celltiter-glo luminescent cell viability assay kit Search Results


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  • 77
    Promega celltiter glo luminescent cell viability assays kit
    An expanded set of contemporary EV-D68 strains infect SH-SY5Y, but HRV strains do not. (A) HeLa and SH-SY5Y cells were infected with 6 different EV-D68 isolates at an MOI of 0.1. Cell culture lysates were collected at various time points, and the viral titer was determined by TCID 50 in HeLa cells. Dotted black lines indicate the limit of detection. Error bars represent SEM from three biological replicates. (B) Similarly, cell viability was determined by quantifying the ATP content of the supernatant with <t>CellTiter</t> <t>Glo</t> (Promega) luminescence. Cell viability was calculated relative to mock-infected cultures. Error bars represent SEM from three replicates. (C) Six different human rhinovirus (HRV) strains and two EV-D68 strains were used to infect HeLa and SH-SY5Y cell cultures at an MOI of 0.1 and were visualized at 72 hpi. Cell viability was calculated as above. (D) Differential cytopathic effects of different EV-D68 isolates in HeLa and SH-SY5Y cells after infection with different EV-D68 isolates at an MOI of 0.1 before visualization at 72 hpi.
    Celltiter Glo Luminescent Cell Viability Assays Kit, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 708 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MEGA Inc celltiter glo luminescent cell viability assay kit
    An expanded set of contemporary EV-D68 strains infect SH-SY5Y, but HRV strains do not. (A) HeLa and SH-SY5Y cells were infected with 6 different EV-D68 isolates at an MOI of 0.1. Cell culture lysates were collected at various time points, and the viral titer was determined by TCID 50 in HeLa cells. Dotted black lines indicate the limit of detection. Error bars represent SEM from three biological replicates. (B) Similarly, cell viability was determined by quantifying the ATP content of the supernatant with <t>CellTiter</t> <t>Glo</t> (Promega) luminescence. Cell viability was calculated relative to mock-infected cultures. Error bars represent SEM from three replicates. (C) Six different human rhinovirus (HRV) strains and two EV-D68 strains were used to infect HeLa and SH-SY5Y cell cultures at an MOI of 0.1 and were visualized at 72 hpi. Cell viability was calculated as above. (D) Differential cytopathic effects of different EV-D68 isolates in HeLa and SH-SY5Y cells after infection with different EV-D68 isolates at an MOI of 0.1 before visualization at 72 hpi.
    Celltiter Glo Luminescent Cell Viability Assay Kit, supplied by MEGA Inc, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiter glo luminescence cell viability assay kit
    Effect of CR8#13 in latently infected cells and its effect in PBMCs A) CYC202, CR8 and CR8#13 were used to treat activated OM10.1 cells. Cells were grown to mid-log phase of growth and treated with TNF-α for 2 hours to induce HIV-1 expression. TNF-α was then removed and cells were subsequently treated with drugs at 0.001, 0.01, 0.1, 1, 10 and 100 µM in complete media. Five days later, sups were collected and processed for p24 HIV-1 Gag ELISA. B) At day five cells were processed for MTT assay. Analysis was carried out with Prizm software using non-liner regression curve-fit of sigmoidal dose-response with variable slope. C D) PHA-activated PBMC (5 × 10 6 ) were infected with THA/92/00 strain (MOI: 0.1). After 8 hours, unadsorbed virus was washed away and cells were treated with 10 or 100 nM of either Flavopiridol or CR8#13. Supernatants were collected every 3 days for RT analysis. Number of live cells was also plotted from each date and treatment (trypan blue exclusion assay). E F) Similar to panel C, where PHA-activated PBMC cells (Lonza) were infected with the THA/92/00 strain and treated with various concentrations of Flavopiridol or CR8#13 cells. Samples were assayed using <t>CellTiter-Glo</t> after either 3 or 16 days.
    Celltiter Glo Luminescence Cell Viability Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiter glo 3d luminescent cell viability assay kits
    Effect of CR8#13 in latently infected cells and its effect in PBMCs A) CYC202, CR8 and CR8#13 were used to treat activated OM10.1 cells. Cells were grown to mid-log phase of growth and treated with TNF-α for 2 hours to induce HIV-1 expression. TNF-α was then removed and cells were subsequently treated with drugs at 0.001, 0.01, 0.1, 1, 10 and 100 µM in complete media. Five days later, sups were collected and processed for p24 HIV-1 Gag ELISA. B) At day five cells were processed for MTT assay. Analysis was carried out with Prizm software using non-liner regression curve-fit of sigmoidal dose-response with variable slope. C D) PHA-activated PBMC (5 × 10 6 ) were infected with THA/92/00 strain (MOI: 0.1). After 8 hours, unadsorbed virus was washed away and cells were treated with 10 or 100 nM of either Flavopiridol or CR8#13. Supernatants were collected every 3 days for RT analysis. Number of live cells was also plotted from each date and treatment (trypan blue exclusion assay). E F) Similar to panel C, where PHA-activated PBMC cells (Lonza) were infected with the THA/92/00 strain and treated with various concentrations of Flavopiridol or CR8#13 cells. Samples were assayed using <t>CellTiter-Glo</t> after either 3 or 16 days.
    Celltiter Glo 3d Luminescent Cell Viability Assay Kits, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 2409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp bioluminescence assay kit celltiter glo luminescent cell viability assay
    Effect of CR8#13 in latently infected cells and its effect in PBMCs A) CYC202, CR8 and CR8#13 were used to treat activated OM10.1 cells. Cells were grown to mid-log phase of growth and treated with TNF-α for 2 hours to induce HIV-1 expression. TNF-α was then removed and cells were subsequently treated with drugs at 0.001, 0.01, 0.1, 1, 10 and 100 µM in complete media. Five days later, sups were collected and processed for p24 HIV-1 Gag ELISA. B) At day five cells were processed for MTT assay. Analysis was carried out with Prizm software using non-liner regression curve-fit of sigmoidal dose-response with variable slope. C D) PHA-activated PBMC (5 × 10 6 ) were infected with THA/92/00 strain (MOI: 0.1). After 8 hours, unadsorbed virus was washed away and cells were treated with 10 or 100 nM of either Flavopiridol or CR8#13. Supernatants were collected every 3 days for RT analysis. Number of live cells was also plotted from each date and treatment (trypan blue exclusion assay). E F) Similar to panel C, where PHA-activated PBMC cells (Lonza) were infected with the THA/92/00 strain and treated with various concentrations of Flavopiridol or CR8#13 cells. Samples were assayed using <t>CellTiter-Glo</t> after either 3 or 16 days.
    Atp Bioluminescence Assay Kit Celltiter Glo Luminescent Cell Viability Assay, supplied by Promega, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime celltiter glo luminescent cell viability assay kit
    Effect of CR8#13 in latently infected cells and its effect in PBMCs A) CYC202, CR8 and CR8#13 were used to treat activated OM10.1 cells. Cells were grown to mid-log phase of growth and treated with TNF-α for 2 hours to induce HIV-1 expression. TNF-α was then removed and cells were subsequently treated with drugs at 0.001, 0.01, 0.1, 1, 10 and 100 µM in complete media. Five days later, sups were collected and processed for p24 HIV-1 Gag ELISA. B) At day five cells were processed for MTT assay. Analysis was carried out with Prizm software using non-liner regression curve-fit of sigmoidal dose-response with variable slope. C D) PHA-activated PBMC (5 × 10 6 ) were infected with THA/92/00 strain (MOI: 0.1). After 8 hours, unadsorbed virus was washed away and cells were treated with 10 or 100 nM of either Flavopiridol or CR8#13. Supernatants were collected every 3 days for RT analysis. Number of live cells was also plotted from each date and treatment (trypan blue exclusion assay). E F) Similar to panel C, where PHA-activated PBMC cells (Lonza) were infected with the THA/92/00 strain and treated with various concentrations of Flavopiridol or CR8#13 cells. Samples were assayed using <t>CellTiter-Glo</t> after either 3 or 16 days.
    Celltiter Glo Luminescent Cell Viability Assay Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 97/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiter glo luminescent cell viability assay atp kit
    Anti-proliferative activity of TA in CRC cell lines HCT116 ( A ) and HT29 ( B ) cells were treated with DMSO (Control) or increasing concentrations of TA (25-100 μM) for 24-72 h. Cell viability was determined using <t>CellTiter-Glo</t> cell viability assay and results are presented as % viable cells (relative to control). Values represent mean ± SEM ( n = 3) and the bars with ‘*’ are significantly different from control ( p
    Celltiter Glo Luminescent Cell Viability Assay Atp Kit, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 531 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiter glo luminiscent cell viability kit
    Anti-proliferative activity of TA in CRC cell lines HCT116 ( A ) and HT29 ( B ) cells were treated with DMSO (Control) or increasing concentrations of TA (25-100 μM) for 24-72 h. Cell viability was determined using <t>CellTiter-Glo</t> cell viability assay and results are presented as % viable cells (relative to control). Values represent mean ± SEM ( n = 3) and the bars with ‘*’ are significantly different from control ( p
    Celltiter Glo Luminiscent Cell Viability Kit, supplied by Promega, used in various techniques. Bioz Stars score: 83/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltitre glo luminescent cell viability kit
    Effects of anosmin-1 in tumor cell proliferation. (A) LN229 cells were treated with anosmin-1 for 6 h with BrdU labeling during the last 2 h in serum-free conditions. In parallel experiments, cells were pre-treated with SU5402 or amiloride for 30 min before stimulation with anosmin-1. Anosmin-1-treated cells (denoted as A) show increased BrdU incorporation (** P =0.0035) compared with the untreated or solvent (DMSO)-treated cells. Pretreatment with SU5402 or amiloride, however, attenuated anosmin-1-induced proliferation (** P =0.0046 and * P =0.0423 respectively). At least five random fields were analysed, which were repeated four times. (B) shRNA-mediated knockdown of KAL1 results in reduced BrdU incorporation in A172. The P values obtained from four independent experiments are 0.0319 (for shRNA673), 0.019 (for shRNA675), and 0.05 (for shRNA676) when compared with the control shRNA. (C) LN229 cells treated with anosmin-1 indicate significantly higher proliferation in <t>CellTitre-Glo</t> luminescent assay, compared with the untreated control at 24 h (* P =0.0103) and 48 h (** P =0.0048) by a two-way ANOVA test. Error bars indicate s.e.m . from five independent experiments.
    Celltitre Glo Luminescent Cell Viability Kit, supplied by Promega, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime celltitre glo luminescent cell viability assay kit
    Effects of anosmin-1 in tumor cell proliferation. (A) LN229 cells were treated with anosmin-1 for 6 h with BrdU labeling during the last 2 h in serum-free conditions. In parallel experiments, cells were pre-treated with SU5402 or amiloride for 30 min before stimulation with anosmin-1. Anosmin-1-treated cells (denoted as A) show increased BrdU incorporation (** P =0.0035) compared with the untreated or solvent (DMSO)-treated cells. Pretreatment with SU5402 or amiloride, however, attenuated anosmin-1-induced proliferation (** P =0.0046 and * P =0.0423 respectively). At least five random fields were analysed, which were repeated four times. (B) shRNA-mediated knockdown of KAL1 results in reduced BrdU incorporation in A172. The P values obtained from four independent experiments are 0.0319 (for shRNA673), 0.019 (for shRNA675), and 0.05 (for shRNA676) when compared with the control shRNA. (C) LN229 cells treated with anosmin-1 indicate significantly higher proliferation in <t>CellTitre-Glo</t> luminescent assay, compared with the untreated control at 24 h (* P =0.0103) and 48 h (** P =0.0048) by a two-way ANOVA test. Error bars indicate s.e.m . from five independent experiments.
    Celltitre Glo Luminescent Cell Viability Assay Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiger glo luminescent cell viability assay kit
    Effects of anosmin-1 in tumor cell proliferation. (A) LN229 cells were treated with anosmin-1 for 6 h with BrdU labeling during the last 2 h in serum-free conditions. In parallel experiments, cells were pre-treated with SU5402 or amiloride for 30 min before stimulation with anosmin-1. Anosmin-1-treated cells (denoted as A) show increased BrdU incorporation (** P =0.0035) compared with the untreated or solvent (DMSO)-treated cells. Pretreatment with SU5402 or amiloride, however, attenuated anosmin-1-induced proliferation (** P =0.0046 and * P =0.0423 respectively). At least five random fields were analysed, which were repeated four times. (B) shRNA-mediated knockdown of KAL1 results in reduced BrdU incorporation in A172. The P values obtained from four independent experiments are 0.0319 (for shRNA673), 0.019 (for shRNA675), and 0.05 (for shRNA676) when compared with the control shRNA. (C) LN229 cells treated with anosmin-1 indicate significantly higher proliferation in <t>CellTitre-Glo</t> luminescent assay, compared with the untreated control at 24 h (* P =0.0103) and 48 h (** P =0.0048) by a two-way ANOVA test. Error bars indicate s.e.m . from five independent experiments.
    Celltiger Glo Luminescent Cell Viability Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiter glo luminescence kit
