Journal: Cell Cycle

Article Title: Identification of the proteome complement of humanTLK1 reveals it binds and phosphorylates NEK1 regulating its activity

doi: 10.1080/15384101.2017.1314421

Figure Lengend Snippet: (A) The activation of ATR (autophosphorylation at T1989) after 6h incubation with hydroxyurea (HU) is strongly reduced by concomitant addition of the TLK1 inhibitor, thioridazine (THD, 10 µM). (B) Activation of ATR (pATR-T1989) is impaired following 1h incubation with 0.2 mM H2O2 in cells expressing NEK1-T141A in contrast to wt-NEK1 expressing cells. Where indicated, siRNA to nek1 (si1) was added 24h before H2O2. (C) Activation of Chk1 (pChk1-S345) is reduced in cells overexpressing NEK1-T141A.

Article Snippet: Antibodies used in this study were: rabbit α-Actin (Ab1801, Abcam), rabbit α-Chk1 phospho-317 (AP3070a, Abgent), rabbit α-Chk1 phospho-345 (sc-17922, Santa Cruz Biotechnology), rabbit α-TLK1 (GTX102891, GeneTex), rabbit αNEK1 Antibody, A304–570A (Bethyl Lab), mouse monoclonal α Nek1 (E-10) (sc-398813 Santa Cruz Biotechnology), mouse monoclonal Anti-polyHistidine antibody (H1029, SIGMA), rabbit αATR antibody (A300–138A, Bethyl Lab), rabbit αpATR (phospho Thr1989) antibody (GTX128145, GeneTex), donkey α-goat IgG-HRP (sc-2020, Santa Cruz Biotechnology), α-rabbit IgG-HRP (7074S, Cell Signaling), α-mouse IgG-HRP (7076, Cell Signaling), mouse α- H2A.X phospho-139 (05–636, Millipore).

Techniques: Activation Assay, Incubation, Expressing

Journal:

Article Title: Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling

doi: 10.1038/sj.emboj.7600463

Figure Lengend Snippet: Depletion of hMCM7 and hRad17 inhibits genotoxic stress-induced hChk1 phosphorylation. (A) HeLa cells were transfected with hMCM7 or hRad17 siRNA, and then treated with 200 J/m2 of UV or 20 Gy IR at 48 h post-transfection. Cellular extracts were harvested at 1 h after UV or IR treatment, and cell extracts (50 μg protein) were immunoblotted with the indicated antibodies. β-Tubulin served as a sample-loading control. (B) hMCM7, hMCM2, or hRad17 siRNA-transfected HeLa cells were treated for 4 h with 200 J/m2 UV light or 1 μM Aph, and cellular extracts were separated by SDS–PAGE and immunoblotted with the indicated antibodies. (C) Cells were transfected with hMCM7 or hMCM2 siRNA, and were treated with 1 μM Aph. Cells were fixed after 4 h and stained with the indicated antibodies. Cell nuclei were stained with DAPI.

Article Snippet: Commercial antibodies were obtained from the following sources (in parentheses): α-hMCM4 (Abcam); α-hMCM7 (Sigma); α-hRad17 and α-hChk1 (Santa Cruz); α-phospho-hChk1 (pSer-317 and pSer-345) and α-Chk2 (Cell Signaling); α-ATR (Affinity Bioreagents); α-γH2AX (Cell Signaling); monoclonal α-FLAG M2 (Sigma); Alexa488-conjugated monoclonal α-BrdU (Molecular Probes); α-HA, clone 12CA5 (BabCo), α-phospho-Chk2 (pT-68) (R&D systems).

Techniques: Transfection, SDS Page, Staining

Journal:

Article Title: Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling

doi: 10.1038/sj.emboj.7600463

Figure Lengend Snippet: Disruption of S-phase checkpoint signaling by ectopic expression of the hMCM7-binding region of hRad17. (A) Binding of ectopically expressed GFP-Rad17-5 to endogenous hMCM7. HeLa cells were transfected with GFP or GFP-Rad17-5 fusion construct (encoding amino acids 421–670 of hRad17). At 24 h post-transfection, the cells were harvested, lysed, and detergent-soluble protein (1 mg) was immunoprecipitated with α-hMCM7 antibody. (B) hChk1 phosphorylation. HeLa cells were transfected with GFP or GFP-Rad17-5, and then treated with 200 J/m2 UV light or 1 μM Aph at 16 h after transfection. Cellular extracts (50 μg protein) were prepared after 2 h, and proteins were immunoblotted with the indicated antibodies. (C) Ectopically expressed GFP-Rad17-5 confers a UVDS phenotype. HeLa cells were transiently transfected with either GFP- or GFP-Rad17-5-encoding plasmids, and, after 24 h, were irradiated with 200 J/m2 UV light. DNA synthesis was determined at 1 h after radiation exposure as described in Materials and methods. (D) hChk2 phosphorylation. HeLa cells were transfected with GFP or GFP-Rad17-5, and then treated with 200 J/m2 UV light or 20 Gy of IR at 16 h after transfection. Cellular extracts (50 μg protein) were prepared after 2 h, and proteins were immunoblotted with the indicated antibodies.

