Journal: iScience

Article Title: Design, synthesis and mechanistic anticancer activity of new acetylated 5-aminosalicylate-thiazolinone hybrid derivatives

doi: 10.1016/j.isci.2023.108659

Figure Lengend Snippet: DNA damage response induced after HH32 and HH33 treatment (A–D) MCF7, HCT-116, HeLa and A549 were treated with 0.5 and 1 μM of HH32 or HH33 for 24h. Upper panels: Western blots detection for γ-H2AX following treatment with HH32 and HH33 . Lower panels: Fold change quantification of γ-H2AX over DMSO -treated control normalized to the β-actin as a loading control. (E–H) Upper panels: Western blots detection for P-ATM, ATM, P- Chk2 and Chk2 after treatment with HH32 and HH33 . Lower panels: P-ATM and P-Chk2 band quantification after normalization to the total protein and the β-actin as a loading control. (I–L) Upper panels: Western blots analysis for P-ATR, ATR, P- Chk1 and Chk1 post HH32 and HH33 treatment. Lower panels: Fold change for P-ATR and P-Chk1 after band quantification and normalization to the total protein and the β-actin as a loading control. Error bars represent SEM of three independent experiments. Data presented as mean ± SEM; n = 3, unpaired Student’s t test, ∗p < 0.05 vs. control (C).

Article Snippet: Anti- p -Chk2 Thr68 , Cell signaling Technology , Cat # 2661, RRID: AB_331479.

Techniques: Western Blot, Control

Journal: iScience

Article Title: Design, synthesis and mechanistic anticancer activity of new acetylated 5-aminosalicylate-thiazolinone hybrid derivatives

doi: 10.1016/j.isci.2023.108659

Figure Lengend Snippet:

Article Snippet: Anti- p -Chk2 Thr68 , Cell signaling Technology , Cat # 2661, RRID: AB_331479.

Techniques: Recombinant, RNA Extraction, Single Cell Gel Electrophoresis, cDNA Synthesis, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: The protein kinase MAP3K19 phosphorylates MAP2Ks and thereby activates ERK and JNK kinases and increases viability of KRAS-mutant lung cancer cells

doi: 10.1074/jbc.RA119.012365

Figure Lengend Snippet: MAP3K19 maintains MEK phosphorylation in the presence of RAF inhibitors. A, 48 h after transfection with EV, MAP3K19-WT, or MLK1-WT HEK293T cells were treated with DMSO vehicle control, 1 μm L779450, 1 μm AZD6244, or a combination of both RAF and MEK inhibitors for 1 h, and Western blotting was performed on cell lysates. B, 48 h after transfection with EV, WT, or KD-MAP3K19, HEK293T cells were treated with DMSO vehicle control or indicated inhibitors: 1 μm vemurafenib (BRAFi), 1 μm selumetinib (S, MEKi), or 500 nm cobimetinib (C, MEKi) for 1 h, and Western blotting was performed on cell lysates. Band density was quantified by ImageJ software. The data are shown as mean phospho:total protein density ± S.D. Dunnett's multiple comparisons test was used for statistical analysis, with EV-DMSO group as control. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: In vitro kinase assay Recombinant human GST-tagged MAP3K19 (YSK4) kinase domain (Thermo Fisher Scientific) or human MLK1 kinase domain (Carna Biosciences) was incubated at room temperature with kinase-inactive MEK1, ERK2, MKK7, JNK1, or JNK2 (Carna Biosciences) in the absence or presence of MAPK pathway inhibitors for 5 min.