    Functional interaction of vFLIP and MAVS is essential for survival of HHV-8-infected PEL cells. (A-B) Co-IP assay using extracts from 293T cells transfected with expression vectors for Flag-MAVS and (A) GST, GST-vFLIP, GST-DED1, or GST-DED2 and (B) GST and GST-vFLIP helices. (C) Immunoblots of extracts from 293T cells transfected with V5-vFLIP and Flag-MAVS together with GST-vFLIP helices. (D) Immunoblots of extracts from BCBL-1 cells treated with 10 μM TAT and TAT-vFLIP DED2-H1 (2H1) peptides twice for 4 days. Asterisk indicates a band of unknown identity. (E) FITC-Annexin V and 7-AAD-based flow cytometric analyses of WT and MAVS KO BCBL-1 cell lines treated with TAT and TAT-2H1 for 2 days. The cells were seeded at low-density (5 x10 4 cells/ml). The percentage of total cells in each quadrant is shown. (F) <t>CellTiter-Glo</t> cell viability assays with the indicated cells treated with TAT and TAT-2H1 for 4 days. Data are represented as mean ± SD of two independent experiments in triplicate. (*p
    Celltiter Glo Luminescence Kit, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiter glo luminescent assay kit
    Effects of MMO on the cell viability of NAFLD model cells. The NAFLD model cells were treated with MMO (10 and 20 mg/mL) for 24 h, and cell viability was determined by <t>CellTiter-Glo</t> assay. Data are presented as mean ± S.D. for three independent experiments with triplicate determination. * p
    Celltiter Glo Luminescent Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega cell viability kit
    Effects of MMO on the cell viability of NAFLD model cells. The NAFLD model cells were treated with MMO (10 and 20 mg/mL) for 24 h, and cell viability was determined by <t>CellTiter-Glo</t> assay. Data are presented as mean ± S.D. for three independent experiments with triplicate determination. * p
    Cell Viability Kit, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiter glo reagent luminescence viability kit
    IFNα-2a increases packaging of HIV-1 viral RNA into EVs. U1 cells were treated with a titration of IFNα-2a (0.5 K, 1 K, 5 K, 10 K, 50 K, and 100 K units) for 5 days. ( a ) EVs were enriched using NT80/82 particles and analyzed for the presence of TAR, TAR- gag , and genomic RNA using RT-qPCR (see Methods). ( b ) The cells responsible for the production of EVs analyzed in panel a were analyzed for the levels of intracellular viral RNA using RT-qPCR. ( c ) AChE activity was measured (relative fluorescent units; RFU) in isolated EVs released from U1 cells that were treated with IFNα-2a and U1 controls. ( d ) U1 cells were treated with a titration of IFNα-2a (0.5K-100K units) for 5 days. Whole cell extracts were analyzed by Western blot for ESCRT pathway proteins [ESCRT-I (TSG101),-II (EAP20, EAP45),-III (CHMP6), and exit (VPS4)] and Actin protein levels. Selected lanes were taken from the same blot with identical exposure settings presented in the figure. Densitometry counts as determined by ImageJ software is shown as increase or decrease of VPS4 or TSG101 relative to the untreated control (set to 100%). (e ) IFNα-2a-treated cells were assessed for cell viability using <t>CellTiter-Glo</t> reagent. ( f–i) U1 cells were treated with cART (45 µM) or IFNα-2a (5 K or 10 K) for 5 days. EVs were enriched from supernatants using immunoprecipitation with EV marker antibodies (CD81, CD9, CD63) or NT80/82 particles. Resulting samples were analyzed by RT-qPCR for TAR, TAR- gag , and genomic RNA (see Methods). Red boxes highlight clinically relevant IFN concentrations. Error bars represent ± S.D. of three technical replicates for all panels. A two-tailed Student’s t- test was used to assess significance: * p
    Celltiter Glo Reagent Luminescence Viability Kit, supplied by Promega, used in various techniques. Bioz Stars score: 81/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega cell viability luminescent assay kit
    IFNα-2a increases packaging of HIV-1 viral RNA into EVs. U1 cells were treated with a titration of IFNα-2a (0.5 K, 1 K, 5 K, 10 K, 50 K, and 100 K units) for 5 days. ( a ) EVs were enriched using NT80/82 particles and analyzed for the presence of TAR, TAR- gag , and genomic RNA using RT-qPCR (see Methods). ( b ) The cells responsible for the production of EVs analyzed in panel a were analyzed for the levels of intracellular viral RNA using RT-qPCR. ( c ) AChE activity was measured (relative fluorescent units; RFU) in isolated EVs released from U1 cells that were treated with IFNα-2a and U1 controls. ( d ) U1 cells were treated with a titration of IFNα-2a (0.5K-100K units) for 5 days. Whole cell extracts were analyzed by Western blot for ESCRT pathway proteins [ESCRT-I (TSG101),-II (EAP20, EAP45),-III (CHMP6), and exit (VPS4)] and Actin protein levels. Selected lanes were taken from the same blot with identical exposure settings presented in the figure. Densitometry counts as determined by ImageJ software is shown as increase or decrease of VPS4 or TSG101 relative to the untreated control (set to 100%). (e ) IFNα-2a-treated cells were assessed for cell viability using <t>CellTiter-Glo</t> reagent. ( f–i) U1 cells were treated with cART (45 µM) or IFNα-2a (5 K or 10 K) for 5 days. EVs were enriched from supernatants using immunoprecipitation with EV marker antibodies (CD81, CD9, CD63) or NT80/82 particles. Resulting samples were analyzed by RT-qPCR for TAR, TAR- gag , and genomic RNA (see Methods). Red boxes highlight clinically relevant IFN concentrations. Error bars represent ± S.D. of three technical replicates for all panels. A two-tailed Student’s t- test was used to assess significance: * p
    Cell Viability Luminescent Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega luminescent assay kits
    IFNα-2a increases packaging of HIV-1 viral RNA into EVs. U1 cells were treated with a titration of IFNα-2a (0.5 K, 1 K, 5 K, 10 K, 50 K, and 100 K units) for 5 days. ( a ) EVs were enriched using NT80/82 particles and analyzed for the presence of TAR, TAR- gag , and genomic RNA using RT-qPCR (see Methods). ( b ) The cells responsible for the production of EVs analyzed in panel a were analyzed for the levels of intracellular viral RNA using RT-qPCR. ( c ) AChE activity was measured (relative fluorescent units; RFU) in isolated EVs released from U1 cells that were treated with IFNα-2a and U1 controls. ( d ) U1 cells were treated with a titration of IFNα-2a (0.5K-100K units) for 5 days. Whole cell extracts were analyzed by Western blot for ESCRT pathway proteins [ESCRT-I (TSG101),-II (EAP20, EAP45),-III (CHMP6), and exit (VPS4)] and Actin protein levels. Selected lanes were taken from the same blot with identical exposure settings presented in the figure. Densitometry counts as determined by ImageJ software is shown as increase or decrease of VPS4 or TSG101 relative to the untreated control (set to 100%). (e ) IFNα-2a-treated cells were assessed for cell viability using <t>CellTiter-Glo</t> reagent. ( f–i) U1 cells were treated with cART (45 µM) or IFNα-2a (5 K or 10 K) for 5 days. EVs were enriched from supernatants using immunoprecipitation with EV marker antibodies (CD81, CD9, CD63) or NT80/82 particles. Resulting samples were analyzed by RT-qPCR for TAR, TAR- gag , and genomic RNA (see Methods). Red boxes highlight clinically relevant IFN concentrations. Error bars represent ± S.D. of three technical replicates for all panels. A two-tailed Student’s t- test was used to assess significance: * p
    Luminescent Assay Kits, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega luminescence assay kit
    IFNα-2a increases packaging of HIV-1 viral RNA into EVs. U1 cells were treated with a titration of IFNα-2a (0.5 K, 1 K, 5 K, 10 K, 50 K, and 100 K units) for 5 days. ( a ) EVs were enriched using NT80/82 particles and analyzed for the presence of TAR, TAR- gag , and genomic RNA using RT-qPCR (see Methods). ( b ) The cells responsible for the production of EVs analyzed in panel a were analyzed for the levels of intracellular viral RNA using RT-qPCR. ( c ) AChE activity was measured (relative fluorescent units; RFU) in isolated EVs released from U1 cells that were treated with IFNα-2a and U1 controls. ( d ) U1 cells were treated with a titration of IFNα-2a (0.5K-100K units) for 5 days. Whole cell extracts were analyzed by Western blot for ESCRT pathway proteins [ESCRT-I (TSG101),-II (EAP20, EAP45),-III (CHMP6), and exit (VPS4)] and Actin protein levels. Selected lanes were taken from the same blot with identical exposure settings presented in the figure. Densitometry counts as determined by ImageJ software is shown as increase or decrease of VPS4 or TSG101 relative to the untreated control (set to 100%). (e ) IFNα-2a-treated cells were assessed for cell viability using <t>CellTiter-Glo</t> reagent. ( f–i) U1 cells were treated with cART (45 µM) or IFNα-2a (5 K or 10 K) for 5 days. EVs were enriched from supernatants using immunoprecipitation with EV marker antibodies (CD81, CD9, CD63) or NT80/82 particles. Resulting samples were analyzed by RT-qPCR for TAR, TAR- gag , and genomic RNA (see Methods). Red boxes highlight clinically relevant IFN concentrations. Error bars represent ± S.D. of three technical replicates for all panels. A two-tailed Student’s t- test was used to assess significance: * p
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    IFNα-2a increases packaging of HIV-1 viral RNA into EVs. U1 cells were treated with a titration of IFNα-2a (0.5 K, 1 K, 5 K, 10 K, 50 K, and 100 K units) for 5 days. ( a ) EVs were enriched using NT80/82 particles and analyzed for the presence of TAR, TAR- gag , and genomic RNA using RT-qPCR (see Methods). ( b ) The cells responsible for the production of EVs analyzed in panel a were analyzed for the levels of intracellular viral RNA using RT-qPCR. ( c ) AChE activity was measured (relative fluorescent units; RFU) in isolated EVs released from U1 cells that were treated with IFNα-2a and U1 controls. ( d ) U1 cells were treated with a titration of IFNα-2a (0.5K-100K units) for 5 days. Whole cell extracts were analyzed by Western blot for ESCRT pathway proteins [ESCRT-I (TSG101),-II (EAP20, EAP45),-III (CHMP6), and exit (VPS4)] and Actin protein levels. Selected lanes were taken from the same blot with identical exposure settings presented in the figure. Densitometry counts as determined by ImageJ software is shown as increase or decrease of VPS4 or TSG101 relative to the untreated control (set to 100%). (e ) IFNα-2a-treated cells were assessed for cell viability using <t>CellTiter-Glo</t> reagent. ( f–i) U1 cells were treated with cART (45 µM) or IFNα-2a (5 K or 10 K) for 5 days. EVs were enriched from supernatants using immunoprecipitation with EV marker antibodies (CD81, CD9, CD63) or NT80/82 particles. Resulting samples were analyzed by RT-qPCR for TAR, TAR- gag , and genomic RNA (see Methods). Red boxes highlight clinically relevant IFN concentrations. Error bars represent ± S.D. of three technical replicates for all panels. A two-tailed Student’s t- test was used to assess significance: * p
    Luciferase Based Atp Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp depletion kit
    Intracellular <t>ATP</t> levels in wild-type and CatE −/− macrophages. Macrophages (1 × 10 5 cells) were cultured on 96-well plates at 37°C for 24 h. After replacement with fresh media, the culture plate was incubated at room temperature (25°C) for 30 min. ATP levels were determined by the manufacturer’s protocol of the <t>CellTiter-Glo</t> assay kit according to the manufacturer’s protocol. The concentration of intracellular ATP was determined by the titration of the control medium without cells plus 0.1−1.0 µM ATP.
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    Intracellular <t>ATP</t> levels in wild-type and CatE −/− macrophages. Macrophages (1 × 10 5 cells) were cultured on 96-well plates at 37°C for 24 h. After replacement with fresh media, the culture plate was incubated at room temperature (25°C) for 30 min. ATP levels were determined by the manufacturer’s protocol of the <t>CellTiter-Glo</t> assay kit according to the manufacturer’s protocol. The concentration of intracellular ATP was determined by the titration of the control medium without cells plus 0.1−1.0 µM ATP.
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    Graphical comparisons of changes in <t>caspase-3/-7</t> activity of normal T cells after 24 (A) and 48 (B) hours incubation with Sutherlandia frutescens extract doses (SFE and SFW). The untreated control was again used as a reference (100%) for all the samples. SFE and SFW extract doses showed a concentration- and time-dependent decrease in decreasing caspase-3/-7 activity, with high doses being more effective. High doses of the SFE extract were more potent in reducing caspase-3/-7 activity over this period and their effect was independent of ethanol ( p
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    Graphical comparisons of changes in <t>caspase-3/-7</t> activity of normal T cells after 24 (A) and 48 (B) hours incubation with Sutherlandia frutescens extract doses (SFE and SFW). The untreated control was again used as a reference (100%) for all the samples. SFE and SFW extract doses showed a concentration- and time-dependent decrease in decreasing caspase-3/-7 activity, with high doses being more effective. High doses of the SFE extract were more potent in reducing caspase-3/-7 activity over this period and their effect was independent of ethanol ( p
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    Graphical comparisons of changes in <t>caspase-3/-7</t> activity of normal T cells after 24 (A) and 48 (B) hours incubation with Sutherlandia frutescens extract doses (SFE and SFW). The untreated control was again used as a reference (100%) for all the samples. SFE and SFW extract doses showed a concentration- and time-dependent decrease in decreasing caspase-3/-7 activity, with high doses being more effective. High doses of the SFE extract were more potent in reducing caspase-3/-7 activity over this period and their effect was independent of ethanol ( p
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    Image Search Results


