Structured Review

Promega atp glo assay
Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent a , V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro . b . c , In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a . d . e , NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml −1 . f were cultured in doxycycline (30 ng ml −1 . g . h , The frequency of coexistent RAS or NF1 alterations in human BRAF mutant melanomas compared to that in NSCLC and colorectal cancers.. The calculation was based on the sample sets as shown in . The P values were calculated by using a paired t -test. N.S., not significant. i , The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j . k , Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the <t>ATP-Glo</t> assay. Graphs were generated using Prism 6 (mean ±s.d. are represented by the dots and error bars, n =8). l . m , The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n . o , Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n =8) Fig. 1d
Atp Glo Assay, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/atp glo assay/product/Promega
Average 80 stars, based on 1 article reviews
Price from $9.99 to $1999.99
atp glo assay - by Bioz Stars, 2020-01
80/100 stars

Images

1) Product Images from "Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS"

Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

Journal:

doi: 10.1038/nature23291

Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent a , V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro . b . c , In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a . d . e , NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml −1 . f were cultured in doxycycline (30 ng ml −1 . g . h , The frequency of coexistent RAS or NF1 alterations in human BRAF mutant melanomas compared to that in NSCLC and colorectal cancers.. The calculation was based on the sample sets as shown in . The P values were calculated by using a paired t -test. N.S., not significant. i , The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j . k , Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the ATP-Glo assay. Graphs were generated using Prism 6 (mean ±s.d. are represented by the dots and error bars, n =8). l . m , The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n . o , Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n =8) Fig. 1d
Figure Legend Snippet: Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent a , V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro . b . c , In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a . d . e , NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml −1 . f were cultured in doxycycline (30 ng ml −1 . g . h , The frequency of coexistent RAS or NF1 alterations in human BRAF mutant melanomas compared to that in NSCLC and colorectal cancers.. The calculation was based on the sample sets as shown in . The P values were calculated by using a paired t -test. N.S., not significant. i , The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j . k , Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the ATP-Glo assay. Graphs were generated using Prism 6 (mean ±s.d. are represented by the dots and error bars, n =8). l . m , The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n . o , Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n =8) Fig. 1d

Techniques Used: Activation Assay, Mutagenesis, Stable Transfection, Isolation, In Vitro, Activity Assay, Expressing, Cell Culture, Glo Assay, Generated, Inhibition

ERK signalling in hypoactive BRAF-mutant cells is sensitive to trametinib a . b , The cell lines as indicated in a were treated with different concentrations of trametinib for 3 days. Cell viability was determined by ATP-Glo assay. Dose-dependent inhibition curves were generated using Prism 6 (mean ± s.d., n =8).
Figure Legend Snippet: ERK signalling in hypoactive BRAF-mutant cells is sensitive to trametinib a . b , The cell lines as indicated in a were treated with different concentrations of trametinib for 3 days. Cell viability was determined by ATP-Glo assay. Dose-dependent inhibition curves were generated using Prism 6 (mean ± s.d., n =8).

Techniques Used: Mutagenesis, Glo Assay, Inhibition, Generated

In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by ATP-Glo assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b
Figure Legend Snippet: In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by ATP-Glo assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b

Techniques Used: Mutagenesis, Inhibition, Isolation, Derivative Assay, Glo Assay, Generated

Related Articles

Concentration Assay:

Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS
Article Snippet: Cells were seeded into 96-well plates at 1,000 cells per well. .. Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 h. For each condition, 8 replicates at each concentration were measured. .. IC50 values were calculated using GraphPad Prizsm 6.

Incubation:

Article Title: Dual targeting of mitochondrial function and mTOR pathway as a therapeutic strategy for diffuse intrinsic pontine glioma
Article Snippet: DIPG cells were plated at a cell density of 10000 cells/well in white opaque 96 well plates and incubated for 24 h. Cells were then treated for 24h with PENAO, temsirolimus, and combination at the indicated doses. .. ATP levels were assessed using the ATP-Glo assay according to the manufacturer’s instructions (Promega).

Growth Assay:

Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS
Article Snippet: Paragraph title: Cell growth assay ... Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 h. For each condition, 8 replicates at each concentration were measured.

