific sites using 5% bovine serum albumin (BSA) (Sigma-Aldrich) or 5% non-fat dry milk, membranes were incubated with the rabbit monoclonal antibody against PARP at a dilution of 1/1000 (cat. # 9532), phospho-ATR (Thr1989) (cat. # 30632), total ATR (cat. # 13934), phospho-CHK1 (Ser345) (cat. # 2348), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cat. #2118), all from Cell Signaling Technology, Inc. (Danvers, MA, USA), and the rabbit anti-CHK1 polyclonal antibody (cat. # STJ92269, St John’s Laboratory).

Techniques: Expressing, Incubation, Standard Deviation, Control

Journal: International Journal of Molecular Sciences

Article Title: PARP Inhibition Increases the Reliance on ATR/CHK1 Checkpoint Signaling Leading to Synthetic Lethality—An Alternative Treatment Strategy for Epithelial Ovarian Cancer Cells Independent from HR Effectiveness

doi: 10.3390/ijms21249715

Figure Lengend Snippet: Proposed model of the molecular and cellular responses to new replication stress inhibitors. ATR/CHK1 stabilizes replication forks and prevents their collapse into DSBs. SSBs can be accurately repaired using the undamaged strand as a template, a process involving the PARP enzyme. SSBs are mainly repaired through the homologous recombination (HR) pathway. BRCA is related to the error-free repair of DSBs by HR. ATRi or CHK1i in monotherapy and in combined treatment with PARPi cause genome instability and leads to the synthetic lethality of ovarian cancer cells.

Article Snippet: After blocking nonspecific sites using 5% bovine serum albumin (BSA) (Sigma-Aldrich) or 5% non-fat dry milk, membranes were incubated with the rabbit monoclonal antibody against PARP at a dilution of 1/1000 (cat. # 9532), phospho-ATR (Thr1989) (cat. # 30632), total ATR (cat. # 13934), phospho-CHK1 (Ser345) (cat. # 2348), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cat. #2118), all from Cell Signaling Technology, Inc. (Danvers, MA, USA), and the rabbit anti-CHK1 polyclonal antibody (cat. # STJ92269, St John’s Laboratory).

Techniques: Homologous Recombination