    An expanded set of contemporary EV-D68 strains infect SH-SY5Y, but HRV strains do not. (A) HeLa and SH-SY5Y cells were infected with 6 different EV-D68 isolates at an MOI of 0.1. Cell culture lysates were collected at various time points, and the viral titer was determined by TCID 50 in HeLa cells. Dotted black lines indicate the limit of detection. Error bars represent SEM from three biological replicates. (B) Similarly, cell viability was determined by quantifying the ATP content of the supernatant with CellTiter Glo (Promega) luminescence. Cell viability was calculated relative to mock-infected cultures. Error bars represent SEM from three replicates. (C) Six different human rhinovirus (HRV) strains and two EV-D68 strains were used to infect HeLa and SH-SY5Y cell cultures at an MOI of 0.1 and were visualized at 72 hpi. Cell viability was calculated as above. (D) Differential cytopathic effects of different EV-D68 isolates in HeLa and SH-SY5Y cells after infection with different EV-D68 isolates at an MOI of 0.1 before visualization at 72 hpi.

    Journal: mBio

    Article Title: Contemporary Circulating Enterovirus D68 Strains Have Acquired the Capacity for Viral Entry and Replication in Human Neuronal Cells

    doi: 10.1128/mBio.01954-18

    Figure Lengend Snippet: An expanded set of contemporary EV-D68 strains infect SH-SY5Y, but HRV strains do not. (A) HeLa and SH-SY5Y cells were infected with 6 different EV-D68 isolates at an MOI of 0.1. Cell culture lysates were collected at various time points, and the viral titer was determined by TCID 50 in HeLa cells. Dotted black lines indicate the limit of detection. Error bars represent SEM from three biological replicates. (B) Similarly, cell viability was determined by quantifying the ATP content of the supernatant with CellTiter Glo (Promega) luminescence. Cell viability was calculated relative to mock-infected cultures. Error bars represent SEM from three replicates. (C) Six different human rhinovirus (HRV) strains and two EV-D68 strains were used to infect HeLa and SH-SY5Y cell cultures at an MOI of 0.1 and were visualized at 72 hpi. Cell viability was calculated as above. (D) Differential cytopathic effects of different EV-D68 isolates in HeLa and SH-SY5Y cells after infection with different EV-D68 isolates at an MOI of 0.1 before visualization at 72 hpi.

    Article Snippet: ATP levels were measured using the CellTiter-Glo luminescent cell viability assay kit (catalog no. G7570; Promega), and cell viability was calculated relative to that of the mock control.

    Techniques: Infection, Cell Culture

    Dynasore has no adverse effect on cell viability. PHHs were treated with Dynasore (80μM) for 0, 1, 2, 4, 16, 24 hours. After removal of Dynasore, cells were incubated for additional 48 hours prior to the CellTiter- Glo cell viability assay (see

    Journal: Virology

    Article Title: The second extracellular loop dictates Occludin-mediated HCV entry

    doi: 10.1016/j.virol.2010.08.009

    Figure Lengend Snippet: Dynasore has no adverse effect on cell viability. PHHs were treated with Dynasore (80μM) for 0, 1, 2, 4, 16, 24 hours. After removal of Dynasore, cells were incubated for additional 48 hours prior to the CellTiter- Glo cell viability assay (see

    Article Snippet: The numbers of viable cells in culture were determined using the CellTiter-Glo Cell Viability Luminescent Assay kit according to the manufacturer’s instruction (Promega).

    Techniques: Incubation, Viability Assay

    Two distinct pathways of necroptosis initiation by TNFα. ( A ) The 661W cells treated with TNFα/zVAD plus either 5Z-7 or CHX to induce necroptosis. Cell viability determined by CellTiter-Glo. ( B ) Scheme for additional tertiary siRNA screen for regulators of necroptosis induced by TNFα/5Z-7/zVAD. ( C ) The 661W cells transfected with siRNAs against APC11, LRRK2, or c-Cbl or nontargeting control (N.C.) siRNA for 72 h and treated with TNFα/5Z-7/zVAD for 8 h to induce necroptosis under RDA conditions. Cell survival assayed by CellTiter-Glo. Knockdown efficiency determined by Western blotting. ( D ) The 661W cells treated with TNFα/5Z-7/zVAD or TNFα/CHX/zVAD to induce necroptosis. Cells fractionated using lysis buffer with Nonidet P-40/TX-100 followed by 6 M urea and analyzed by Western blotting with indicated antibodies; pRIPK1 is RIPK1-pS166, pMLKL is MLKL-pS345. Concentrations of reagents: TNFα, 1 ng/mL ( C ), 10 ng/mL ( A and D ). All data shown are mean ± SD of three or more independent experiments. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 by one-way ANOVA.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of a distinct activated RIPK1 intermediate bridging complex I and complex II in TNFα-mediated apoptosis

    doi: 10.1073/pnas.1806973115

    Figure Lengend Snippet: Two distinct pathways of necroptosis initiation by TNFα. ( A ) The 661W cells treated with TNFα/zVAD plus either 5Z-7 or CHX to induce necroptosis. Cell viability determined by CellTiter-Glo. ( B ) Scheme for additional tertiary siRNA screen for regulators of necroptosis induced by TNFα/5Z-7/zVAD. ( C ) The 661W cells transfected with siRNAs against APC11, LRRK2, or c-Cbl or nontargeting control (N.C.) siRNA for 72 h and treated with TNFα/5Z-7/zVAD for 8 h to induce necroptosis under RDA conditions. Cell survival assayed by CellTiter-Glo. Knockdown efficiency determined by Western blotting. ( D ) The 661W cells treated with TNFα/5Z-7/zVAD or TNFα/CHX/zVAD to induce necroptosis. Cells fractionated using lysis buffer with Nonidet P-40/TX-100 followed by 6 M urea and analyzed by Western blotting with indicated antibodies; pRIPK1 is RIPK1-pS166, pMLKL is MLKL-pS345. Concentrations of reagents: TNFα, 1 ng/mL ( C ), 10 ng/mL ( A and D ). All data shown are mean ± SD of three or more independent experiments. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 by one-way ANOVA.

    Article Snippet: CellTiter-Glo luminescent cell viability kit was from Promega.

    Techniques: Transfection, Western Blot, Lysis

    Induction of RDA by TNFα leads to rapid and elevated activation of RIPK1 in a detergent-insoluble cellular compartment before formation of complex II. ( A ) The 661W cells treated with TNFα/5Z-7 to induce RDA, TNFα/CHX to induce RIPK1-independent apoptosis, or TNFα/CHX/zVAD to induce necroptosis in the presence or absence of Nec-1s. Cell viability determined by CellTiter-Glo. ( B ) Primary neuronal cultures treated with TNFα/5Z-7 to induce RDA for 8 h and cell death measured by Toxilight. ( C ) Primary astrocytes treated for 5 h and cell death measured by Toxilight. ( D ) Primary microglia treated for 5 h and cell survival measured by CellTiter-Glo. ( E – J ) Cells treated as indicated and samples analyzed by Western blotting using indicated antibodies; pRIPK1 is RIPK1-pS166. ( E ) The 661W cells treated with TNFα/5Z-7 to induce RDA. Lysates immunoprecipitated with RIPK1-pS166 ab. ( F ) WT MEFs lysates immunoprecipitated with RIPK1-pS166 ab. ( G ) The 661W cells treated with TNFα/5Z-7 to induce RDA, harvested with Nonidet P-40 buffer, and cleared by centrifugation to yield the Nonidet P-40 fraction. Pellet resuspended in Triton X-100 buffer and cleared by centrifugation to yield the TX-100 fraction. Resulting pellet was resuspended in buffer with 6 M urea and cleared by centrifugation to yield the urea fraction. ( H ) The 661W cells harvested as in G ; Nonidet P-40 fraction marked as detergent “soluble” fraction, 6 M urea fraction marked as detergent “insoluble” fraction ( Top ). Urea fraction used for immunoprecipitation with anti-linear ubiquitin (UBQ1) or anti-K11, -K48, or -K63 ubiquitin antibodies ( Bottom ). ( I ) Tak1 −/− MEFs treated with TNFα to induce RDA and harvested as in H . Nonidet P-40 soluble lysate used for complex II immunoprecipitation with anti-FADD (M-19). ( J ) The 661W cells harvested as in H . Concentrations of reagents: TNFα, 10 ng/mL ( A – G , I , and J ), 1 ng/mL ( H ); CHX, 1–2 μg/mL; 5Z-7, 500 nM; Nec-1s, 10 μM; zVAD, 20 μM; SM-164, 500 nM. Same concentrations of these compounds were used in subsequent experiments unless noted. All data shown are mean ± SD of three or more independent experiments. ** P ≤ 0.01 and *** P ≤ 0.001 by one-way ANOVA ( A – C ) or Student’s t test ( D ).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of a distinct activated RIPK1 intermediate bridging complex I and complex II in TNFα-mediated apoptosis

