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  • 99
    Thermo Fisher alamarblue cell viability reagent
    Analysis of cell activities by <t>alamarBlue</t> assay. Each data point is the mean ± SD of 6 samples. ** p
    Alamarblue Cell Viability Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1938 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alamarblue cell viability reagent/product/Thermo Fisher
    Average 99 stars, based on 1938 article reviews
    Price from $9.99 to $1999.99
    alamarblue cell viability reagent - by Bioz Stars, 2020-10
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    99
    Thermo Fisher alamarblue
    The tc aptamer controlled CD20 expression. ( A ) Schematic representation of the suicide system. When treated with the therapeutic antibody rituximab (RTX), cell death was induced in cells expressing CD20. As a positive control, CD20 was stably integrated into HeLa HF1–3 cells (cell line HF1–3CD20). The aptamer-controlled exon 2 along with the neighboring introns from construct L2 was inserted into the CD20 coding region, which resulted in the cell line HF1–3C2. In the presence of tc, exon 2 was skipped, the coding sequence of CD20 was restored, and apoptosis was triggered. Without tc, exon 2 was retained, which destroyed the CD20 reading frame and resulted in cell survival. ( B, C ) Cell lines were incubated with and without 50 μM tc for 2 days. Afterwards, RT-PCR and a western blot were performed. Primers used for RT-PCR bind to the coding sequence of CD20, which spans the alternatively spliced exon 2. HSP60 was used as a loading control for western blot. ( D ) Viability assay. The cell lines were incubated with and without RTX and 50 μM tc or doxycycline (dox) for 3 days. Afterwards, cell viability was analyzed by an <t>AlamarBlue</t> ® .
    Alamarblue, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3924 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alamarblue/product/Thermo Fisher
    Average 99 stars, based on 3924 article reviews
    Price from $9.99 to $1999.99
    alamarblue - by Bioz Stars, 2020-10
    99/100 stars
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    93
    Bio-Rad alamarblue reagent
    Domain swapping analysis of ZNT2 and ZNT3 on the ZNT3 backbone. ( A ) Expression of ZNT3 (ZNT2Nter) , ZNT3 (ZNT2Loop) , or ZNT3 (ZNT2Cter) in znt1 −/− mt −/− znt4 −/− cells did not result in zinc resistance. ( B ) Cells expressing ZNT3 (ZNT2Nter , ZNT2Loop) or ZNT3 (ZNT2Nter , ZNT2Cter) , with two grafted cytosolic segments, showed almost no resistance to high zinc concentrations. In ( A , B ), <t>alamarBlue</t> assay was performed as shown in Fig. 2 . Stable expression of WT and mutant ZNT2 in znt1 −/− mt −/− znt4 −/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control.
    Alamarblue Reagent, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alamarblue reagent/product/Bio-Rad
    Average 93 stars, based on 217 article reviews
    Price from $9.99 to $1999.99
    alamarblue reagent - by Bioz Stars, 2020-10
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    91
    Bio-Rad alamarblue cell viability reagent
    DNA methylation directly suppresses SE activity and affects ES cell state. (A) Experimental setup for assessing the causal role of SE DNA methylation suppresses H3K27ac. FACS (DNA methylation), RT-qPCR ( Mir290–295 ) and ChIP-qPCR (H3K27ac) were co-assessed from the same pool of cells from each sample. (B) Loss of DNA methylation in MIR290-SE-TG T−G− cells 8 days post Dnmt1 and Uhrf1 sgRNA transfection as compared to controls. (C) H3K27ac ChIP-qPCR at the Mir290 SE from the experimental groups in (B), respectively. Data are represented as mean ± SD. (D) . Mir290–295 pri-miRNA level from the experimental groups in (B). Data are represented as mean ± SD. (E) Summary of the dynamic regulation and functional impact of allelic SE methylation. (F) Colony formation assays in “2i” starting from 100 sorted cells. Data are represented as mean ± SD . (G) Growth curves measured by <t>AlamarBlue</t> Cell Viability Reagent. Data are represented as mean ± SD . (H) .
    Alamarblue Cell Viability Reagent, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alamarblue cell viability reagent/product/Bio-Rad
    Average 91 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    alamarblue cell viability reagent - by Bioz Stars, 2020-10
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    Image Search Results


    Analysis of cell activities by alamarBlue assay. Each data point is the mean ± SD of 6 samples. ** p

    Journal: ACS Omega

    Article Title: Sacrificial Alginate-Assisted Microfluidic Engineering of Cell-Supportive Protein Microfibers for Hydrogel-Based Cell Encapsulation

    doi: 10.1021/acsomega.0c02385

    Figure Lengend Snippet: Analysis of cell activities by alamarBlue assay. Each data point is the mean ± SD of 6 samples. ** p

    Article Snippet: Fetal bovine serum (FBS), green fluorescent microbeads (diameter of 0.5 μm), and alamarBlue cell viability reagent were obtained from Thermo Fisher Scientific (MA, USA).