Techniques: Transfection, Western Blot, Software

Journal: The Journal of Biological Chemistry

Article Title: The protein kinase MAP3K19 phosphorylates MAP2Ks and thereby activates ERK and JNK kinases and increases viability of KRAS-mutant lung cancer cells

doi: 10.1074/jbc.RA119.012365

Figure Lengend Snippet: MAP3K19 directly phosphorylates MAP2Ks. A, MAP3K19 was immunoprecipitated (IP) from HEK293T cells and subjected to a kinase assay with kinase-inactive MEK1. B and C, in vitro kinase assay using recombinant MAP3K19 protein and kinase-inactive MEK1 or ERK2, respectively. D, kinase-inactive MEK1 and purified GST-MAP3K19 or GST-MLK1 kinase domain were subjected to in vitro kinase assay in the presence or absence of inhibitors: 5 μm L779450, 1 μm PLX4032, 5 μm U0126, or 2 μm AZD6244. E and F, in vitro kinase assay using recombinant MAP3K19 protein and kinase-inactive MKK7 or JNK1/2, respectively. The data are shown as mean phospho:total protein density ± S.D. Dunnett's multiple comparisons test was used for statistical analysis, with samples in the first lane as control. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ♢, kinase-inactive.

Article Snippet: In vitro kinase assay Recombinant human GST-tagged MAP3K19 (YSK4) kinase domain (Thermo Fisher Scientific) or human MLK1 kinase domain (Carna Biosciences) was incubated at room temperature with kinase-inactive MEK1, ERK2, MKK7, JNK1, or JNK2 (Carna Biosciences) in the absence or presence of MAPK pathway inhibitors for 5 min.

Techniques: Immunoprecipitation, Kinase Assay, In Vitro, Recombinant, Purification

Journal: Journal of Biological Chemistry

Article Title: TTK/hMps1 Participates in the Regulation of DNA Damage Checkpoint Response by Phosphorylating CHK2 on Threonine 68

doi: 10.1074/jbc.m410152200

Figure Lengend Snippet: FIG. 1. TTK interacts with CHK2 in vitro and in vivo. A, sche- matic diagram of full-length TTK and two truncated constructs (d2, and d2C), with the first and last amino acids shown in parentheses. For in vitro binding assays, these cDNAs were cloned into pGEX vectors and expressed as GST fusion proteins. B, GST pull-down assays were per- formed by incubating 35S-labeled in vitro translated CHK2 with GST- fused d2C, d2, or d2KD (D647A). The proteins pulled down by gluta- thione-Sepharose beads were analyzed by SDS-PAGE followed by autoradiography (right panel). The silver-stained input GST-fused pro- teins are shown in the left panel. C, overexpressed CHK2 and TTK interact in 293T cells. 293T cells were transfected with HA-tagged TTK-WT or TTK-KD alone or together with myc-tagged CHK2-WT or CHK2-KD, then lysates were immunoprecipitated (IP) with anti-HA antibody and analyzed by Western blotting using anti-myc antibody to detect CHK2. D, coimmunoprecipitation of endogenous CHK2 and TTK. HeLa cells were untreated or treated with 8 Gy x-rays and then col- lected at the indicated times. CHK2 was immunoprecipitated from HeLa WCEs using anti-CHK2, and TTK in the immune complex was then detected by Western blotting using anti-TTK.

Article Snippet: Antibodies against CHK2 (sc-9064), p-CHK2 (Thr-68) (sc-16297-R), Cdc2 (sc-54), and cyclin B1 (sc-245) were purchased from Santa Cruz Biotechnology.

Techniques: In Vitro, In Vivo, Construct, Binding Assay, Clone Assay, Labeling, SDS Page, Autoradiography, Staining, Transfection, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: TTK/hMps1 Participates in the Regulation of DNA Damage Checkpoint Response by Phosphorylating CHK2 on Threonine 68