ATP Assay:

Article Title: Dual targeting of mitochondrial function and mTOR pathway as a therapeutic strategy for diffuse intrinsic pontine glioma
Article Snippet: Paragraph title: ATP assay measurements ... ATP levels were assessed using the ATP-Glo assay according to the manufacturer’s instructions (Promega).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 80
    Promega atp glo assay
    Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent a , V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro . b . c , In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a . d . e , NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml −1 . f were cultured in doxycycline (30 ng ml −1 . g . h , The frequency of coexistent RAS or NF1 alterations in human BRAF mutant melanomas compared to that in NSCLC and colorectal cancers.. The calculation was based on the sample sets as shown in . The P values were calculated by using a paired t -test. N.S., not significant. i , The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j . k , Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the <t>ATP-Glo</t> assay. Graphs were generated using Prism 6 (mean ±s.d. are represented by the dots and error bars, n =8). l . m , The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n . o , Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n =8) Fig. 1d
    Atp Glo Assay, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp glo assay/product/Promega
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atp glo assay - by Bioz Stars, 2020-01
    80/100 stars
      Buy from Supplier

    79
    Promega atp based celltiter glo luminescent cell viability assay solution
    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using <t>Caspase-Glo</t> 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic <t>ATP</t> levels were measured using ATP-based <t>CellTiter-Glo</t> Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P
    Atp Based Celltiter Glo Luminescent Cell Viability Assay Solution, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp based celltiter glo luminescent cell viability assay solution/product/Promega
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atp based celltiter glo luminescent cell viability assay solution - by Bioz Stars, 2020-01
    79/100 stars
      Buy from Supplier

    99
    Promega adp glo kinase assay kit
    Pictorial representation of <t>ADP-Glo™</t> Kinase Assay with Leishmania donovani strain AG83 lysate . The assay is broadly composed of two steps. a) Preparation of parasite lysate to generate active kinases (enzymes’ source) heat activated proteins (substrate source), followed by protein quantification. b) Kinase or ATPase reaction. ADP thus formed by active kinases present in the cell lysate is subsequently utilized by ADP-Glo™ Kinase Assay kit to produce light, which is subsequently detected by luminometry.
    Adp Glo Kinase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/adp glo kinase assay kit/product/Promega
    Average 99 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    adp glo kinase assay kit - by Bioz Stars, 2020-01
    99/100 stars
      Buy from Supplier

    Image Search Results


    Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent a , V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro . b . c , In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a . d . e , NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml −1 . f were cultured in doxycycline (30 ng ml −1 . g . h , The frequency of coexistent RAS or NF1 alterations in human BRAF mutant melanomas compared to that in NSCLC and colorectal cancers.. The calculation was based on the sample sets as shown in . The P values were calculated by using a paired t -test. N.S., not significant. i , The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j . k , Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the ATP-Glo assay. Graphs were generated using Prism 6 (mean ±s.d. are represented by the dots and error bars, n =8). l . m , The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n . o , Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n =8) Fig. 1d

    Journal:

    Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

    doi: 10.1038/nature23291

    Figure Lengend Snippet: Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent a , V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro . b . c , In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a . d . e , NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml −1 . f were cultured in doxycycline (30 ng ml −1 . g . h , The frequency of coexistent RAS or NF1 alterations in human BRAF mutant melanomas compared to that in NSCLC and colorectal cancers.. The calculation was based on the sample sets as shown in . The P values were calculated by using a paired t -test. N.S., not significant. i , The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j . k , Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the ATP-Glo assay. Graphs were generated using Prism 6 (mean ±s.d. are represented by the dots and error bars, n =8). l . m , The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n . o , Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n =8) Fig. 1d

    Article Snippet: Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 h. For each condition, 8 replicates at each concentration were measured.

    Techniques: Activation Assay, Mutagenesis, Stable Transfection, Isolation, In Vitro, Activity Assay, Expressing, Cell Culture, Glo Assay, Generated, Inhibition

    ERK signalling in hypoactive BRAF-mutant cells is sensitive to trametinib a . b , The cell lines as indicated in a were treated with different concentrations of trametinib for 3 days. Cell viability was determined by ATP-Glo assay. Dose-dependent inhibition curves were generated using Prism 6 (mean ± s.d., n =8).