    doi: 10.1073/pnas.1806973115

    Figure Lengend Snippet: Induction of RDA by TNFα leads to rapid and elevated activation of RIPK1 in a detergent-insoluble cellular compartment before formation of complex II. ( A ) The 661W cells treated with TNFα/5Z-7 to induce RDA, TNFα/CHX to induce RIPK1-independent apoptosis, or TNFα/CHX/zVAD to induce necroptosis in the presence or absence of Nec-1s. Cell viability determined by CellTiter-Glo. ( B ) Primary neuronal cultures treated with TNFα/5Z-7 to induce RDA for 8 h and cell death measured by Toxilight. ( C ) Primary astrocytes treated for 5 h and cell death measured by Toxilight. ( D ) Primary microglia treated for 5 h and cell survival measured by CellTiter-Glo. ( E – J ) Cells treated as indicated and samples analyzed by Western blotting using indicated antibodies; pRIPK1 is RIPK1-pS166. ( E ) The 661W cells treated with TNFα/5Z-7 to induce RDA. Lysates immunoprecipitated with RIPK1-pS166 ab. ( F ) WT MEFs lysates immunoprecipitated with RIPK1-pS166 ab. ( G ) The 661W cells treated with TNFα/5Z-7 to induce RDA, harvested with Nonidet P-40 buffer, and cleared by centrifugation to yield the Nonidet P-40 fraction. Pellet resuspended in Triton X-100 buffer and cleared by centrifugation to yield the TX-100 fraction. Resulting pellet was resuspended in buffer with 6 M urea and cleared by centrifugation to yield the urea fraction. ( H ) The 661W cells harvested as in G ; Nonidet P-40 fraction marked as detergent “soluble” fraction, 6 M urea fraction marked as detergent “insoluble” fraction ( Top ). Urea fraction used for immunoprecipitation with anti-linear ubiquitin (UBQ1) or anti-K11, -K48, or -K63 ubiquitin antibodies ( Bottom ). ( I ) Tak1 −/− MEFs treated with TNFα to induce RDA and harvested as in H . Nonidet P-40 soluble lysate used for complex II immunoprecipitation with anti-FADD (M-19). ( J ) The 661W cells harvested as in H . Concentrations of reagents: TNFα, 10 ng/mL ( A – G , I , and J ), 1 ng/mL ( H ); CHX, 1–2 μg/mL; 5Z-7, 500 nM; Nec-1s, 10 μM; zVAD, 20 μM; SM-164, 500 nM. Same concentrations of these compounds were used in subsequent experiments unless noted. All data shown are mean ± SD of three or more independent experiments. ** P ≤ 0.01 and *** P ≤ 0.001 by one-way ANOVA ( A – C ) or Student’s t test ( D ).

    Article Snippet: CellTiter-Glo luminescent cell viability kit was from Promega.

    Techniques: Activation Assay, Western Blot, Immunoprecipitation, Centrifugation

    Tertiary screens identify LRRK2 and c-Cbl as specific positive regulators of RDA. ( A ) Scheme of tertiary siRNA screens for regulators of RIPK1-independent apoptosis induced by TNFα/CHX and necroptosis induced by TNFα/CHX/zVAD in 661W cells. Evaluation of hits was performed by normalizing values to nontargeting control (N.C.) and averaging three replicates. ( B ) Euler diagram of screen results. ( C – F ) The 661W cells transfected with siRNAs against LRRK2, c-Cbl, CYLD, RIPK1 (positive control), or N.C. siRNA for 72 h. ( C ) Cells treated with TNFα/5Z-7 and cell death determined by Toxilight. ( D ) Knockdown efficiency for C , E , and F determined by Western blotting of total lysates. ( E and F ) The 661W cells treated as indicated for 8 h and cell survival determined by CellTiter-Glo. ( G and H ) Matching ( G ) c-Cbl WT or KO or ( H ) Lrrk2 WT or KO MEFs treated as indicated and cell survival determined using CellTiter-Glo. Western blots show loss of protein in mutant MEFs. Concentrations of reagents: TNFα, 1 ng/mL for TNFα/5Z-7; 10 ng/mL for TNFα/CHX and TNFα/CHX/zVAD. All data shown are mean ± SD of three or more independent experiments. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 by one-way ANOVA.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of a distinct activated RIPK1 intermediate bridging complex I and complex II in TNFα-mediated apoptosis

    doi: 10.1073/pnas.1806973115

    Figure Lengend Snippet: Tertiary screens identify LRRK2 and c-Cbl as specific positive regulators of RDA. ( A ) Scheme of tertiary siRNA screens for regulators of RIPK1-independent apoptosis induced by TNFα/CHX and necroptosis induced by TNFα/CHX/zVAD in 661W cells. Evaluation of hits was performed by normalizing values to nontargeting control (N.C.) and averaging three replicates. ( B ) Euler diagram of screen results. ( C – F ) The 661W cells transfected with siRNAs against LRRK2, c-Cbl, CYLD, RIPK1 (positive control), or N.C. siRNA for 72 h. ( C ) Cells treated with TNFα/5Z-7 and cell death determined by Toxilight. ( D ) Knockdown efficiency for C , E , and F determined by Western blotting of total lysates. ( E and F ) The 661W cells treated as indicated for 8 h and cell survival determined by CellTiter-Glo. ( G and H ) Matching ( G ) c-Cbl WT or KO or ( H ) Lrrk2 WT or KO MEFs treated as indicated and cell survival determined using CellTiter-Glo. Western blots show loss of protein in mutant MEFs. Concentrations of reagents: TNFα, 1 ng/mL for TNFα/5Z-7; 10 ng/mL for TNFα/CHX and TNFα/CHX/zVAD. All data shown are mean ± SD of three or more independent experiments. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 by one-way ANOVA.

    Article Snippet: CellTiter-Glo luminescent cell viability kit was from Promega.

    Techniques: Transfection, Positive Control, Western Blot, Mutagenesis

    NEK1 restricts while APC11 promotes RIPK1 activation and iuRIPK1 formation. ( A ) The 661W cells transfected with siRNAs targeting NEK1, A20 (positive control), or nontargeting control (N.C.) siRNA for 72 h and treated with TNFα/5Z-7 for 6 h to induce RDA. Cell survival determined using CellTiter-Glo. Knockdown efficiency shown in B . ( B – F ) Cells transfected and/or treated as indicated and samples analyzed by Western blotting using indicated antibodies; pRIPK1 is RIPK1-pS166. ( B ) The 661W cells fractionated using lysis buffer with Nonidet P-40/TX-100 followed by 6 M urea. ( C ) Activated RIPK1 isolated from 661W cells treated with TNFα/5Z-7/zVAD by immunoprecipitation with RIPK1-pS166 ab. ( D – F ) The 661W cells transfected with siRNAs against APC11, CYLD (positive control) or N.C. siRNA for 72 h. ( D ) Cells treated with TNFα/5Z-7 for 8 h to induce RDA. Cell survival determined using CellTiter-Glo. ( E ) Immunoprecipitation of complex I with anti-TNFR1 from soluble lysates. ( F ) Cells fractionated using lysis buffer with Nonidet P-40/TX-100 followed by 6 M urea. Concentrations of reagents: TNFα, 1 ng/mL ( A , B , and D – F ), 10 ng/mL ( C ). All data shown are mean ± SD of three or more independent experiments. ** P ≤ 0.01 by one-way ANOVA.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of a distinct activated RIPK1 intermediate bridging complex I and complex II in TNFα-mediated apoptosis

    doi: 10.1073/pnas.1806973115

    Figure Lengend Snippet: NEK1 restricts while APC11 promotes RIPK1 activation and iuRIPK1 formation. ( A ) The 661W cells transfected with siRNAs targeting NEK1, A20 (positive control), or nontargeting control (N.C.) siRNA for 72 h and treated with TNFα/5Z-7 for 6 h to induce RDA. Cell survival determined using CellTiter-Glo. Knockdown efficiency shown in B . ( B – F ) Cells transfected and/or treated as indicated and samples analyzed by Western blotting using indicated antibodies; pRIPK1 is RIPK1-pS166. ( B ) The 661W cells fractionated using lysis buffer with Nonidet P-40/TX-100 followed by 6 M urea. ( C ) Activated RIPK1 isolated from 661W cells treated with TNFα/5Z-7/zVAD by immunoprecipitation with RIPK1-pS166 ab. ( D – F ) The 661W cells transfected with siRNAs against APC11, CYLD (positive control) or N.C. siRNA for 72 h. ( D ) Cells treated with TNFα/5Z-7 for 8 h to induce RDA. Cell survival determined using CellTiter-Glo. ( E ) Immunoprecipitation of complex I with anti-TNFR1 from soluble lysates. ( F ) Cells fractionated using lysis buffer with Nonidet P-40/TX-100 followed by 6 M urea. Concentrations of reagents: TNFα, 1 ng/mL ( A , B , and D – F ), 10 ng/mL ( C ). All data shown are mean ± SD of three or more independent experiments. ** P ≤ 0.01 by one-way ANOVA.

    Article Snippet: CellTiter-Glo luminescent cell viability kit was from Promega.

    Techniques: Activation Assay, Transfection, Positive Control, Western Blot, Lysis, Isolation, Immunoprecipitation

    Cultured primary neurons derived from ALDH2*2/*2 mice are more sensitive to ethanol-induced toxicity relative to primary neurons of WT mice. a ) LDH release a measure of cell death, was determined using LDH assay kit in primary neurons in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM); Veh – Vehicle; A – Alda-1. b ) Cellular ROS production was determined using 2,7 dichloro- fluorescein diacetate (DCFDA) in primary neurons, treated as in a; Veh – Vehicle; A – Alda-1. c ) Measurement of cellular ATP levels using CellTiter-Glo Luminescent Cell Viability kit; Veh – Vehicle; A – Alda-1. d ) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in primary neurons treated as in a; Veh – Vehicle; A – Alda-1. e ) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux in primary neuron, treated as in a; Veh – Vehicle; A – Alda-1. f ) Levels of LC3B, TOM20, p53 and phosphorylation of JNK (Thr183/Tyr185) were determined in primary neurons, treated as in a, by immunoblotting. β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/ fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Journal: Acta Neuropathologica Communications

    Article Title: Aldehyde dehydrogenase 2 activity and aldehydic load contribute to neuroinflammation and Alzheimer’s disease related pathology

    doi: 10.1186/s40478-019-0839-7

    Figure Lengend Snippet: Cultured primary neurons derived from ALDH2*2/*2 mice are more sensitive to ethanol-induced toxicity relative to primary neurons of WT mice. a ) LDH release a measure of cell death, was determined using LDH assay kit in primary neurons in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM); Veh – Vehicle; A – Alda-1. b ) Cellular ROS production was determined using 2,7 dichloro- fluorescein diacetate (DCFDA) in primary neurons, treated as in a; Veh – Vehicle; A – Alda-1. c ) Measurement of cellular ATP levels using CellTiter-Glo Luminescent Cell Viability kit; Veh – Vehicle; A – Alda-1. d ) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in primary neurons treated as in a; Veh – Vehicle; A – Alda-1. e ) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux in primary neuron, treated as in a; Veh – Vehicle; A – Alda-1. f ) Levels of LC3B, TOM20, p53 and phosphorylation of JNK (Thr183/Tyr185) were determined in primary neurons, treated as in a, by immunoblotting. β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/ fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Article Snippet: ATP measurements Relative intracellular ATP levels were determined using ATP-based CellTiter-Glo Luminescent Cell Viability kit (Promega), which causes cell lysis and generates a luminescent signal proportional to the amount of ATP present.

    Techniques: Cell Culture, Derivative Assay, Mouse Assay, Lactate Dehydrogenase Assay, Quantitation Assay, Standard Deviation

    Cultured primary astrocyte derived from ALDH2*2/*2 mice are more activated in response to ethanol relative to primary astrocytes of WT mice. a ) Measurement of mitochondrial ROS using MitoSOX™ in primary astrocytes in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. b ) Heat map representation of qPCR analysis of genes associated with inflammation, apoptosis, autophagy and mitochondrial health; Veh – Vehicle; A – Alda-1. c ) Nitrite levels were determined in primary astrocytes using Griess reagent kit in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. d ) Measurement of cellular ATP levels using CellTiter-Glo Luminescent Cell Viability kit in primary astrocytes in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. e ) Levels of cellular COX-2 and interleukin-1β release at 6 h were determined by immunoblotting in primary astrocytes in the presence or absence of Alda-1 (20 μM; 50 mM Ethanol). β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Journal: Acta Neuropathologica Communications

    Article Title: Aldehyde dehydrogenase 2 activity and aldehydic load contribute to neuroinflammation and Alzheimer’s disease related pathology

    doi: 10.1186/s40478-019-0839-7

    Figure Lengend Snippet: Cultured primary astrocyte derived from ALDH2*2/*2 mice are more activated in response to ethanol relative to primary astrocytes of WT mice. a ) Measurement of mitochondrial ROS using MitoSOX™ in primary astrocytes in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. b ) Heat map representation of qPCR analysis of genes associated with inflammation, apoptosis, autophagy and mitochondrial health; Veh – Vehicle; A – Alda-1. c ) Nitrite levels were determined in primary astrocytes using Griess reagent kit in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. d ) Measurement of cellular ATP levels using CellTiter-Glo Luminescent Cell Viability kit in primary astrocytes in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. e ) Levels of cellular COX-2 and interleukin-1β release at 6 h were determined by immunoblotting in primary astrocytes in the presence or absence of Alda-1 (20 μM; 50 mM Ethanol). β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Article Snippet: ATP measurements Relative intracellular ATP levels were determined using ATP-based CellTiter-Glo Luminescent Cell Viability kit (Promega), which causes cell lysis and generates a luminescent signal proportional to the amount of ATP present.