doi: 10.1074/jbc.m410152200

Figure Lengend Snippet: FIG. 2. TTK directly phosphorylates CHK2 on Thr-68. A, TTK phosphorylates both the N-terminal and the C-terminal domains of CHK2. In vitro kinase assays were performed by incubating purified GST-d2C, GST-d2, or GST-d2KD (D647A) with purified His-tagged full-length CHK2 or the truncated fragments, CHK2N (amino acids 1–243) or CHK2C (244–543), in the presence of [-32P]ATP, then the products were separated on SDS gels and phosphorylation visualized by autoradiography. B, TTK specifically phosphorylates CHK2 on Thr-68 in vitro. In vitro kinase assays were performed by incubating purified recombinant full-length GST-WT or KD TTK with purified His-CHK2 in the presence of unlabeled ATP. Phosphorylation was detected by Western blotting using anti-phospho-Thr-68-specific CHK2 antibody. C, in vitro kinase assays were conducted as described in B, and phosphorylation on CHK2 Ser-19 or Thr-68 was detected with the corresponding antibodies. Lysates prepared from x-ray-irradiated 293T cells were used as positive controls. D and E, immunoprecipitated (IP) endogenous TTK phosphorylates CHK2 on Thr-68. Immunoprecipitation/kinase assays were performed using TTK immunoprecipitated from LNCaP (D) or MCF7 (E) cells that were either untreated () or treated () with 8 Gy x-rays and harvested 30 min after. Normal rabbit IgG (NR, lane 1) was used as an immunoprecipitation control. The amounts of Thr-68 phosphorylation on the added substrate (His-CHK2) in lanes 2 and 3 were quantified by densitometry and are shown in F. Although the overall phosphorylation as revealed by 32P incorporation was unchanged before and after IR, the specific phosphorylation on Thr-68 was enhanced by IR. G, expression of TTK-KD in cells diminishes the induction of Thr-68 phosphorylation on endogenous CHK2 by DNA damage. 293T cells were transiently transfected with HA-tagged TTK-KD or mock-transfected for about 45 h before irradiation with x-rays (left panels) or UV light (right panels). Induction of CHK2 Thr-68 phosphorylation was compared at the indicated time points by Western blotting.

Article Snippet: Antibodies against CHK2 (sc-9064), p-CHK2 (Thr-68) (sc-16297-R), Cdc2 (sc-54), and cyclin B1 (sc-245) were purchased from Santa Cruz Biotechnology.

Techniques: In Vitro, Purification, Phospho-proteomics, Autoradiography, Recombinant, Western Blot, Irradiation, Immunoprecipitation, Control, Expressing, Transfection

Journal: Journal of Biological Chemistry

Article Title: TTK/hMps1 Participates in the Regulation of DNA Damage Checkpoint Response by Phosphorylating CHK2 on Threonine 68

doi: 10.1074/jbc.m410152200

Figure Lengend Snippet: FIG. 3. Impaired checkpoint response in stable cell line expressing TTK-KD. HeLa 10-3 (control) and HeLa 6 (TTK-KD) cells were irradiated with either x-rays (A and B) or UV light (C), lysed at the indicated times after irradiation, and analyzed by Western blotting for CHK2, CHK2 Thr-68 phosphorylation (pT68), Cdc2, Cdc2 Tyr-15 phosphorylation (pY15), or cyclin B1 (B). Actin was used as loading control. Doxycycline (Dox) was used to control the expression of HA-TTK KD in B. D, DNA damage-induced G2/M arrest is compromised in cells expressing TTK-KD. HeLa 10-3 or HeLa 6 cells were irradiated with 8 Gy x-rays or 30 J/m2 UV light and collected at the indicated time for FACS analysis. Shown are cell cycle profiles of cells collected at 12 h after IR or 24 h after UV irradiation. E, bar graphs depicting results of time course experiments showing the G2/G1 ratio before and after IR treatment and the G2/M increase after UV treatment. Data presented are the averages of three experiments. We used two-way analysis of variance to test whether the differences between HeLa 10-3 and HeLa 6 were significant. The results show a p value of 0.0015 for the IR experiments and a p 0.0001 for the UV experiments, indicating that expression of TTK-KD in HeLa 6 cells significantly compromised the execution of checkpoint control.

Article Snippet: Antibodies against CHK2 (sc-9064), p-CHK2 (Thr-68) (sc-16297-R), Cdc2 (sc-54), and cyclin B1 (sc-245) were purchased from Santa Cruz Biotechnology.

