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Promega atp glo assay
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 <t>ATP-Glo</t> assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b
Atp Glo Assay, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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: Nature

doi: 10.1038/nature23291

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

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

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 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 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

Related Articles

Concentration Assay:

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

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.

Article Title: RAF inhibitor PLX8394 selectively disrupts BRAF-dimers and RAS-independent BRAF mutant-driven signaling
Article Snippet: Paragraph title: Cell growth assay ... Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 hrs.

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).

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  • 93
    Promega celltiter glo luminescent atp assay kit
    Effects of APE1 overexpression on mitochondrial function in H/R-treated H9c2 cells. H9c2 cells were transfected with LV-APE1 or LV-Scr and subjected to H/R. (A) The mitochondrial membrane potential was detected using JC-1 staining. (B) Complex I, (C) complex II, (D) complex III and (E) complex IV activities were detected using microplate assay kits. (F) <t>ATP</t> production was assessed using the <t>CellTiter-Glo</t> luminescent ATP assay kit. (G) Bax/Bcl-2 ratio was measured using western blotting and (H) quantified. *P
    Celltiter Glo Luminescent Atp Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/celltiter glo luminescent atp assay kit/product/Promega
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    celltiter glo luminescent atp assay kit - by Bioz Stars, 2020-04
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    93
    Promega atp glo assay
    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 <t>ATP-Glo</t> assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b
    Atp Glo Assay, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp glo assay/product/Promega
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    atp glo assay - by Bioz Stars, 2020-04
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    99
    Promega celltiter glo atp assay
    Sendai virus Y1/Y2 proteins are required for necroptosis. ( a ) Infection of L929 cells with ultraviolet-inactivated (UV) SeV rescues cell death as monitored by <t>CellTiter-Glo</t> (experiment repeated three times). Right panel: western blot of L929 cells infected with live or UV-inactivated SeV (10 HA U/ml) after 18 h (performed once) (41). ( b ) Schematic representation of SeV proteins. Six structural proteins (NP, P, M, F, HN, L) and accessory proteins (V, W, X, C, C', Y1, Y2). ( c ) L929 cells stably expressing a control (Ctl) shRNA or shRNA against P and accessory proteins (PVC) were treated with zVAD and infected with SeV (10 HA U/ml) and cell survival monitored. Right panel: knockdown was monitored by western blot using antibodies against the C proteins of SeV. ( d ) L929 cells were infected with Cantell strain of SeV, Z strain of SeV (SeV-Z), or mutants (d2Y-Z, V[-]-Z). 16–18 h post-infection, mRNA was isolated and used for quantitative RT-PCR using oligonucleotides specific for the SeV NP or γ -actin gene. Expression was plotted on a log scale relative to mock-infected samples after normalization to γ -actin. Standard error of the mean was calculated from three independent experiments. ( e ) Survival of cells infected with Cantell strain of SeV (SeV) or Z strain (SeV-Z, d2Y-Z, V[-]-Z) as measured by CellTiter-Glo assay. ( f ) Lysates from SeV-infected cells at various time points (M: mock-infected) were probed in a western blot analysis using SeV C protein or β-actin specific antibodies (experiment repeated twice). For 3A, 3C, 3D, 3E, data are averages of triplicates from a single experiment, which is representative of at least three independent experiments. *** P
    Celltiter Glo Atp Assay, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega atp based cell viability assay celltiter glo
    Effect of HSA levels on the in vitro sensitivity of JAK2wt K562 and JAK2V617F mutated HEL and SET-2 cells to ruxolitinib. JAK2wt K562 (circles) and JAK2V617F + HEL (squares) and SET-2 (triangles) myeloid cell lines were cultured in 10% FBS medium or serum-free medium containing HSA (0–80 µM) and treated with the indicated doses of ruxolitinib for 72 hours. Cell viability was determined by the <t>ATP-based</t> cell-viability assay <t>CellTiter-Glo.</t> Data are the percent of untreated control cells. All data are presented as means of three independent experiments ± SD.
    Atp Based Cell Viability Assay Celltiter Glo, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp based cell viability assay celltiter glo/product/Promega
    Average 93 stars, based on 1 article reviews
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    atp based cell viability assay celltiter glo - by Bioz Stars, 2020-04
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    Effects of APE1 overexpression on mitochondrial function in H/R-treated H9c2 cells. H9c2 cells were transfected with LV-APE1 or LV-Scr and subjected to H/R. (A) The mitochondrial membrane potential was detected using JC-1 staining. (B) Complex I, (C) complex II, (D) complex III and (E) complex IV activities were detected using microplate assay kits. (F) ATP production was assessed using the CellTiter-Glo luminescent ATP assay kit. (G) Bax/Bcl-2 ratio was measured using western blotting and (H) quantified. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Apurinic/apyrimidinic endonuclease/redox factor 1 (APE1) alleviates myocardial hypoxia-reoxygenation injury by inhibiting oxidative stress and ameliorating mitochondrial dysfunction