    Techniques: Cell Culture, Derivative Assay, Mouse Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    ALDH2*2 mutation is associated with increased oxidative stress in patient-derived fibroblasts with familial Alzheimer’s Disease (AD). a ) Genotyping of a Japanese AD patient-derived fibroblasts identifies heterozygous ALDH2*2/*1 mutation. ALDH2 protein expression in these cells was measured by immunoblotting three different culture passage numbers. b ) The levels of ALDH2 protein were determined in total lysates by immunoblotting of control (healthy subject; H)- and 2*2/*1 AD patient-derived fibroblasts. β-actin was used as loading control. c ) ALDH2 protein levels were quantified and represented as fold change of control. d ) Enzymatic activity of ALDH2 in lysates from ALDH2*2/*1 AD patient-derived fibroblasts relative to fibroblasts from control was measured over 5 min. e ) 4-HNE levels in control and AD patient-derived fibroblasts were measured using 4-HNE assay kit in the presence or absence of Alda-1 (20 μM/48 h) cultured in glucose free and galactose supplemented media. f ) Mitochondrial ROS measured by MitoSOX™ in control- and patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/36 h) cultured in glucose free and galactose supplemented media. g ) Cellular ATP levels measured using CellTiter-Glo Luminescent Cell Viability kit in control- and patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) cultured in glucose free and galactose supplemented media. h ) Mitochondrial ROS measured using MitoSOX™ in 2 control- and 2 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. Each data point represents an average of 3 independent biological replicates from each subject. i ) Cellular ATP levels measured using CellTiter-Glo Luminescent Cell Viability kit in 2 control- and 2 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. Each data point represents an average of 3 independent biological replicates from each subject. j ) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in one control- and one AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. k ) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux as in panel j. Data information: Mean, standard deviation, and p -values are shown. Results are presented as fold of control. n = 3 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Journal: Acta Neuropathologica Communications

    Article Title: Aldehyde dehydrogenase 2 activity and aldehydic load contribute to neuroinflammation and Alzheimer’s disease related pathology

    doi: 10.1186/s40478-019-0839-7

    Figure Lengend Snippet: ALDH2*2 mutation is associated with increased oxidative stress in patient-derived fibroblasts with familial Alzheimer’s Disease (AD). a ) Genotyping of a Japanese AD patient-derived fibroblasts identifies heterozygous ALDH2*2/*1 mutation. ALDH2 protein expression in these cells was measured by immunoblotting three different culture passage numbers. b ) The levels of ALDH2 protein were determined in total lysates by immunoblotting of control (healthy subject; H)- and 2*2/*1 AD patient-derived fibroblasts. β-actin was used as loading control. c ) ALDH2 protein levels were quantified and represented as fold change of control. d ) Enzymatic activity of ALDH2 in lysates from ALDH2*2/*1 AD patient-derived fibroblasts relative to fibroblasts from control was measured over 5 min. e ) 4-HNE levels in control and AD patient-derived fibroblasts were measured using 4-HNE assay kit in the presence or absence of Alda-1 (20 μM/48 h) cultured in glucose free and galactose supplemented media. f ) Mitochondrial ROS measured by MitoSOX™ in control- and patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/36 h) cultured in glucose free and galactose supplemented media. g ) Cellular ATP levels measured using CellTiter-Glo Luminescent Cell Viability kit in control- and patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) cultured in glucose free and galactose supplemented media. h ) Mitochondrial ROS measured using MitoSOX™ in 2 control- and 2 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. Each data point represents an average of 3 independent biological replicates from each subject. i ) Cellular ATP levels measured using CellTiter-Glo Luminescent Cell Viability kit in 2 control- and 2 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. Each data point represents an average of 3 independent biological replicates from each subject. j ) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in one control- and one AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. k ) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux as in panel j. Data information: Mean, standard deviation, and p -values are shown. Results are presented as fold of control. n = 3 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Article Snippet: ATP measurements Relative intracellular ATP levels were determined using ATP-based CellTiter-Glo Luminescent Cell Viability kit (Promega), which causes cell lysis and generates a luminescent signal proportional to the amount of ATP present.

    Techniques: Mutagenesis, Derivative Assay, Expressing, Activity Assay, Cell Culture, Transfection, Quantitation Assay, Standard Deviation

    Ethanol increases metabolic dysfunction of Alzheimer’s disease (AD) patient-derived fibroblasts that is rescued by ALDH2 activation. a ) Measurement of mitochondrial ROS using MitoSOX™ in 4 control (healthy subject; H)- and 4 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. b ) 4-HNE levels were measured using 4-HNE Assay Kit in control (healthy subject; H)- and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. c ) Cellular ATP levels were analyzed using CellTiter-Glo Luminescent Cell Viability kit in control (healthy subject; H)- and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/36 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. d ) Cellular ROS production was measured using 2,7 dichloro-fluorescein diacetate (DCFDA) in control (healthy subject; H) and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/fold of control. n = 3 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Journal: Acta Neuropathologica Communications

    Article Title: Aldehyde dehydrogenase 2 activity and aldehydic load contribute to neuroinflammation and Alzheimer’s disease related pathology

    doi: 10.1186/s40478-019-0839-7

    Figure Lengend Snippet: Ethanol increases metabolic dysfunction of Alzheimer’s disease (AD) patient-derived fibroblasts that is rescued by ALDH2 activation. a ) Measurement of mitochondrial ROS using MitoSOX™ in 4 control (healthy subject; H)- and 4 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. b ) 4-HNE levels were measured using 4-HNE Assay Kit in control (healthy subject; H)- and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. c ) Cellular ATP levels were analyzed using CellTiter-Glo Luminescent Cell Viability kit in control (healthy subject; H)- and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/36 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. d ) Cellular ROS production was measured using 2,7 dichloro-fluorescein diacetate (DCFDA) in control (healthy subject; H) and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/fold of control. n = 3 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Article Snippet: ATP measurements Relative intracellular ATP levels were determined using ATP-based CellTiter-Glo Luminescent Cell Viability kit (Promega), which causes cell lysis and generates a luminescent signal proportional to the amount of ATP present.

    Techniques: Derivative Assay, Activation Assay, Standard Deviation

    HGF rescues MET-amplified lung cancer cells from MET inhibition EBC-1 (A) and H1993 (B) MET-amplified lung cancer cells, and A549 cells (C) which do not have amplified MET were treated with JNJ38877605 (JNJ) (25 nM) or HGF neutralizing antibody (α-HGF Ab). Cell viability was determined after 72 h by CellTiter Glo ® . (D and E) EBC-1 and H1993 cells were treated with JNJ38877605 (25 nM), HGF (100 nM) or pro-HGF (100 nM) as indicated. Cell viability was determined by CellTiter Glo ® . (F) Representative images of EBC-1 cells treated with JNJ38877605 alone or with HGF or pro-HGF after 72 h of treatment.

    Journal: Oncotarget

    Article Title: Targeting the tumor-promoting microenvironment in MET-amplified NSCLC cells with a novel inhibitor of pro-HGF activation

    doi: 10.18632/oncotarget.18260

    Figure Lengend Snippet: HGF rescues MET-amplified lung cancer cells from MET inhibition EBC-1 (A) and H1993 (B) MET-amplified lung cancer cells, and A549 cells (C) which do not have amplified MET were treated with JNJ38877605 (JNJ) (25 nM) or HGF neutralizing antibody (α-HGF Ab). Cell viability was determined after 72 h by CellTiter Glo ® . (D and E) EBC-1 and H1993 cells were treated with JNJ38877605 (25 nM), HGF (100 nM) or pro-HGF (100 nM) as indicated. Cell viability was determined by CellTiter Glo ® . (F) Representative images of EBC-1 cells treated with JNJ38877605 alone or with HGF or pro-HGF after 72 h of treatment.

    Article Snippet: Viability assays CellTiter Glo® luminescent assay kit from Promega was used to assess cell viability.

    Techniques: Amplification, Inhibition

    Inhibition of pro-HGF activation overcomes fibroblast-mediated resistance to MET tyrosine kinase inhibition ( A) EBC-1 cells were treated with JNJ38877605 (25 nM) alone or in the presence of conditioned medium (CM) from WI38 fibroblasts (FIB). CM was also prepared from fibroblasts with silenced HGF (HGF −/− FIB), or from fibroblasts cultured with HGF neutralizing antibody (α-HGF Ab) or SRI31215 (10 μM) as indicated. Cell viability was determined by CellTiter Glo ® 72 h after treatment. (B) H1993 cells were treated with JNJ38877605 (25 nM), fibroblast CM (FIB) and SRI31215 (10 μM) as indicated and cell viability was determined after 72 h. (C) Serum-starved EBC-1 cells were treated with JNJ38877605, recombinant HGF (100 nM), FIB and SRI31215 (10 μM) as indicated for 6 hours. Cell lysates were analyzed by immunoblotting for phospho- and total MET, EGFR, Gab1, AKT and ERK 1/2. *, p

    Journal: Oncotarget

    Article Title: Targeting the tumor-promoting microenvironment in MET-amplified NSCLC cells with a novel inhibitor of pro-HGF activation

    doi: 10.18632/oncotarget.18260

    Figure Lengend Snippet: Inhibition of pro-HGF activation overcomes fibroblast-mediated resistance to MET tyrosine kinase inhibition ( A) EBC-1 cells were treated with JNJ38877605 (25 nM) alone or in the presence of conditioned medium (CM) from WI38 fibroblasts (FIB). CM was also prepared from fibroblasts with silenced HGF (HGF −/− FIB), or from fibroblasts cultured with HGF neutralizing antibody (α-HGF Ab) or SRI31215 (10 μM) as indicated. Cell viability was determined by CellTiter Glo ® 72 h after treatment. (B) H1993 cells were treated with JNJ38877605 (25 nM), fibroblast CM (FIB) and SRI31215 (10 μM) as indicated and cell viability was determined after 72 h. (C) Serum-starved EBC-1 cells were treated with JNJ38877605, recombinant HGF (100 nM), FIB and SRI31215 (10 μM) as indicated for 6 hours. Cell lysates were analyzed by immunoblotting for phospho- and total MET, EGFR, Gab1, AKT and ERK 1/2. *, p

    Article Snippet: Viability assays CellTiter Glo® luminescent assay kit from Promega was used to assess cell viability.

    Techniques: Inhibition, Activation Assay, Cell Culture, Recombinant

    Conditioned media from C5a-treated Müller cells promote human REC proliferation. Culture supernatants collected 48 hours after C5a stimulation from the above studies were added into hREC cultures (1:1 dilution), and REC proliferation was assessed 72 hours later using a CellTiter-Glo Luminescent Cell Viability Assay Kit ( A ). To verify the role of IL-6 and VEGF in the culture supernatant in promoting hREC proliferation, the experiments were repeated with IL-6 and VEGF neutralization mAbs ( B ). * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Modulation of Retinal M?ller Cells by Complement Receptor C5aR

    doi: 10.1167/iovs.13-12428

    Figure Lengend Snippet: Conditioned media from C5a-treated Müller cells promote human REC proliferation. Culture supernatants collected 48 hours after C5a stimulation from the above studies were added into hREC cultures (1:1 dilution), and REC proliferation was assessed 72 hours later using a CellTiter-Glo Luminescent Cell Viability Assay Kit ( A ). To verify the role of IL-6 and VEGF in the culture supernatant in promoting hREC proliferation, the experiments were repeated with IL-6 and VEGF neutralization mAbs ( B ). * P

    Article Snippet: Cell proliferation was determined using the CellTiter-Glo Luminescent Cell Assay kit (Promega, Madison, WI) following the manufacturer's protocol, and normalized against the controls.