Techniques: Stable Transfection, Expressing, Control, Irradiation, Western Blot, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: TTK/hMps1 Participates in the Regulation of DNA Damage Checkpoint Response by Phosphorylating CHK2 on Threonine 68

doi: 10.1074/jbc.m410152200

Figure Lengend Snippet: FIG. 4. Disruption of TTK or CHK2 expression by siRNA in HeLa 10-3 cells results in defective G2/M arrest after IR or UV irradiation. A, down- regulation of TTK and CHK2 by TK1 and 2–3 siRNA, respectively, but not by the control siRNA. The sequence of TK1 was scrambled and used as control. B, induc- tion of CHK2 Thr-68 phosphorylation was reduced in TK1 siRNA-transfected cells. Cells were transfected with control or TK1 siRNA, irradiated with 8 Gy x-rays or 40 J/m2 UV light, and harvested for Western analysis at the indicated times. C, FACS analyses of the control, TK1, or 2-3 siRNA-transfected HeLa 10-3 cells af- ter 8 Gy x-ray or 30 J/m2 UV irradiation.

Article Snippet: Antibodies against CHK2 (sc-9064), p-CHK2 (Thr-68) (sc-16297-R), Cdc2 (sc-54), and cyclin B1 (sc-245) were purchased from Santa Cruz Biotechnology.

Techniques: Disruption, Expressing, Irradiation, Control, Sequencing, Phospho-proteomics, Transfection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: TTK/hMps1 Participates in the Regulation of DNA Damage Checkpoint Response by Phosphorylating CHK2 on Threonine 68

doi: 10.1074/jbc.m410152200

Figure Lengend Snippet: FIG. 5. Down-regulation of TTK but not MAD2 by siRNA diminishes DNA damage-induced CHK2 Thr-68 phosphorylation and checkpoint response in U2OS cells. A and B, impaired CHK2 Thr-68 phosphorylation in TTK-ablated cells. Cells were transfected with either control or TTK siRNA duplex (TK1) and incubated for 40 h before irradiation with 4 Gy x-rays (A) or 30 J/m2 UV light (B). Lysates were collected at the indicated times and analyzed by Western blotting for levels of TTK, CHK2, and CHK2 Thr-68 phosphorylation (pT68). Nucleolin was used as a loading control. C, CHK2 Thr-68 phosphorylation is not affected in MAD2-knock-down cells. U2OS cells were transfected with MAD2-targeting siRNA and irradiated with x-rays or UV light as described above. Cell lysates were then analyzed by Western blotting. D, cell cycle profiles of U2OS cells with down-regulated TTK or MAD2 after IR. TK1 or MAD2 siRNA-transfected cells were irradiated with 4 Gy x-rays, collected at 12 or 16 h after irradiation, and processed for flow cytometry. E, impaired cell cycle arrest in cells with down-regulated TTK but not in cells with down-regulated MAD2. U2OS cells were treated as described in D. Data represent the averages of two independent experiments (one shown in D), and the error bars correspond to the S.E. values. Based on the analysis of variance, the G2/G1 ratios of the TK1-transfected cells after IR are statistically different from those of the MAD2 siRNA-transfected cells (p 0.03). On the other hand, the G2/G1 ratios are statistically indistinguishable between the control and the MAD2 siRNA-transfected cells (p 0.46).

Article Snippet: Antibodies against CHK2 (sc-9064), p-CHK2 (Thr-68) (sc-16297-R), Cdc2 (sc-54), and cyclin B1 (sc-245) were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Transfection, Control, Incubation, Irradiation, Western Blot, Knockdown, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: TTK/hMps1 Participates in the Regulation of DNA Damage Checkpoint Response by Phosphorylating CHK2 on Threonine 68

doi: 10.1074/jbc.m410152200

Figure Lengend Snippet: FIG. 6. Model depicting the possible role of TTK in DNA damage-induced checkpoint responses. After DNA dam- age, TTK phosphorylates CHK2 on Thr- 68, which leads to activation of CHK2 and phosphorylation of the downstream tar- gets, Cdc25 and p53. The phosphatase ac- tivity of Cdc25C is inactivated by CHK2 phosphorylation, leading to accumulation of Cdc2 Tyr-15 phosphorylation and Cdk inactivation. CHK2 may also regulate the activity of p53 and consequently the ex- pression of its downstream target genes, such as p21 and AIP1, leading to either growth arrest or apoptosis.