    doi: 10.3892/etm.2019.7212

    Figure Lengend Snippet: Effects of APE1 overexpression on mitochondrial function in H/R-treated H9c2 cells. H9c2 cells were transfected with LV-APE1 or LV-Scr and subjected to H/R. (A) The mitochondrial membrane potential was detected using JC-1 staining. (B) Complex I, (C) complex II, (D) complex III and (E) complex IV activities were detected using microplate assay kits. (F) ATP production was assessed using the CellTiter-Glo luminescent ATP assay kit. (G) Bax/Bcl-2 ratio was measured using western blotting and (H) quantified. *P

    Article Snippet: ATP generation was detected using a CellTiter-Glo luminescent ATP assay kit (Promega Corp., Madison, WI, USA) according to the manufacturer's protocol.

    Techniques: Over Expression, Transfection, Staining, ATP Assay, Western Blot

    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: Nature

    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

    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: Nature

    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

    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 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: Nature

    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 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

    Sendai virus Y1/Y2 proteins are required for necroptosis. ( a ) Infection of L929 cells with ultraviolet-inactivated (UV) SeV rescues cell death as monitored by CellTiter-Glo (experiment repeated three times). Right panel: western blot of L929 cells infected with live or UV-inactivated SeV (10 HA U/ml) after 18 h (performed once) (41). ( b ) Schematic representation of SeV proteins. Six structural proteins (NP, P, M, F, HN, L) and accessory proteins (V, W, X, C, C', Y1, Y2). ( c ) L929 cells stably expressing a control (Ctl) shRNA or shRNA against P and accessory proteins (PVC) were treated with zVAD and infected with SeV (10 HA U/ml) and cell survival monitored. Right panel: knockdown was monitored by western blot using antibodies against the C proteins of SeV. ( d ) L929 cells were infected with Cantell strain of SeV, Z strain of SeV (SeV-Z), or mutants (d2Y-Z, V[-]-Z). 16–18 h post-infection, mRNA was isolated and used for quantitative RT-PCR using oligonucleotides specific for the SeV NP or γ -actin gene. Expression was plotted on a log scale relative to mock-infected samples after normalization to γ -actin. Standard error of the mean was calculated from three independent experiments. ( e ) Survival of cells infected with Cantell strain of SeV (SeV) or Z strain (SeV-Z, d2Y-Z, V[-]-Z) as measured by CellTiter-Glo assay. ( f ) Lysates from SeV-infected cells at various time points (M: mock-infected) were probed in a western blot analysis using SeV C protein or β-actin specific antibodies (experiment repeated twice). For 3A, 3C, 3D, 3E, data are averages of triplicates from a single experiment, which is representative of at least three independent experiments. *** P

    Journal: Cell Death and Differentiation

    Article Title: Induction of necroptotic cell death by viral activation of the RIG-I or STING pathway