    Techniques: Cell Viability Assay, Neutralization

    Effect of CR8#13 in latently infected cells and its effect in PBMCs A) CYC202, CR8 and CR8#13 were used to treat activated OM10.1 cells. Cells were grown to mid-log phase of growth and treated with TNF-α for 2 hours to induce HIV-1 expression. TNF-α was then removed and cells were subsequently treated with drugs at 0.001, 0.01, 0.1, 1, 10 and 100 µM in complete media. Five days later, sups were collected and processed for p24 HIV-1 Gag ELISA. B) At day five cells were processed for MTT assay. Analysis was carried out with Prizm software using non-liner regression curve-fit of sigmoidal dose-response with variable slope. C D) PHA-activated PBMC (5 × 10 6 ) were infected with THA/92/00 strain (MOI: 0.1). After 8 hours, unadsorbed virus was washed away and cells were treated with 10 or 100 nM of either Flavopiridol or CR8#13. Supernatants were collected every 3 days for RT analysis. Number of live cells was also plotted from each date and treatment (trypan blue exclusion assay). E F) Similar to panel C, where PHA-activated PBMC cells (Lonza) were infected with the THA/92/00 strain and treated with various concentrations of Flavopiridol or CR8#13 cells. Samples were assayed using CellTiter-Glo after either 3 or 16 days.

    Journal: Virology

    Article Title: Use of ATP analogs to inhibit HIV-1 transcription

    doi: 10.1016/j.virol.2012.06.007

    Figure Lengend Snippet: Effect of CR8#13 in latently infected cells and its effect in PBMCs A) CYC202, CR8 and CR8#13 were used to treat activated OM10.1 cells. Cells were grown to mid-log phase of growth and treated with TNF-α for 2 hours to induce HIV-1 expression. TNF-α was then removed and cells were subsequently treated with drugs at 0.001, 0.01, 0.1, 1, 10 and 100 µM in complete media. Five days later, sups were collected and processed for p24 HIV-1 Gag ELISA. B) At day five cells were processed for MTT assay. Analysis was carried out with Prizm software using non-liner regression curve-fit of sigmoidal dose-response with variable slope. C D) PHA-activated PBMC (5 × 10 6 ) were infected with THA/92/00 strain (MOI: 0.1). After 8 hours, unadsorbed virus was washed away and cells were treated with 10 or 100 nM of either Flavopiridol or CR8#13. Supernatants were collected every 3 days for RT analysis. Number of live cells was also plotted from each date and treatment (trypan blue exclusion assay). E F) Similar to panel C, where PHA-activated PBMC cells (Lonza) were infected with the THA/92/00 strain and treated with various concentrations of Flavopiridol or CR8#13 cells. Samples were assayed using CellTiter-Glo after either 3 or 16 days.

    Article Snippet: Cell viability was measured using CellTiter-Glo Cell Luminescence Viability kit (Promega) as per manufacturer’s instructions.

    Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay, MTT Assay, Software, Trypan Blue Exclusion Assay

    Effect of CR8#13 on cdk9 and Pol II occupancy on the HIV-1 genome A) Inhibition of loading of cdk9 and Pol II onto the HIV-1 genome when using CR8#13 or CR8#1 (negative control). Following induction of OM10.1, cells were treated with 50 nM of either drug. Forty eight hours later samples were collected and treated for ChIP analysis and PCR using LTR and Env primers. Percent of live cells is included from treatment prior to ChIP assay using any of the two antibodies. Live cell numbers and percentages were calculated based on a trypan blue exclusion assay. B) ChIP samples were diluted 1:2 and run on an agarose gel (2%) at three concentrations. Specific band intensities were counted using a phosphorimager (Molecular Dynamics software) and plotted for both LTR and Env products. C) Induced OM10.1 samples were treated with CR8#1 or CR8#13 (similar to Panel A) and carried out to 48 hours. Samples were processed for viability using CellTiter-Glo assay. D) OM10.1 activated cells were either untreated or treated with DRB (100 µM) or CR8#13 (50 nM) concentrations for 48 hours (lanes 2 and 4). The cytosolic extracts were prepared and the nuclei were separated using centrifugation. Samples were run on a 4–20% gel and western blotting was performed with 1/5 of the samples and the fraction of cdk9 in the cytosolic extracts (CE) and nuclear pellets (NP) were determined. The top panel of DRB treated cells was used for western blot with α-cdk9 antibody. The CR8#13 treated cells were further processed for the presence of cdk9, cyclin T1, cdk7 and cyclin H.

    Journal: Virology

    Article Title: Use of ATP analogs to inhibit HIV-1 transcription

    doi: 10.1016/j.virol.2012.06.007

    Figure Lengend Snippet: Effect of CR8#13 on cdk9 and Pol II occupancy on the HIV-1 genome A) Inhibition of loading of cdk9 and Pol II onto the HIV-1 genome when using CR8#13 or CR8#1 (negative control). Following induction of OM10.1, cells were treated with 50 nM of either drug. Forty eight hours later samples were collected and treated for ChIP analysis and PCR using LTR and Env primers. Percent of live cells is included from treatment prior to ChIP assay using any of the two antibodies. Live cell numbers and percentages were calculated based on a trypan blue exclusion assay. B) ChIP samples were diluted 1:2 and run on an agarose gel (2%) at three concentrations. Specific band intensities were counted using a phosphorimager (Molecular Dynamics software) and plotted for both LTR and Env products. C) Induced OM10.1 samples were treated with CR8#1 or CR8#13 (similar to Panel A) and carried out to 48 hours. Samples were processed for viability using CellTiter-Glo assay. D) OM10.1 activated cells were either untreated or treated with DRB (100 µM) or CR8#13 (50 nM) concentrations for 48 hours (lanes 2 and 4). The cytosolic extracts were prepared and the nuclei were separated using centrifugation. Samples were run on a 4–20% gel and western blotting was performed with 1/5 of the samples and the fraction of cdk9 in the cytosolic extracts (CE) and nuclear pellets (NP) were determined. The top panel of DRB treated cells was used for western blot with α-cdk9 antibody. The CR8#13 treated cells were further processed for the presence of cdk9, cyclin T1, cdk7 and cyclin H.

    Article Snippet: Cell viability was measured using CellTiter-Glo Cell Luminescence Viability kit (Promega) as per manufacturer’s instructions.

    Techniques: Inhibition, Negative Control, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Trypan Blue Exclusion Assay, Agarose Gel Electrophoresis, Software, Glo Assay, Centrifugation, Western Blot

    Anti-proliferative activity of TA in CRC cell lines HCT116 ( A ) and HT29 ( B ) cells were treated with DMSO (Control) or increasing concentrations of TA (25-100 μM) for 24-72 h. Cell viability was determined using CellTiter-Glo cell viability assay and results are presented as % viable cells (relative to control). Values represent mean ± SEM ( n = 3) and the bars with ‘*’ are significantly different from control ( p

    Journal: Oncotarget

    Article Title: Combination of Tolfenamic acid and curcumin induces colon cancer cell growth inhibition through modulating specific transcription factors and reactive oxygen species

    doi: 10.18632/oncotarget.6553

    Figure Lengend Snippet: Anti-proliferative activity of TA in CRC cell lines HCT116 ( A ) and HT29 ( B ) cells were treated with DMSO (Control) or increasing concentrations of TA (25-100 μM) for 24-72 h. Cell viability was determined using CellTiter-Glo cell viability assay and results are presented as % viable cells (relative to control). Values represent mean ± SEM ( n = 3) and the bars with ‘*’ are significantly different from control ( p

    Article Snippet: Cell proliferation assay The effect of TA and Cur on cell proliferation was determined by measuring cellular ATP levels as an indication of viable cells using the CellTiter-Glo (luminescent cell viability assay kit from Promega, Madison, WI).

    Techniques: Activity Assay, Viability Assay

    Effects of anosmin-1 in tumor cell proliferation. (A) LN229 cells were treated with anosmin-1 for 6 h with BrdU labeling during the last 2 h in serum-free conditions. In parallel experiments, cells were pre-treated with SU5402 or amiloride for 30 min before stimulation with anosmin-1. Anosmin-1-treated cells (denoted as A) show increased BrdU incorporation (** P =0.0035) compared with the untreated or solvent (DMSO)-treated cells. Pretreatment with SU5402 or amiloride, however, attenuated anosmin-1-induced proliferation (** P =0.0046 and * P =0.0423 respectively). At least five random fields were analysed, which were repeated four times. (B) shRNA-mediated knockdown of KAL1 results in reduced BrdU incorporation in A172. The P values obtained from four independent experiments are 0.0319 (for shRNA673), 0.019 (for shRNA675), and 0.05 (for shRNA676) when compared with the control shRNA. (C) LN229 cells treated with anosmin-1 indicate significantly higher proliferation in CellTitre-Glo luminescent assay, compared with the untreated control at 24 h (* P =0.0103) and 48 h (** P =0.0048) by a two-way ANOVA test. Error bars indicate s.e.m . from five independent experiments.

    Journal: Endocrine-Related Cancer

    Article Title: Anosmin-1 contributes to brain tumor malignancy through integrin signal pathways

    doi: 10.1530/ERC-13-0181

    Figure Lengend Snippet: Effects of anosmin-1 in tumor cell proliferation. (A) LN229 cells were treated with anosmin-1 for 6 h with BrdU labeling during the last 2 h in serum-free conditions. In parallel experiments, cells were pre-treated with SU5402 or amiloride for 30 min before stimulation with anosmin-1. Anosmin-1-treated cells (denoted as A) show increased BrdU incorporation (** P =0.0035) compared with the untreated or solvent (DMSO)-treated cells. Pretreatment with SU5402 or amiloride, however, attenuated anosmin-1-induced proliferation (** P =0.0046 and * P =0.0423 respectively). At least five random fields were analysed, which were repeated four times. (B) shRNA-mediated knockdown of KAL1 results in reduced BrdU incorporation in A172. The P values obtained from four independent experiments are 0.0319 (for shRNA673), 0.019 (for shRNA675), and 0.05 (for shRNA676) when compared with the control shRNA. (C) LN229 cells treated with anosmin-1 indicate significantly higher proliferation in CellTitre-Glo luminescent assay, compared with the untreated control at 24 h (* P =0.0103) and 48 h (** P =0.0048) by a two-way ANOVA test. Error bars indicate s.e.m . from five independent experiments.

    Article Snippet: Cell proliferation assay Serum-starved cells were treated with 10 nM recombinant anosmin-1 and the number of viable cells was assessed using CellTitre-Glo Luminescent Cell Viability kit (Promega) following the manufacturer's protocol.