Article Snippet: Commercial antibodies were obtained from the following sources (in parentheses): α-hMCM4 (Abcam); α-hMCM7 (Sigma); α-hRad17 and α-hChk1 (Santa Cruz); α-phospho-hChk1 (pSer-317 and pSer-345) and α-Chk2 (Cell Signaling); α-ATR (Affinity Bioreagents); α-γH2AX (Cell Signaling); monoclonal α-FLAG M2 (Sigma); Alexa488-conjugated monoclonal α-BrdU (Molecular Probes); α-HA, clone 12CA5 (BabCo), α-phospho-Chk2 (pT-68) (R&D systems).

Techniques: Expressing, Binding Assay, Transfection, Construct, Immunoprecipitation, Irradiation, DNA Synthesis

Journal: Cell Death and Differentiation

Article Title: CHK1 dosage in germinal center B cells controls humoral immunity

doi: 10.1038/s41418-019-0318-5

Figure Lengend Snippet: CHK1-mediated survival of in vitro-activated B cells depends on the nature of the mitogen. a Murine splenic wild-type B cells were loaded with eFluor450 cell proliferation dye and stimulated with the indicated mitogens inducing BCR-signaling or mimicking T cell help, respectively. After 72 h, cells were analyzed for proliferation by flow cytometry as indicated by the division-dependent loss of the proliferation dye over time. Filled gray peaks represent un-stimulated (non-dividing) cells that were cultured for 72 h. Data are representative for three mice per genotype. b Contour plots depict intracellular flow cytometry staining for DNA content by TO-PRO-3 and for pATR within the S–G2–M population of the cultures described in a. Data are representative for three mice per genotype. c qRT-PCR analysis of CHK1 mRNA expression in wild-type B cells directly after isolation (naïve ex vivo) or after 48 h of cultivation without (naïve in vitro) or with mitogenic stimulation as indicated. The data are presented as means ± SD and represent 3–6 mice per cell population. d Immunoblot analysis for CHK1 and pCHK1 in wild-type B cells directly after isolation (naïve ex vivo) or after 48 h of cultivation without (naïve in vitro) or with mitogenic stimulation as indicated. Western blot is representative of four independent experiments. e Splenic wild-type B cells were left untreated or stimulated with the indicated mitogens. After 48 h, the cells were treated with vehicle or graded doses of either of the two CHK1-inhibitors PF-477736 (25, 100, 400, 1600, and 6400 nM) or CHIR-124 (0.38, 1.5, 6.25, 25, 100, and 400 nM), and analyzed 24 h later for Annexin V−/TO-PRO-3− viable cells by flow cytometry. Survival is depicted normalized to the survival of the vehicle-treated culture, and hence termed “survival (% of control)”. Data are cumulative from three experiments with three mice per concentration, and presented as log(inhibitor) vs. response curves

Article Snippet: The following primary antibodies were diluted in 5% skim milk in PBS-T: α-HSP90 (Santa Cruz, Dallas, TX, USA, 13119, 1:200), α-CHK1 (Cell Signaling 2360, 1:1000), α-Caspase-3 (Cell Signaling 9665, 1:1000), α-BIM (ENZO ALX-804-527, 1:500), α-MCL1 (Rockland 600-401-394 S, 1:500), α-BCL2 (BioLegend 633502, 1:500), α-BCLX L (Cell Signaling 2764, 1:500), α-BCL2a1 (clone 6D6 [ 57 ], 1:500). α-pCHK1 Ser-345 (Cell Signaling 2345, 1:500) was diluted in 5% BSA in PBS-T.

Techniques: In Vitro, Flow Cytometry, Cell Culture, Staining, Quantitative RT-PCR, Expressing, Isolation, Ex Vivo, Western Blot, Concentration Assay