Article Snippet: Antibodies against CHK2 (sc-9064), p-CHK2 (Thr-68) (sc-16297-R), Cdc2 (sc-54), and cyclin B1 (sc-245) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Activity Assay

Journal: Cell

Article Title: FBXO44 promotes DNA replication-coupled repetitive element silencing in cancer cells

doi: 10.1016/j.cell.2020.11.042

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-human p-CHK2 T68 , Cell Signaling , Cat# 2197; RRID:AB_2080501.

Techniques: Control, Marker, Microarray, Recombinant, Plasmid Preparation, Imaging, Staining, Extraction, Immunoprecipitation, Cell Isolation, Knockdown, Software

Journal: Oncology Letters

Article Title: MLN4924 neddylation inhibitor promotes cell death in paclitaxel-resistant human lung adenocarcinoma cells

doi: 10.3892/ol.2017.7314

Figure Lengend Snippet: MLN4924 induces apoptosis and DNA damage in PTX-resistant non-small cell lung cancer cells. (A) A549/PTX and H460/PTX cells were treated with DMSO or 1, 5 and 10 µM MLN4924 for 24, 48 and 72 h. The cells were double-stained with AV and PI, apoptosis was analyzed using flow cytometry. The percentage of apoptotic cells were presented as the mean ± standard deviation. The A549/PTX and H460/PTX cells were treated with vehicle or 10 µM MLN4924 for 12, 24 and 48 h. The cell lysates were collected and detected using western blot analysis. (B) Western blot analysis for caspase-3, PARP and β-actin. (C) Western blot analysis for p-H2AX, H2AX, p-CHK2, CHK2, CDT1 and β-actin. (D) Western blot analysis for cullin1, p21, p27, REDD1, Wee1 and β-actin. All experiments were performed 3 times. **P<0.01, ***P<0.001. PI, propidium iodide; AV, annexin V; PTX, paclitaxel; PARP, poly(ADP-ribose) polymerase; p-, phosphorylated; H2AX, histone 2AX; CHK2, checkpoint kinase 2; CDT1, chromatin licensing and DNA replication factor 1; REDD1, regulated in development and DNA damage responses 1.

Article Snippet: The anti-checkpoint kinase 2 (CHK2; cat. no. ab109413), anti-Histone H2AX (H2AX) (cat. no. ab124781) and anti-p21 (cat. no. ab109199) antibodies were obtained from Abcam (Cambridge, UK).

Techniques: Staining, Flow Cytometry, Standard Deviation, Western Blot

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Predicted MST4 inhibitors as determined by computational modelling

Article Snippet: TR-FRET kinase assay LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques:

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hypoxia model confirms the effects of selected inhibitor candidates on cell survival under severe hypoxia. A, immunoblot analysis of MST4 protein levels in normal pituitary and pituitary adenomas [gonadotrope, prolactin (PRL), ACTH, and growth hormone]. GAPDH was used as a loading control. B, immunoblot analysis shows overexpression of MST4 in pcDNA3-MST4–stable transfectants. C, percentage of nonviable cells in vector control and MST4 cells in the presence of the mammalian target of rapamycin (mTOR) inhibitor PKI-587 (0, 0.1, 1, 10, 100, and 1,000 nmol/L). D, percentage of nonviable cells in vector control and MST4 cells in the presence of the Polo-like kinase 1 (PLK-1) inhibitor volasertib (0, 0.1, 1, 10, 100, and 1,000 nmol/L). E, percentage of nonviable cells in control and MST4 transfectants in the presence of hesperadin (0, 0.1, 1, 10, 100, and 1000 nmol/L). *, P < 0.01; #, P < 0.0001, MST4 transfectants compared with pcDNA3 vector control cells.