    Techniques: Labeling, BrdU Incorporation Assay, shRNA, Luminescence Assay

    Functional interaction of vFLIP and MAVS is essential for survival of HHV-8-infected PEL cells. (A-B) Co-IP assay using extracts from 293T cells transfected with expression vectors for Flag-MAVS and (A) GST, GST-vFLIP, GST-DED1, or GST-DED2 and (B) GST and GST-vFLIP helices. (C) Immunoblots of extracts from 293T cells transfected with V5-vFLIP and Flag-MAVS together with GST-vFLIP helices. (D) Immunoblots of extracts from BCBL-1 cells treated with 10 μM TAT and TAT-vFLIP DED2-H1 (2H1) peptides twice for 4 days. Asterisk indicates a band of unknown identity. (E) FITC-Annexin V and 7-AAD-based flow cytometric analyses of WT and MAVS KO BCBL-1 cell lines treated with TAT and TAT-2H1 for 2 days. The cells were seeded at low-density (5 x10 4 cells/ml). The percentage of total cells in each quadrant is shown. (F) CellTiter-Glo cell viability assays with the indicated cells treated with TAT and TAT-2H1 for 4 days. Data are represented as mean ± SD of two independent experiments in triplicate. (*p

    Journal: PLoS Pathogens

    Article Title: Peroxisomes support human herpesvirus 8 latency by stabilizing the viral oncogenic protein vFLIP via the MAVS-TRAF complex

    doi: 10.1371/journal.ppat.1007058

    Figure Lengend Snippet: Functional interaction of vFLIP and MAVS is essential for survival of HHV-8-infected PEL cells. (A-B) Co-IP assay using extracts from 293T cells transfected with expression vectors for Flag-MAVS and (A) GST, GST-vFLIP, GST-DED1, or GST-DED2 and (B) GST and GST-vFLIP helices. (C) Immunoblots of extracts from 293T cells transfected with V5-vFLIP and Flag-MAVS together with GST-vFLIP helices. (D) Immunoblots of extracts from BCBL-1 cells treated with 10 μM TAT and TAT-vFLIP DED2-H1 (2H1) peptides twice for 4 days. Asterisk indicates a band of unknown identity. (E) FITC-Annexin V and 7-AAD-based flow cytometric analyses of WT and MAVS KO BCBL-1 cell lines treated with TAT and TAT-2H1 for 2 days. The cells were seeded at low-density (5 x10 4 cells/ml). The percentage of total cells in each quadrant is shown. (F) CellTiter-Glo cell viability assays with the indicated cells treated with TAT and TAT-2H1 for 4 days. Data are represented as mean ± SD of two independent experiments in triplicate. (*p

    Article Snippet: For cell viability assays, the CellTiter-Glo® Luminescence kit (Promega) was used.

    Techniques: Functional Assay, Infection, Co-Immunoprecipitation Assay, Transfection, Expressing, Western Blot, Flow Cytometry

    Increased death of HHV-8-infected MAVS-deficient cells. (A-B) Growth curve of wild-type (WT) and MAVS knockout (KO) BCBL-1 cell lines (A) and BJAB cells infected or uninfected with HHV-8 BAC16 virus (B). Cell viability was assessed by determining ATP levels using CellTiter-Glo for 4 days. MAVS and GFP expression were determined on day 0 by immunoblot as shown next to each graph. Data are presented as mean ± SD of triplicate samples. Probability (p) values at day 4 are depicted; * p

    Journal: PLoS Pathogens

    Article Title: Peroxisomes support human herpesvirus 8 latency by stabilizing the viral oncogenic protein vFLIP via the MAVS-TRAF complex

    doi: 10.1371/journal.ppat.1007058

    Figure Lengend Snippet: Increased death of HHV-8-infected MAVS-deficient cells. (A-B) Growth curve of wild-type (WT) and MAVS knockout (KO) BCBL-1 cell lines (A) and BJAB cells infected or uninfected with HHV-8 BAC16 virus (B). Cell viability was assessed by determining ATP levels using CellTiter-Glo for 4 days. MAVS and GFP expression were determined on day 0 by immunoblot as shown next to each graph. Data are presented as mean ± SD of triplicate samples. Probability (p) values at day 4 are depicted; * p

    Article Snippet: For cell viability assays, the CellTiter-Glo® Luminescence kit (Promega) was used.

    Techniques: Infection, Knock-Out, Expressing

    Optimization of nucleofection conditions. A. Percentage of GFP-positive cells. One million DF19 cells were transfected with 2 µg of the pmaxGFP plasmid using the Nucleofector 2b device and different Nucleofection solutions and programs. Two days post transfection, the percentage of GFP positive cells was determined using FACS. B. FACS profile of cells nucleofected using Solution I and program B016. The gray line represents transfected cells and the black graph untransfected cells. C. Cell viability. Cell viability was determined using Promega's CellTiter-Glo kit. The viability of control cells that were not nucleofected but otherwise treated in the same way was taken as 100%.

    Journal: PLoS ONE

    Article Title: Targeting of Herpes Simplex Virus 1 Thymidine Kinase Gene Sequences into the OCT4 Locus of Human Induced Pluripotent Stem Cells

    doi: 10.1371/journal.pone.0081131

    Figure Lengend Snippet: Optimization of nucleofection conditions. A. Percentage of GFP-positive cells. One million DF19 cells were transfected with 2 µg of the pmaxGFP plasmid using the Nucleofector 2b device and different Nucleofection solutions and programs. Two days post transfection, the percentage of GFP positive cells was determined using FACS. B. FACS profile of cells nucleofected using Solution I and program B016. The gray line represents transfected cells and the black graph untransfected cells. C. Cell viability. Cell viability was determined using Promega's CellTiter-Glo kit. The viability of control cells that were not nucleofected but otherwise treated in the same way was taken as 100%.

    Article Snippet: One of the cell aliquots was analyzed by FACS to determine the percentage of GFP positive cells; and the other aliquot was analyzed for cell viability using the CellTiter-Glo luminescence assay kit (Promega, Madison, WI).

    Techniques: Transfection, Plasmid Preparation, FACS

    Depletion of c-Myc inhibits growth of glioma cancer stem cells. (A) T3359, (B) T3832, and (C) T4597 CD133− and CD133+ cells were isolated and infected as described. Cells were then plated in 96-well plates in triplicate at 5000 cells per well for CD133− cells or 1000 cells per well for CD133+ cells. Total viable cell numbers were then determined by the CellTiter-Glo Luminescent Cell Viability Assay (Promega) daily. *, p

    Journal: PLoS ONE

    Article Title: c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells

    doi: 10.1371/journal.pone.0003769

    Figure Lengend Snippet: Depletion of c-Myc inhibits growth of glioma cancer stem cells. (A) T3359, (B) T3832, and (C) T4597 CD133− and CD133+ cells were isolated and infected as described. Cells were then plated in 96-well plates in triplicate at 5000 cells per well for CD133− cells or 1000 cells per well for CD133+ cells. Total viable cell numbers were then determined by the CellTiter-Glo Luminescent Cell Viability Assay (Promega) daily. *, p

    Article Snippet: Cell number was measured for 5 consecutive days using the CellTiter-Glo assay kit (Promega, Madison, MI).

    Techniques: Isolation, Infection, Cell Viability Assay

    Depletion of c-Myc induces apoptosis in glioma cancer stem cells. (A, B) T3359 and (C, D) T4597 CD133− and CD133+ cells were isolated and infected as described. (A, C) Six days after infection, cells were trypsinized and quantified for apoptosis using the Annexin V-FITC Apoptosis Detection kit (Calbiochem). The percentage of FITC positive cells was determined by FACS analysis, and dead cells were excluded by propidium iodide staining. (B, D) These cells were also plated in 96-well plates at 5000 cells per well for CD133− cells and 1000 cells per well for CD133+ cells after infection and selection. Six days after infection, combined activities of caspase 3 and caspase 7 were determined by the Caspase 3/7 Luminescence Assay kit (Promega), and were normalized by the viable cell numbers determined by the CellTiter-Glo assays as described in Figure 4 . *, p

    Journal: PLoS ONE

    Article Title: c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells

    doi: 10.1371/journal.pone.0003769

    Figure Lengend Snippet: Depletion of c-Myc induces apoptosis in glioma cancer stem cells. (A, B) T3359 and (C, D) T4597 CD133− and CD133+ cells were isolated and infected as described. (A, C) Six days after infection, cells were trypsinized and quantified for apoptosis using the Annexin V-FITC Apoptosis Detection kit (Calbiochem). The percentage of FITC positive cells was determined by FACS analysis, and dead cells were excluded by propidium iodide staining. (B, D) These cells were also plated in 96-well plates at 5000 cells per well for CD133− cells and 1000 cells per well for CD133+ cells after infection and selection. Six days after infection, combined activities of caspase 3 and caspase 7 were determined by the Caspase 3/7 Luminescence Assay kit (Promega), and were normalized by the viable cell numbers determined by the CellTiter-Glo assays as described in Figure 4 . *, p

    Article Snippet: Cell number was measured for 5 consecutive days using the CellTiter-Glo assay kit (Promega, Madison, MI).

    Techniques: Isolation, Infection, FACS, Staining, Selection, Luminescence Assay

    Effects of MMO on the cell viability of NAFLD model cells. The NAFLD model cells were treated with MMO (10 and 20 mg/mL) for 24 h, and cell viability was determined by CellTiter-Glo assay. Data are presented as mean ± S.D. for three independent experiments with triplicate determination. * p

    Journal: Marine Drugs

    Article Title: Protective Effects and Mechanism of Meretrix meretrix Oligopeptides against Nonalcoholic Fatty Liver Disease

    doi: 10.3390/md15020031

    Figure Lengend Snippet: Effects of MMO on the cell viability of NAFLD model cells. The NAFLD model cells were treated with MMO (10 and 20 mg/mL) for 24 h, and cell viability was determined by CellTiter-Glo assay. Data are presented as mean ± S.D. for three independent experiments with triplicate determination. * p

    Article Snippet: Cell Viability Assay The number of viable cells in culture based on the quantitation of the ATP present produced by metabolically active cells were determined using the CellTiter-Glo (CTG) luminescent assay kit (Promega, Fitchburg, WI, USA), as per the manufacturer’s instructions.

    Techniques: Glo Assay

    IFNα-2a increases packaging of HIV-1 viral RNA into EVs. U1 cells were treated with a titration of IFNα-2a (0.5 K, 1 K, 5 K, 10 K, 50 K, and 100 K units) for 5 days. ( a ) EVs were enriched using NT80/82 particles and analyzed for the presence of TAR, TAR- gag , and genomic RNA using RT-qPCR (see Methods). ( b ) The cells responsible for the production of EVs analyzed in panel a were analyzed for the levels of intracellular viral RNA using RT-qPCR. ( c ) AChE activity was measured (relative fluorescent units; RFU) in isolated EVs released from U1 cells that were treated with IFNα-2a and U1 controls. ( d ) U1 cells were treated with a titration of IFNα-2a (0.5K-100K units) for 5 days. Whole cell extracts were analyzed by Western blot for ESCRT pathway proteins [ESCRT-I (TSG101),-II (EAP20, EAP45),-III (CHMP6), and exit (VPS4)] and Actin protein levels. Selected lanes were taken from the same blot with identical exposure settings presented in the figure. Densitometry counts as determined by ImageJ software is shown as increase or decrease of VPS4 or TSG101 relative to the untreated control (set to 100%). (e ) IFNα-2a-treated cells were assessed for cell viability using CellTiter-Glo reagent. ( f–i) U1 cells were treated with cART (45 µM) or IFNα-2a (5 K or 10 K) for 5 days. EVs were enriched from supernatants using immunoprecipitation with EV marker antibodies (CD81, CD9, CD63) or NT80/82 particles. Resulting samples were analyzed by RT-qPCR for TAR, TAR- gag , and genomic RNA (see Methods). Red boxes highlight clinically relevant IFN concentrations. Error bars represent ± S.D. of three technical replicates for all panels. A two-tailed Student’s t- test was used to assess significance: * p

    Journal: Scientific Reports

    Article Title: Antiretroviral Drugs Alter the Content of Extracellular Vesicles from HIV-1-Infected Cells

    doi: 10.1038/s41598-018-25943-2

    Figure Lengend Snippet: IFNα-2a increases packaging of HIV-1 viral RNA into EVs. U1 cells were treated with a titration of IFNα-2a (0.5 K, 1 K, 5 K, 10 K, 50 K, and 100 K units) for 5 days. ( a ) EVs were enriched using NT80/82 particles and analyzed for the presence of TAR, TAR- gag , and genomic RNA using RT-qPCR (see Methods). ( b ) The cells responsible for the production of EVs analyzed in panel a were analyzed for the levels of intracellular viral RNA using RT-qPCR. ( c ) AChE activity was measured (relative fluorescent units; RFU) in isolated EVs released from U1 cells that were treated with IFNα-2a and U1 controls. ( d ) U1 cells were treated with a titration of IFNα-2a (0.5K-100K units) for 5 days. Whole cell extracts were analyzed by Western blot for ESCRT pathway proteins [ESCRT-I (TSG101),-II (EAP20, EAP45),-III (CHMP6), and exit (VPS4)] and Actin protein levels. Selected lanes were taken from the same blot with identical exposure settings presented in the figure. Densitometry counts as determined by ImageJ software is shown as increase or decrease of VPS4 or TSG101 relative to the untreated control (set to 100%). (e ) IFNα-2a-treated cells were assessed for cell viability using CellTiter-Glo reagent. ( f–i) U1 cells were treated with cART (45 µM) or IFNα-2a (5 K or 10 K) for 5 days. EVs were enriched from supernatants using immunoprecipitation with EV marker antibodies (CD81, CD9, CD63) or NT80/82 particles. Resulting samples were analyzed by RT-qPCR for TAR, TAR- gag , and genomic RNA (see Methods). Red boxes highlight clinically relevant IFN concentrations. Error bars represent ± S.D. of three technical replicates for all panels. A two-tailed Student’s t- test was used to assess significance: * p

    Article Snippet: Cells were incubated for 5 days and assessed for cell viability using CellTiter-Glo reagent Luminescence Viability kit (Promega) according to the manufacturer’s instructions.