    Journal:

    Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

    doi: 10.1038/nature23291

    Figure Lengend Snippet: ERK signalling in hypoactive BRAF-mutant cells is sensitive to trametinib a . b , The cell lines as indicated in a were treated with different concentrations of trametinib for 3 days. Cell viability was determined by ATP-Glo assay. Dose-dependent inhibition curves were generated using Prism 6 (mean ± s.d., n =8).

    Article Snippet: Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 h. For each condition, 8 replicates at each concentration were measured.

    Techniques: Mutagenesis, Glo Assay, Inhibition, Generated

    In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by ATP-Glo assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b

    Journal:

    Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

    doi: 10.1038/nature23291

    Figure Lengend Snippet: In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by ATP-Glo assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b

    Article Snippet: Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 h. For each condition, 8 replicates at each concentration were measured.

    Techniques: Mutagenesis, Inhibition, Isolation, Derivative Assay, Glo Assay, Generated

    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: MTT Assay, Expressing, Western Blot, Activity Assay, Double Staining, FACS, Fluorescence, Staining

    Treatment with a ROS scavenger suppresses doxorubicin (DOX)-induced cytosolic adenosine triphosphate (cATP) production, DNA damage, mitochondrial hyper-activation, and necrosis. HK-2 cells were treated with a ROS scavenger (N-acetyl cysteine, NAC, 5 mM), DOX (1 μM), or both DOX and NAC for 72 h. ( A , B ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, staining, respectively, and signals were detected by FACS analysis. ( C ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), and H2AX were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. The gels were electrophoresed under the same experimental conditions. ( D , E ) Mitochondrial respiration and cATP production were measured using MitoTracker Red CMXRos staining and ATP-based CellTiter-Glo Luminescent Cell Viability Assays, and signals were detected using FACS analysis or analysis on a microplate reader, respectively. ( F ) Morphological changes were measured using phase-contrast microscopy (original magnification, 100×). ( G ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected using FACS analysis. The histogram shows the percentage of cells in each group, as the average of the results of three independent experiments (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Treatment with a ROS scavenger suppresses doxorubicin (DOX)-induced cytosolic adenosine triphosphate (cATP) production, DNA damage, mitochondrial hyper-activation, and necrosis. HK-2 cells were treated with a ROS scavenger (N-acetyl cysteine, NAC, 5 mM), DOX (1 μM), or both DOX and NAC for 72 h. ( A , B ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, staining, respectively, and signals were detected by FACS analysis. ( C ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), and H2AX were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. The gels were electrophoresed under the same experimental conditions. ( D , E ) Mitochondrial respiration and cATP production were measured using MitoTracker Red CMXRos staining and ATP-based CellTiter-Glo Luminescent Cell Viability Assays, and signals were detected using FACS analysis or analysis on a microplate reader, respectively. ( F ) Morphological changes were measured using phase-contrast microscopy (original magnification, 100×). ( G ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected using FACS analysis. The histogram shows the percentage of cells in each group, as the average of the results of three independent experiments (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: Activation Assay, Staining, FACS, Expressing, Western Blot, Microscopy, Double Staining