    doi: 10.1038/cdd.2016.153

    Figure Lengend Snippet: Sendai virus Y1/Y2 proteins are required for necroptosis. ( a ) Infection of L929 cells with ultraviolet-inactivated (UV) SeV rescues cell death as monitored by CellTiter-Glo (experiment repeated three times). Right panel: western blot of L929 cells infected with live or UV-inactivated SeV (10 HA U/ml) after 18 h (performed once) (41). ( b ) Schematic representation of SeV proteins. Six structural proteins (NP, P, M, F, HN, L) and accessory proteins (V, W, X, C, C', Y1, Y2). ( c ) L929 cells stably expressing a control (Ctl) shRNA or shRNA against P and accessory proteins (PVC) were treated with zVAD and infected with SeV (10 HA U/ml) and cell survival monitored. Right panel: knockdown was monitored by western blot using antibodies against the C proteins of SeV. ( d ) L929 cells were infected with Cantell strain of SeV, Z strain of SeV (SeV-Z), or mutants (d2Y-Z, V[-]-Z). 16–18 h post-infection, mRNA was isolated and used for quantitative RT-PCR using oligonucleotides specific for the SeV NP or γ -actin gene. Expression was plotted on a log scale relative to mock-infected samples after normalization to γ -actin. Standard error of the mean was calculated from three independent experiments. ( e ) Survival of cells infected with Cantell strain of SeV (SeV) or Z strain (SeV-Z, d2Y-Z, V[-]-Z) as measured by CellTiter-Glo assay. ( f ) Lysates from SeV-infected cells at various time points (M: mock-infected) were probed in a western blot analysis using SeV C protein or β-actin specific antibodies (experiment repeated twice). For 3A, 3C, 3D, 3E, data are averages of triplicates from a single experiment, which is representative of at least three independent experiments. *** P

    Article Snippet: 16–18 hours post-infection, cell survival was measured by CellTiter-Glo ATP Assay (Promega).

    Techniques: Infection, Western Blot, Stable Transfection, Expressing, CTL Assay, shRNA, Isolation, Quantitative RT-PCR, Glo Assay

    Sendai virus and MHV68 cause RIP1- and RIP3-dependent necroptosis. ( a ) L929 cells were treated with zVAD (10 μ M) and infected with MCMV (MOI 5), HSV-1 (MOI 5), LCMV (MOI 5), MHV68 (MOI 5), SeV (10 HA U/ml), KSHV (titre giving 70% infection) or WSN (MOI 5) and % cell survival was determined by the CellTiter-Glo assay. ( b ) Cell death of L929 cells treated with zVAD (10 μ M), Necrostatin-1 (20 μM) and infected with SeV (10 HA U/ml) or MHV68 (MOI 5). ( c ) L929 cells stably expressing mouse shRIP3 or control shRNA were treated with or without zVAD (5 μ M) in the absence or presence of SeV (10 HA U/ml) or MHV68 (MOI 5). Right panel: cell lysates of the shRNA retrovirally infected cells were probed by RIP3- or actin-specific antibodies. ( d ) L929 cells were mock-treated or treated with Necrostatin-1 (Nec-1) at 20 μM and infected with indicated titres of SeV. Cell death was monitored 18–20 h post-infection by CellTiter-Glo. (e ) Increased relative expression of viral NP protein from RIP3 shRNA knockdown cells as compared to control shRNA knockdown cells by quantitative RT-PCR. Expression is relative to mock-infected samples after normalization to γ -actin. ( f ) TNF ELISA from supernatants of L929 cells treated with zVAD and infected with SeV (10 or 100 HA U/ml) or MHV68 (MOI 5 or 10). ( g ) Death of cells treated with control or anti-TNF neutralizing antibody, zVAD (10 μ M and 100 μ M), TNF (60 pg/ml), SeV (100 HA U/ml) or MHV68 (MOI 5). ( h ) LA-4 cells were treated with zVAD (20 μ M), TNF (100 ng/ml) or Nec-1 (20 μ M) and infected with SeV (100 HA U/ml) or MHV68 (MOI 10). Cell survival was monitored 24 h post-infection. ( i ) LA-4 cells were treated with anti-control or anti-TNF neutralizing antibodies and treated with zVAD, TNF and infected with SeV (100 HA U/ml) before cell survival was assayed. Experiment repeated twice. Data are averages of triplicates from a single experiment, which is representative of at least three independent experiments (unless otherwise stated). *** P