Journal: Cell Death and Differentiation

Article Title: CHK1 dosage in germinal center B cells controls humoral immunity

doi: 10.1038/s41418-019-0318-5

Figure Lengend Snippet: CHK1 expression levels tune the GC reaction. a qRT-PCR analysis of CHK1 mRNA expression in FACS-sorted wild-type developing B cells in the bone marrow (pro B cells, large pre B cells, small pre B cells, immature IgM+ B cells), immature splenic B cells (T (transitional) 1 B cells, T2 B cells, T3 B cells), mature splenic B cells (Fo (follicular) B cells, MZ (marginal zone) B cells), and splenic antigen-experienced B cells (GC B cells, plasmablasts/plasma cells (PC), DZ centroblasts, LZ centrocytes). GC B cells, PC, DZ, and LZ GC B cells were from wild-type mice that had been challenged with sheep RBCs 14 days prior FACS-sorting, all other cell subsets were sorted from unchallenged wild-type mice. Data are means ± SD for three mice per cell population. b Immunoblot analysis for CHK1, pCHK1, and caspase-3 expression in wild-type naïve (Fo) follicular and GC B cells FACS-sorted from spleens 14 days post immunization with sheep RBCs. Cells from six mice were pooled per lane. c Mice of the indicated genotypes were immunized at the age of 8–10 weeks with NP-CGG and analyzed 14 days later. Flow cytometry analysis of splenic single-cell suspensions identified the fractions of B cells (B220+CD19+), GC B cells (B220+CD19+CD138−CD95hiCD38lo/−) and NP+IgG1+ GC B cells within total splenocytes. Data are cumulative from two experiments with 5–6 mice per genotype, with each symbol representing one mouse, and shown as mean ± SEM. d Representative dot plots for the data shown in c. Numbers adjacent to regions indicate the % corresponding population within the respective parent population, and red arrows indicate the gating strategy. e Spleen sections from the indicated genotypes 14 days post immunization with NP-CGG were analyzed for Ki67 (proliferating cells) and for the ability of cells to bind PNA (characteristic of GC B cells). The brown precipitate indicates positive cells. Specimens were counterstained with hematoxylin. One representative mouse out of three per group is shown. Bars: 200 μm. f qRT-PCR analysis for CHK1, BCL6, and AID mRNA expression in FACS-sorted Fo (naïve) and GC splenic B cells of the indicated genotypes 10 days post immunization with NP-CGG. Data are shown as mean ± SD for three mice per group. g Flow cytometry analysis of Peyer’s patch single-cell suspensions in non-challenged 10-week-old mice identified the fractions of B cells (B220+CD19+), GC B cells (B220+CD19+CD95hiCD38lo/−), and class-switched IgG1+ or IgA+ B cells within total Peyer’s patch cells. Data are cumulative from two experiments with 3–6 mice per genotype, with each symbol representing one mouse, and shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001 (one-way ANOVA followed by Tukey post-hoc test)

Article Snippet: The following primary antibodies were diluted in 5% skim milk in PBS-T: α-HSP90 (Santa Cruz, Dallas, TX, USA, 13119, 1:200), α-CHK1 (Cell Signaling 2360, 1:1000), α-Caspase-3 (Cell Signaling 9665, 1:1000), α-BIM (ENZO ALX-804-527, 1:500), α-MCL1 (Rockland 600-401-394 S, 1:500), α-BCL2 (BioLegend 633502, 1:500), α-BCLX L (Cell Signaling 2764, 1:500), α-BCL2a1 (clone 6D6 [ 57 ], 1:500). α-pCHK1 Ser-345 (Cell Signaling 2345, 1:500) was diluted in 5% BSA in PBS-T.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Cell & Bioscience

Article Title: Dapagliflozin attenuates hypoxia/reoxygenation-caused cardiac dysfunction and oxidative damage through modulation of AMPK

doi: 10.1186/s13578-021-00547-y

Figure Lengend Snippet: Dapagliflozin (DAPA) prevents the upregulation of ROS via AMPK/ PKC/ NADPH oxidase signaling. Representative Western blot images ( a ) and relative densitometric bar graphs of Nox-2/Na/K ATPase ( b ) and Rac-1/Na/K ATPase ( c ) in H9c2 cells exposed to hypoxia for 1 h and reoxygenation for 4 h (H1R4) were shown. Protein expression levels of Nox-2 and β-actin in ventricular tissue from sham control, ischemia/reperfusion (I/R), and I/R plus DAPA treatment animals ( d , e ), eight animals in each group, were examined. Activity of NADPH oxidase in H9c2 cells ( f ) and primary cardiomyocytes ( g ) was measured. ROS generation was measured using a flow cytometry to examine the fluorescent intensity of H9c2 cells ( h ) and primary cardiomyocytes ( i ). For in vitro experiments, the data were presented as the mean ± SD of three biological replicates at three separate times. (* indicating p < 0.05 compared with the control group; # indicating p < 0.05 compared to H1R4 condition or I/R without DAPA treatment; & indicating p < 0.05 compared with the DAPA-treated cells in H1R4 condition)

Article Snippet: Anti-β-actin (sc-47,778; 1:50,000), anti-AMPK (sc-74,461; 1:1000), anti-Nox-2 (sc-74,514; 1:1000), anti-phospho‐PKC (sc-377,565; 1:1000), anti-PKC (sc-8393; 1:1000), anti-phospho-p53 (sc-51,690; 1:1000), anti-Bax (sc-20,067; 1:2000), anti-Bcl2 (sc-27,382; 1:1000), anti-cytochrome c (sc-13,156; 1:1000), anti-Na/K ATPase (sc-48,345; 1:10,000), and anti‐PGC-1α (sc-518,025; 1:1000) were obtained from Santa Cruz Biotechnology ( CA, USA).

Techniques: Western Blot, Expressing, Control, Activity Assay, Flow Cytometry, In Vitro