    Inhibition of PARP1 suppresses doxorubicin (DOX)-induced DNA damage, mitochondrial hyper-activation, and necrosis. ( A–J ) HK-2 cells were treated with a pharmacological PARP1 inhibitor (PJ-34, 10 μM), DOX (1 μM), or both PJ-34 and DOX for 72 h. (A) Expressions of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), H2AX (15 kDa), 53BP1 (350 kDa), p-ATM ser1981 (350 kDa), ATM (350 kDa), p-CHK2 Thr68 (62 kDa), and CHK2 (62 kDa) proteins were measured by western blot in whole cell extracts. ( B ) Levels of p-ATM ser1981 and γ-H2AX ser139 in the nucleus were measured using immunofluorescence staining and Cellomics ArrayScan HCS Reader. ( C ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected by FACS. ( D ) Mitochondrial respiration levels were measured using MitoTracker Red CMXRos and signals were detected using FACS. ( E ) Genomic DNA was analyzed for mtDNA copy numbers using ND1- , ND4- , and GAPDH- specific primers and real-time PCR. ND1 and ND4 copy numbers were normalized to GAPDH copy numbers. ( F ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. ( G , H ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, respectively, and signals were detected by FACS. (I and J) Cytosolic NO and secreted NO levels were measured using DAF-FM and an NO detection kit. ( K , N ) HK-2 cells were transfected with SC-siRNA (5 nM) or PARP1 -siRNA (5 nM) and treated with DOX for 72 h. ( K ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assay. ( L ) Cytosolic ROS levels were measured using DCF-DA and were detected using FACS. ( M ) Cell viability was measured using MTT assay, and signals were detected by a microplate reader. ( N ) Necrotic cell death was measured using PI staining, and signals were detected by FACS. (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Inhibition of PARP1 suppresses doxorubicin (DOX)-induced DNA damage, mitochondrial hyper-activation, and necrosis. ( A–J ) HK-2 cells were treated with a pharmacological PARP1 inhibitor (PJ-34, 10 μM), DOX (1 μM), or both PJ-34 and DOX for 72 h. (A) Expressions of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), H2AX (15 kDa), 53BP1 (350 kDa), p-ATM ser1981 (350 kDa), ATM (350 kDa), p-CHK2 Thr68 (62 kDa), and CHK2 (62 kDa) proteins were measured by western blot in whole cell extracts. ( B ) Levels of p-ATM ser1981 and γ-H2AX ser139 in the nucleus were measured using immunofluorescence staining and Cellomics ArrayScan HCS Reader. ( C ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected by FACS. ( D ) Mitochondrial respiration levels were measured using MitoTracker Red CMXRos and signals were detected using FACS. ( E ) Genomic DNA was analyzed for mtDNA copy numbers using ND1- , ND4- , and GAPDH- specific primers and real-time PCR. ND1 and ND4 copy numbers were normalized to GAPDH copy numbers. ( F ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. ( G , H ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, respectively, and signals were detected by FACS. (I and J) Cytosolic NO and secreted NO levels were measured using DAF-FM and an NO detection kit. ( K , N ) HK-2 cells were transfected with SC-siRNA (5 nM) or PARP1 -siRNA (5 nM) and treated with DOX for 72 h. ( K ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assay. ( L ) Cytosolic ROS levels were measured using DCF-DA and were detected using FACS. ( M ) Cell viability was measured using MTT assay, and signals were detected by a microplate reader. ( N ) Necrotic cell death was measured using PI staining, and signals were detected by FACS. (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: Inhibition, Activation Assay, Western Blot, Immunofluorescence, Staining, Double Staining, FACS, Real-time Polymerase Chain Reaction, Transfection, Cell Viability Assay, MTT Assay

    Adenosine triphosphate (ATP) treatment induces DNA damage and necrosis at the early stage and apoptosis at the late stage, but does not alter cytosolic reactive oxygen species (cROS) and mitochondrial reactive oxygen species (mROS) levels. HK-2 cells were treated with ATP (1 mM) for 24, 48, and 72 h. ( A ) Cell viability was measured using MTT assays. HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Cytosolic ATP was measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. Signals were detected using a microplate reader. ( B ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa and 55 kDa), γ-H2AX ser139 (15 kDa), and H2AX (15 kDa) proteins were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Caspase 3/7 activity was measured by Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. Representative images are shown. ( D , E ) cROS, mROS, and cytosolic NO levels were measured using DCF-DA, MitoSOX, and DAF-FM staining, respectively, and signals were detected by FACS analysis. ( F ) Cell death, including necrosis (PI-positive cells) and apoptosis (Annexin V-positive cells), was measured using Annexin V or PI staining. Signals were detected by FACS analysis. The histogram shows the percentage of PI- and Annexin V-positive cells, as the average of the results of three independent experiments (*P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Adenosine triphosphate (ATP) treatment induces DNA damage and necrosis at the early stage and apoptosis at the late stage, but does not alter cytosolic reactive oxygen species (cROS) and mitochondrial reactive oxygen species (mROS) levels. HK-2 cells were treated with ATP (1 mM) for 24, 48, and 72 h. ( A ) Cell viability was measured using MTT assays. HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Cytosolic ATP was measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. Signals were detected using a microplate reader. ( B ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa and 55 kDa), γ-H2AX ser139 (15 kDa), and H2AX (15 kDa) proteins were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Caspase 3/7 activity was measured by Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. Representative images are shown. ( D , E ) cROS, mROS, and cytosolic NO levels were measured using DCF-DA, MitoSOX, and DAF-FM staining, respectively, and signals were detected by FACS analysis. ( F ) Cell death, including necrosis (PI-positive cells) and apoptosis (Annexin V-positive cells), was measured using Annexin V or PI staining. Signals were detected by FACS analysis. The histogram shows the percentage of PI- and Annexin V-positive cells, as the average of the results of three independent experiments (*P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: MTT Assay, Expressing, Western Blot, Activity Assay, Staining, FACS