Article Snippet: TR-FRET kinase assay LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Western Blot, Over Expression, Plasmid Preparation

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hesperadin is identified as a potent inhibitor of MST4. A, the chemical structure of hesperadin. B, small molecule docking depicting the predicted interaction of hesperadin (cyan) with the ATP-binding domain of MST4. C, in vitro TR-FRET recombinant kinase assay demonstrates direct inhibition of the MST4 kinase by hesperadin at low nanomolar concentrations. PKI-587 and volasertib were also screened in the TR-FRET recombinant kinase assay and show no direct inhibition of MST4.

Article Snippet: TR-FRET kinase assay LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Binding Assay, In Vitro, Recombinant, Kinase Assay, Inhibition

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hesperadin blocks the effects of MST4 on cell survival under acute hypoxia (1% O2). A, representative immunocytochemistry of apoptotic cells as assessed by TUNEL in the presence or absence of hesperadin (40 nmol/L) under hypoxia for 17 hours. B, rates of apoptosis were expressed as a percentage of TUNEL-positive cells to total cells in the absence or presence of various concentrations of hesperadin (5, 10, 20, and 40 nmol/L). *, P = 0.01 (at 10 nmol/L); #, P = 0.04 (at 20 nmol/L); **, P < 0.001, MST4 transfectants compared with pcDNA3 control cells.

Article Snippet: TR-FRET kinase assay LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Immunocytochemistry, TUNEL Assay

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hesperadin blocks the effects of MST4 on proliferation and colony formation under chronic hypoxic stress (5% O2). A, representative immunocytochemistry of the rates of BrdUrd incorporation in pcDNA3 control and MST4 transfectants in the absence and presence of hesperadin (40 nmol/L) under chronic hypoxia (5% O2) for 7 days. B, hesperadin abolishes MST4 increased cell proliferation. In the absence or presence of various doses of hesperadin (0, 5, 10, 20, and 40 nmol/L), cell proliferation was measured by BrdUrd after exposure to chronic hypoxia (5% O2) for 7 days. C, photomicrograph of colony formation in vector and MST4 transfectants incubated with DMSO or hesperadin (20 nmol/L). D, hesperadin decreases the ability of MST4 to promote increased colony formation. Numbers of pcDNA3 and MST4 transfectant colonies were counted after exposure to chronic hypoxia (5%O2) for 7 days. *, P = 0.002; **, P < 0.001, MST4 transfectants compared with pcDNA3 vector cells; #, P < 0.01, MST4 transfectants with hesperadin treatment (20 nmol/L) compared with the cells treated with DMSO.

Article Snippet: TR-FRET kinase assay LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Immunocytochemistry, Plasmid Preparation, Incubation, Transfection

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: A, illustration of MST4 signaling pathways in response to hypoxic stress. B, hesperadin blocks MST4 downstream signaling effectors. Phosphorylation of AKT, p38 MAPK, and ERK were determined by immunoblot in the presence or absence of hesperadin (0, 20, and 40 nmol/L). C, MST4-induced HIF-1 activity is blocked by hesperadin. Control or MST4 cells were transfected with HRE-luciferase reporter constructs. After 24 hours of transfection, cells were subjected to normoxia or hypoxia (1% O2) for 17 hours. HRE-luciferase values were detected and normalized to Renilla control luciferase values. *, P < 0.001, MST4 transfectants compared with pcDNA3 vector cells; #, P = 0.002, MST4 transfectants with hesperadin treatment compared with DMSO-treated controls.

Article Snippet: TR-FRET kinase assay LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Western Blot, Activity Assay, Transfection, Luciferase, Construct, Plasmid Preparation