    Journal: Cell Death and Differentiation

    Article Title: Induction of necroptotic cell death by viral activation of the RIG-I or STING pathway

    doi: 10.1038/cdd.2016.153

    Figure Lengend Snippet: Sendai virus and MHV68 cause RIP1- and RIP3-dependent necroptosis. ( a ) L929 cells were treated with zVAD (10 μ M) and infected with MCMV (MOI 5), HSV-1 (MOI 5), LCMV (MOI 5), MHV68 (MOI 5), SeV (10 HA U/ml), KSHV (titre giving 70% infection) or WSN (MOI 5) and % cell survival was determined by the CellTiter-Glo assay. ( b ) Cell death of L929 cells treated with zVAD (10 μ M), Necrostatin-1 (20 μM) and infected with SeV (10 HA U/ml) or MHV68 (MOI 5). ( c ) L929 cells stably expressing mouse shRIP3 or control shRNA were treated with or without zVAD (5 μ M) in the absence or presence of SeV (10 HA U/ml) or MHV68 (MOI 5). Right panel: cell lysates of the shRNA retrovirally infected cells were probed by RIP3- or actin-specific antibodies. ( d ) L929 cells were mock-treated or treated with Necrostatin-1 (Nec-1) at 20 μM and infected with indicated titres of SeV. Cell death was monitored 18–20 h post-infection by CellTiter-Glo. (e ) Increased relative expression of viral NP protein from RIP3 shRNA knockdown cells as compared to control shRNA knockdown cells by quantitative RT-PCR. Expression is relative to mock-infected samples after normalization to γ -actin. ( f ) TNF ELISA from supernatants of L929 cells treated with zVAD and infected with SeV (10 or 100 HA U/ml) or MHV68 (MOI 5 or 10). ( g ) Death of cells treated with control or anti-TNF neutralizing antibody, zVAD (10 μ M and 100 μ M), TNF (60 pg/ml), SeV (100 HA U/ml) or MHV68 (MOI 5). ( h ) LA-4 cells were treated with zVAD (20 μ M), TNF (100 ng/ml) or Nec-1 (20 μ M) and infected with SeV (100 HA U/ml) or MHV68 (MOI 10). Cell survival was monitored 24 h post-infection. ( i ) LA-4 cells were treated with anti-control or anti-TNF neutralizing antibodies and treated with zVAD, TNF and infected with SeV (100 HA U/ml) before cell survival was assayed. Experiment repeated twice. Data are averages of triplicates from a single experiment, which is representative of at least three independent experiments (unless otherwise stated). *** P

    Article Snippet: 16–18 hours post-infection, cell survival was measured by CellTiter-Glo ATP Assay (Promega).

    Techniques: Infection, Glo Assay, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Effect of HSA levels on the in vitro sensitivity of JAK2wt K562 and JAK2V617F mutated HEL and SET-2 cells to ruxolitinib. JAK2wt K562 (circles) and JAK2V617F + HEL (squares) and SET-2 (triangles) myeloid cell lines were cultured in 10% FBS medium or serum-free medium containing HSA (0–80 µM) and treated with the indicated doses of ruxolitinib for 72 hours. Cell viability was determined by the ATP-based cell-viability assay CellTiter-Glo. Data are the percent of untreated control cells. All data are presented as means of three independent experiments ± SD.

    Journal: Scientific Reports

    Article Title: Ruxolitinib binding to human serum albumin: bioinformatics, biochemical and functional characterization in JAK2V617F+ cell models

    doi: 10.1038/s41598-019-52852-9

    Figure Lengend Snippet: Effect of HSA levels on the in vitro sensitivity of JAK2wt K562 and JAK2V617F mutated HEL and SET-2 cells to ruxolitinib. JAK2wt K562 (circles) and JAK2V617F + HEL (squares) and SET-2 (triangles) myeloid cell lines were cultured in 10% FBS medium or serum-free medium containing HSA (0–80 µM) and treated with the indicated doses of ruxolitinib for 72 hours. Cell viability was determined by the ATP-based cell-viability assay CellTiter-Glo. Data are the percent of untreated control cells. All data are presented as means of three independent experiments ± SD.

    Article Snippet: Cell viability The viability of cells was determined using the ATP-based cell-viability assay CellTiter-Glo (Promega, Madison, WI, USA) according to the manufacturer’s instruction.

    Techniques: In Vitro, Cell Culture, Viability Assay