    Pictorial representation of ADP-Glo™ Kinase Assay with Leishmania donovani strain AG83 lysate . The assay is broadly composed of two steps. a) Preparation of parasite lysate to generate active kinases (enzymes’ source) heat activated proteins (substrate source), followed by protein quantification. b) Kinase or ATPase reaction. ADP thus formed by active kinases present in the cell lysate is subsequently utilized by ADP-Glo™ Kinase Assay kit to produce light, which is subsequently detected by luminometry.

    Journal: MethodsX

    Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay

    doi: 10.1016/j.mex.2018.12.003

    Figure Lengend Snippet: Pictorial representation of ADP-Glo™ Kinase Assay with Leishmania donovani strain AG83 lysate . The assay is broadly composed of two steps. a) Preparation of parasite lysate to generate active kinases (enzymes’ source) heat activated proteins (substrate source), followed by protein quantification. b) Kinase or ATPase reaction. ADP thus formed by active kinases present in the cell lysate is subsequently utilized by ADP-Glo™ Kinase Assay kit to produce light, which is subsequently detected by luminometry.

    Article Snippet: The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101).

    Techniques: Kinase Assay

    Evaluation of ADP-Glo™ kinase assay linearity and implementation of the assay for parasite lysate . Panel A . ATP to ADP conversion curves were prepared at the indicated “ATP + ADP” concentration in 20 μl of 1× reaction buffer. Kinase assay was performed as described in methods section. There is a linear relationship observed between the luminescent signal and amount of ADP present in the reaction mix. Panel B . ADP-Glo™ Kinase Assay was utilized to detect functional activity of kinases in the parasite lysate. Reactions were setup by taking varying amounts of the lysate in a total volume of 20 μl per reaction, as described in methods section. Kinase reactions were initiated by adding 1 μM ATP and allowed to take place at 30 °C for 1 h, followed by ADP-Glo™ kinase assay. The luminescence signal thus produced increases with the amount of lysate in a kinase reaction. Luminescence values represent the mean of two replicates. Abbreviation: RLU, Relative Lights Unit.

    Journal: MethodsX

    Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay

    doi: 10.1016/j.mex.2018.12.003

    Figure Lengend Snippet: Evaluation of ADP-Glo™ kinase assay linearity and implementation of the assay for parasite lysate . Panel A . ATP to ADP conversion curves were prepared at the indicated “ATP + ADP” concentration in 20 μl of 1× reaction buffer. Kinase assay was performed as described in methods section. There is a linear relationship observed between the luminescent signal and amount of ADP present in the reaction mix. Panel B . ADP-Glo™ Kinase Assay was utilized to detect functional activity of kinases in the parasite lysate. Reactions were setup by taking varying amounts of the lysate in a total volume of 20 μl per reaction, as described in methods section. Kinase reactions were initiated by adding 1 μM ATP and allowed to take place at 30 °C for 1 h, followed by ADP-Glo™ kinase assay. The luminescence signal thus produced increases with the amount of lysate in a kinase reaction. Luminescence values represent the mean of two replicates. Abbreviation: RLU, Relative Lights Unit.