    Techniques: Titration, Quantitative RT-PCR, Activity Assay, Isolation, Western Blot, Software, Immunoprecipitation, Marker, Two Tailed Test

    Oxytetracycline lowers the levels of EV-associated, short, non-coding, HIV-1 RNAs. Some U1 cells were pre-treated with cART (45 µM) for 3 days followed by an additional dose of cART, Oxytetracyline (0.1, 1, 10 nM), or a combination of both drugs for 5 days. EVs were enriched from supernatants using NT80/82 with overnight incubation at 4 °C. ( a–b ) EV-associated RNA was isolated and quantified using RT-qPCR with primers specific for HIV-1 TAR, TAR- gag , and genomic RNA (See Methods). Percent decreases of viral RNAs compared to untreated controls are indicated. ( c ) AChE activity was measured (relative fluorescent units; RFU) from EVs isolated as in panels a–b. (d ) Treated cells were assessed for cell viability using CellTiter-Glo reagent. Error bars represent ± S.D. of three technical replicates for all panels. A two-tailed Student’s t- test was used to assess significance: * p

    Journal: Scientific Reports

    Article Title: Antiretroviral Drugs Alter the Content of Extracellular Vesicles from HIV-1-Infected Cells

    doi: 10.1038/s41598-018-25943-2

    Figure Lengend Snippet: Oxytetracycline lowers the levels of EV-associated, short, non-coding, HIV-1 RNAs. Some U1 cells were pre-treated with cART (45 µM) for 3 days followed by an additional dose of cART, Oxytetracyline (0.1, 1, 10 nM), or a combination of both drugs for 5 days. EVs were enriched from supernatants using NT80/82 with overnight incubation at 4 °C. ( a–b ) EV-associated RNA was isolated and quantified using RT-qPCR with primers specific for HIV-1 TAR, TAR- gag , and genomic RNA (See Methods). Percent decreases of viral RNAs compared to untreated controls are indicated. ( c ) AChE activity was measured (relative fluorescent units; RFU) from EVs isolated as in panels a–b. (d ) Treated cells were assessed for cell viability using CellTiter-Glo reagent. Error bars represent ± S.D. of three technical replicates for all panels. A two-tailed Student’s t- test was used to assess significance: * p

    Article Snippet: Cells were incubated for 5 days and assessed for cell viability using CellTiter-Glo reagent Luminescence Viability kit (Promega) according to the manufacturer’s instructions.

    Techniques: Incubation, Isolation, Quantitative RT-PCR, Activity Assay, Two Tailed Test

    The tetracycline family exhibit different efficacies in the presence and absence of cART. ( a ) HIV-1-infected U1 cells were treated with tetracycline class antibiotics (10 nM) for 5 days, followed by EV enrichment with NT80/82 particles and RT-qPCR analysis using primers specific for HIV-1 TAR, TAR- gag , and genomic RNA (See Methods). ( b ) U1 cells were treated (as described in 6a) and assessed for cell viability using CellTiter-Glo reagent. (RLU = Relative Luminescent Units) ( c ) U1 cells were pretreated with cART (45 µM) for 3 days followed by an additional dose of cART and tetracycline antibiotics (10 nM) for 5 days. EVs were enriched using NT80/82 particles and analyzed via RT-qPCR as in panel a. ( d ) Drug-treated cells (as described in panel c) were assessed for cell viability using CellTiter-Glo reagent. Error bars represent ± S.D. of three technical replicates for all panels. A two-tailed Student’s t- test was used to assess significance: * p

    Journal: Scientific Reports

    Article Title: Antiretroviral Drugs Alter the Content of Extracellular Vesicles from HIV-1-Infected Cells

    doi: 10.1038/s41598-018-25943-2

    Figure Lengend Snippet: The tetracycline family exhibit different efficacies in the presence and absence of cART. ( a ) HIV-1-infected U1 cells were treated with tetracycline class antibiotics (10 nM) for 5 days, followed by EV enrichment with NT80/82 particles and RT-qPCR analysis using primers specific for HIV-1 TAR, TAR- gag , and genomic RNA (See Methods). ( b ) U1 cells were treated (as described in 6a) and assessed for cell viability using CellTiter-Glo reagent. (RLU = Relative Luminescent Units) ( c ) U1 cells were pretreated with cART (45 µM) for 3 days followed by an additional dose of cART and tetracycline antibiotics (10 nM) for 5 days. EVs were enriched using NT80/82 particles and analyzed via RT-qPCR as in panel a. ( d ) Drug-treated cells (as described in panel c) were assessed for cell viability using CellTiter-Glo reagent. Error bars represent ± S.D. of three technical replicates for all panels. A two-tailed Student’s t- test was used to assess significance: * p

    Article Snippet: Cells were incubated for 5 days and assessed for cell viability using CellTiter-Glo reagent Luminescence Viability kit (Promega) according to the manufacturer’s instructions.

    Techniques: Infection, Quantitative RT-PCR, Two Tailed Test

    Intracellular ATP levels in wild-type and CatE −/− macrophages. Macrophages (1 × 10 5 cells) were cultured on 96-well plates at 37°C for 24 h. After replacement with fresh media, the culture plate was incubated at room temperature (25°C) for 30 min. ATP levels were determined by the manufacturer’s protocol of the CellTiter-Glo assay kit according to the manufacturer’s protocol. The concentration of intracellular ATP was determined by the titration of the control medium without cells plus 0.1−1.0 µM ATP.

    Journal: PLoS ONE

    Article Title: Cathepsin E Deficiency Impairs Autophagic Proteolysis in Macrophages

    doi: 10.1371/journal.pone.0082415

    Figure Lengend Snippet: Intracellular ATP levels in wild-type and CatE −/− macrophages. Macrophages (1 × 10 5 cells) were cultured on 96-well plates at 37°C for 24 h. After replacement with fresh media, the culture plate was incubated at room temperature (25°C) for 30 min. ATP levels were determined by the manufacturer’s protocol of the CellTiter-Glo assay kit according to the manufacturer’s protocol. The concentration of intracellular ATP was determined by the titration of the control medium without cells plus 0.1−1.0 µM ATP.

    Article Snippet: The ATP assay kit (CellTiter-Gio Luminescent cell viability assay) was purchased from Promega (Madison, WI, USA).

    Techniques: Cell Culture, Incubation, Glo Assay, Concentration Assay, Titration

    BNTX sensitizes human pancreatic cancer cells to TRAIL-induced apoptosis (A) Assay data for a screening analysis of AsPC-1 cells pre-incubated with various test compounds (5 μM) and were then exposed to TRAIL (50 ng/ml). Cell survival was measured as a percentage of the control sample. The arrow indicates BNTX (5 μM). (B) AsPC-1 cells were exposed to various concentrations of BNTX with or without TRAIL (25 ng/ml) for 24 h. Cell viability was measured by a luminescent CellTiter-Glo assay, and relative cell survival was determined by assaying ATP levels. (C) Relative survival of three pancreatic cancer cell lines (AsPC-1, PANC-1, and MIA PaCa-2) exposed to TRAIL (25 ng/ml) with or without BNTX (2.5 μM) for 24 h. Cell viability was measured by a luminescent CellTiter-Glo assay. (D) Apoptotic activity in AsPC-1 cells exposed to TRAIL (25 ng/ml) with or without BNTX (2.5 μM) for 24 h. Cells were analyzed by annexin V/PI double-stained flow cytometry. (E) The percentage of apoptotic cells. Values are the mean ± SD from two independent experiments performed in duplicate. Asterisks indicate significant differences compared with the control ( *** P

    Journal: Oncotarget

    Article Title: Downregulation of X-linked inhibitor of apoptosis protein by ‘7-Benzylidenenaltrexone maleate’ sensitizes pancreatic cancer cells to TRAIL-induced apoptosis

    doi: 10.18632/oncotarget.17841

    Figure Lengend Snippet: BNTX sensitizes human pancreatic cancer cells to TRAIL-induced apoptosis (A) Assay data for a screening analysis of AsPC-1 cells pre-incubated with various test compounds (5 μM) and were then exposed to TRAIL (50 ng/ml). Cell survival was measured as a percentage of the control sample. The arrow indicates BNTX (5 μM). (B) AsPC-1 cells were exposed to various concentrations of BNTX with or without TRAIL (25 ng/ml) for 24 h. Cell viability was measured by a luminescent CellTiter-Glo assay, and relative cell survival was determined by assaying ATP levels. (C) Relative survival of three pancreatic cancer cell lines (AsPC-1, PANC-1, and MIA PaCa-2) exposed to TRAIL (25 ng/ml) with or without BNTX (2.5 μM) for 24 h. Cell viability was measured by a luminescent CellTiter-Glo assay. (D) Apoptotic activity in AsPC-1 cells exposed to TRAIL (25 ng/ml) with or without BNTX (2.5 μM) for 24 h. Cells were analyzed by annexin V/PI double-stained flow cytometry. (E) The percentage of apoptotic cells. Values are the mean ± SD from two independent experiments performed in duplicate. Asterisks indicate significant differences compared with the control ( *** P

    Article Snippet: Cell viability assay and flow cytometry Cells were seeded at 2∼3 × 103 cells/well in 96-well plates and were pre-treated with BNTX at indicated concentrations for 0.5 h followed by TRAIL (25 ng/ml) for an additional 24 h. Cell viability assays were performed using a cellular ATP-based luminescence assay kit (CellTiter-Glo; Promega, Madison, WI).

    Techniques: Incubation, Glo Assay, Activity Assay, Staining, Flow Cytometry, Cytometry

    Graphical comparisons of changes in caspase-3/-7 activity of normal T cells after 24 (A) and 48 (B) hours incubation with Sutherlandia frutescens extract doses (SFE and SFW). The untreated control was again used as a reference (100%) for all the samples. SFE and SFW extract doses showed a concentration- and time-dependent decrease in decreasing caspase-3/-7 activity, with high doses being more effective. High doses of the SFE extract were more potent in reducing caspase-3/-7 activity over this period and their effect was independent of ethanol ( p

    Journal: African Journal of Traditional, Complementary, and Alternative Medicines

    Article Title: Effects of Sutherlandia Frutescens Extracts on Normal T-Lymphocytes In Vitro

    doi:

    Figure Lengend Snippet: Graphical comparisons of changes in caspase-3/-7 activity of normal T cells after 24 (A) and 48 (B) hours incubation with Sutherlandia frutescens extract doses (SFE and SFW). The untreated control was again used as a reference (100%) for all the samples. SFE and SFW extract doses showed a concentration- and time-dependent decrease in decreasing caspase-3/-7 activity, with high doses being more effective. High doses of the SFE extract were more potent in reducing caspase-3/-7 activity over this period and their effect was independent of ethanol ( p

    Article Snippet: Promega CellTiter-Glo™ Luminescent Cell Viability and Promega Caspase 3/7™ assay kits were products of Promega (SA).

    Techniques: Activity Assay, Incubation, Concentration Assay