    Article Snippet: The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101).

    Techniques: Kinase Assay, Concentration Assay, Functional Assay, Activity Assay, Produced

    Applications of ADP-Glo™ kinase assay with parasite lysate . Panel A . To Elucidate proportion of calcium-dependent protein kinases in the parasite kinome, kinase reaction was performed using 10 ng of parasite lysate in the presence and absence of 2.5 mM Ca 2+ , as described in methods section. ˜30% of kinome activity has been reported to impart largely because of calcium-dependent protein kinases. Panel B . To check if the assay can be applied for HTS, thio-oxo-imidazolidinone derivative, MCR03 was tested for any effect on the activity of kinases. For this, 10 ng of parasite lysate was allowed to pre-incubate in the reaction buffer with 100 μM of the test compound in the presence and absence of calcium. Once the reactions were complete, the ADP-Glo™ Kinase Assay was performed as described in methods section. MCR 03 significantly exhibited differential inhibition in the presence and absence of calcium. Percent activity represent the mean of two replicates.

    Journal: MethodsX

    Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay

    doi: 10.1016/j.mex.2018.12.003

    Figure Lengend Snippet: Applications of ADP-Glo™ kinase assay with parasite lysate . Panel A . To Elucidate proportion of calcium-dependent protein kinases in the parasite kinome, kinase reaction was performed using 10 ng of parasite lysate in the presence and absence of 2.5 mM Ca 2+ , as described in methods section. ˜30% of kinome activity has been reported to impart largely because of calcium-dependent protein kinases. Panel B . To check if the assay can be applied for HTS, thio-oxo-imidazolidinone derivative, MCR03 was tested for any effect on the activity of kinases. For this, 10 ng of parasite lysate was allowed to pre-incubate in the reaction buffer with 100 μM of the test compound in the presence and absence of calcium. Once the reactions were complete, the ADP-Glo™ Kinase Assay was performed as described in methods section. MCR 03 significantly exhibited differential inhibition in the presence and absence of calcium. Percent activity represent the mean of two replicates.

    Article Snippet: The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101).

    Techniques: Kinase Assay, Activity Assay, Inhibition

    The sensitivity of cancer cell lines to Nek2 inhibitors is correlated with their proteasome activity. (a) Cell lines sensitive to HCI-2184 treatment had, on average, higher proteasome activity compared to resistant cell lines. (b) Cell lines sensitive to HCI-2389 treatment had, on average, higher proteasome activity compared to resistant cell lines. These cancer cell lines were selected from the 150 cell lines in our lab, based on whether or not they were sensitive to both HCI-2184 or HCI-2389. “Sensitive” was defined as an IC 50 value of 1 μ M or lower. The 26S proteasomes were isolated by ultracentrifugation and proteasome activity measured by Proteasome-Glo Assay.

    Journal: BioMed Research International

    Article Title: Inhibition of Nek2 by Small Molecules Affects Proteasome Activity

    doi: 10.1155/2014/273180

    Figure Lengend Snippet: The sensitivity of cancer cell lines to Nek2 inhibitors is correlated with their proteasome activity. (a) Cell lines sensitive to HCI-2184 treatment had, on average, higher proteasome activity compared to resistant cell lines. (b) Cell lines sensitive to HCI-2389 treatment had, on average, higher proteasome activity compared to resistant cell lines. These cancer cell lines were selected from the 150 cell lines in our lab, based on whether or not they were sensitive to both HCI-2184 or HCI-2389. “Sensitive” was defined as an IC 50 value of 1 μ M or lower. The 26S proteasomes were isolated by ultracentrifugation and proteasome activity measured by Proteasome-Glo Assay.

    Article Snippet: Compounds were incubated with human Nek2 kinase (Invitrogen) and then kinase activity was examined by the Kinase-Glo Luminescence Kinase Assay (Promega).

    Techniques: Activity Assay, Isolation, Glo Assay

    Nek2 overexpression elevates the proteasome activity in multiple cancer cell lines. (a) Proteasome activity is significantly increased in Nek2 overexpressed HeLa cells compared to GFP-transfected control. Proteasome activity was also significantly elevated in H929 (b), KMS28PE (c), and ARP-1 (d) cell lines compared to empty vector transfected (control). For the ARP-1 cell line, Nek-2-OE, NEK-2-KD, and bortezomib-resistant clones were tested in addition to wild-type cells. The 26S proteasome was isolated by ultracentrifugation and the proteasome activity was determined by Proteasome-Glo Assay. (d) For ARP1 cells, the bortezomib-resistant cells (third column in (d)) showed higher proteasome activity. For Figures 2(a) – 2(d) , * P

    Journal: BioMed Research International

    Article Title: Inhibition of Nek2 by Small Molecules Affects Proteasome Activity

    doi: 10.1155/2014/273180

    Figure Lengend Snippet: Nek2 overexpression elevates the proteasome activity in multiple cancer cell lines. (a) Proteasome activity is significantly increased in Nek2 overexpressed HeLa cells compared to GFP-transfected control. Proteasome activity was also significantly elevated in H929 (b), KMS28PE (c), and ARP-1 (d) cell lines compared to empty vector transfected (control). For the ARP-1 cell line, Nek-2-OE, NEK-2-KD, and bortezomib-resistant clones were tested in addition to wild-type cells. The 26S proteasome was isolated by ultracentrifugation and the proteasome activity was determined by Proteasome-Glo Assay. (d) For ARP1 cells, the bortezomib-resistant cells (third column in (d)) showed higher proteasome activity. For Figures 2(a) – 2(d) , * P

    Article Snippet: Compounds were incubated with human Nek2 kinase (Invitrogen) and then kinase activity was examined by the Kinase-Glo Luminescence Kinase Assay (Promega).

    Techniques: Over Expression, Activity Assay, Transfection, Plasmid Preparation, Clone Assay, Isolation, Glo Assay

    Novel Nek2 Inhibitors significantly Inhibit Nek2's activity. (a), (b), and (c), three compounds, HCI-2184, HCI-2388, and HCI-2389 were designed by virtual screening. Synthesized compounds were validated by NMR and MS. The abilities of the three compounds to inhibit Nek2 kinase were tested by Kinase-Glo Assay. (d) HCI-2389 acts as an irreversible Nek2 inhibitor. A 0.5 hr incubation of HCI-2389 and Nek2 kinase increased the ability of HCI-2389 to inhibit Nek2. This effect was more pronounced when HCI-2389 was incubated with Nek2 kinase for 1 hr. (e) 10 nM HCI-2389 treatment for 72 hours greatly decreased the level of phosphorylated PP1-α in both Nek2 overexpressed HeLa cells and GFP controls. The effect was equal to or greater than treatment with 5 nM Nek-2 siRNA.

    Journal: BioMed Research International

    Article Title: Inhibition of Nek2 by Small Molecules Affects Proteasome Activity

    doi: 10.1155/2014/273180

    Figure Lengend Snippet: Novel Nek2 Inhibitors significantly Inhibit Nek2's activity. (a), (b), and (c), three compounds, HCI-2184, HCI-2388, and HCI-2389 were designed by virtual screening. Synthesized compounds were validated by NMR and MS. The abilities of the three compounds to inhibit Nek2 kinase were tested by Kinase-Glo Assay. (d) HCI-2389 acts as an irreversible Nek2 inhibitor. A 0.5 hr incubation of HCI-2389 and Nek2 kinase increased the ability of HCI-2389 to inhibit Nek2. This effect was more pronounced when HCI-2389 was incubated with Nek2 kinase for 1 hr. (e) 10 nM HCI-2389 treatment for 72 hours greatly decreased the level of phosphorylated PP1-α in both Nek2 overexpressed HeLa cells and GFP controls. The effect was equal to or greater than treatment with 5 nM Nek-2 siRNA.

    Article Snippet: Compounds were incubated with human Nek2 kinase (Invitrogen) and then kinase activity was examined by the Kinase-Glo Luminescence Kinase Assay (Promega).

    Techniques: Activity Assay, Synthesized, Nuclear Magnetic Resonance, Mass Spectrometry, Glo Assay, Incubation