    Techniques: Alamar Blue Assay

    Effect of ethyl pyruvate and ethyl lactate on T. brucei cells proliferation. A cell proliferation/viability assay was conducted in 96-well cell culture plates containing 2x104 cells, medium and ethyl pyruvate in a volume of 200 μl. After 24 hrs of incubation the AlamarBlue cell proliferation reagent (20 μl) was added and the absorbance was read: (A) Ethyl pyruvate; (B) Ethyl lactate; (C) For the time-dependency test 2x105 cells/ml were cultured in 24-well plates in the presence of different concentrations of ethyl pyruvate at 0 mM (■), 1 mM (▲), 2.5 mM (x) and 5 mM (□). After certain time intervals aliquots were removed and the number of vital cells was counted. (D) For cell-recovery test 2x105 cells/ml were cultured in the presence of ethyl pyruvate at 0 mM (■), 1 mM (▲), 2.5 mM (▼), 5 mM (□) for 3 hrs in 24-well plates. Then, the medium was removed and the cells were replenished with fresh medium without the ethyl pyruvate and further cultured for 24 hrs. At the indicated time points aliquots were removed and vital cells were counted. Cells were cultured at 37°C containing 5% CO2 in a 100% humidified environment, * p

    Journal: PLoS ONE

    Article Title: Ethyl Pyruvate Emerges as a Safe and Fast Acting Agent against Trypanosoma brucei by Targeting Pyruvate Kinase Activity

    doi: 10.1371/journal.pone.0137353

    Figure Lengend Snippet: Effect of ethyl pyruvate and ethyl lactate on T. brucei cells proliferation. A cell proliferation/viability assay was conducted in 96-well cell culture plates containing 2x104 cells, medium and ethyl pyruvate in a volume of 200 μl. After 24 hrs of incubation the AlamarBlue cell proliferation reagent (20 μl) was added and the absorbance was read: (A) Ethyl pyruvate; (B) Ethyl lactate; (C) For the time-dependency test 2x105 cells/ml were cultured in 24-well plates in the presence of different concentrations of ethyl pyruvate at 0 mM (■), 1 mM (▲), 2.5 mM (x) and 5 mM (□). After certain time intervals aliquots were removed and the number of vital cells was counted. (D) For cell-recovery test 2x105 cells/ml were cultured in the presence of ethyl pyruvate at 0 mM (■), 1 mM (▲), 2.5 mM (▼), 5 mM (□) for 3 hrs in 24-well plates. Then, the medium was removed and the cells were replenished with fresh medium without the ethyl pyruvate and further cultured for 24 hrs. At the indicated time points aliquots were removed and vital cells were counted. Cells were cultured at 37°C containing 5% CO2 in a 100% humidified environment, * p

    Article Snippet: Cell proliferation/viability assay The AlamarBlue cell proliferation assay (Thermo Fisher Scientific, USA) has been used to measure the metabolically active cells.

    Techniques: Viability Assay, Cell Culture, Incubation

    Wild-type CHCHD10 ameliorates and FTD/ALS CHCHD10 mutations potentiate TDP-43-induced apoptosis and synaptic impairment. ( a , b ) HT22 neuroblastoma cells transfected with TDP-43-tomato and/or Flag-CHCHD10 variants, then subjected to Annexin V staining and direct TDP-43-tomato fluorescence. Cells also subjected to alamarBlue staining and quantification of OD570/600 ratio. Scale bar, 100 μm. ( b ) Quantification of Annexin V+ cells in RFP+ or TDP-43-tomato+ cells normalized to control (upper graph: one-way ANOVA, post hoc Tukey, ** P

    Journal: Nature Communications

    Article Title: Loss of function CHCHD10 mutations in cytoplasmic TDP-43 accumulation and synaptic integrity

    doi: 10.1038/ncomms15558

    Figure Lengend Snippet: Wild-type CHCHD10 ameliorates and FTD/ALS CHCHD10 mutations potentiate TDP-43-induced apoptosis and synaptic impairment. ( a , b ) HT22 neuroblastoma cells transfected with TDP-43-tomato and/or Flag-CHCHD10 variants, then subjected to Annexin V staining and direct TDP-43-tomato fluorescence. Cells also subjected to alamarBlue staining and quantification of OD570/600 ratio. Scale bar, 100 μm. ( b ) Quantification of Annexin V+ cells in RFP+ or TDP-43-tomato+ cells normalized to control (upper graph: one-way ANOVA, post hoc Tukey, ** P

    Article Snippet: AlamarBlue cell viability assays (Invitrogen) were performed per the manufacturer's instructions in a microtiter plate reader with absorbance reading at 570 nm, using 600 nm as a reference wavelength for normalization.

    Techniques: Transfection, Staining, Fluorescence

    The tc aptamer controlled CD20 expression. ( A ) Schematic representation of the suicide system. When treated with the therapeutic antibody rituximab (RTX), cell death was induced in cells expressing CD20. As a positive control, CD20 was stably integrated into HeLa HF1–3 cells (cell line HF1–3CD20). The aptamer-controlled exon 2 along with the neighboring introns from construct L2 was inserted into the CD20 coding region, which resulted in the cell line HF1–3C2. In the presence of tc, exon 2 was skipped, the coding sequence of CD20 was restored, and apoptosis was triggered. Without tc, exon 2 was retained, which destroyed the CD20 reading frame and resulted in cell survival. ( B, C ) Cell lines were incubated with and without 50 μM tc for 2 days. Afterwards, RT-PCR and a western blot were performed. Primers used for RT-PCR bind to the coding sequence of CD20, which spans the alternatively spliced exon 2. HSP60 was used as a loading control for western blot. ( D ) Viability assay. The cell lines were incubated with and without RTX and 50 μM tc or doxycycline (dox) for 3 days. Afterwards, cell viability was analyzed by an AlamarBlue ® .

    Journal: Nucleic Acids Research

    Article Title: A small, portable RNA device for the control of exon skipping in mammalian cells

    doi: 10.1093/nar/gky062

    Figure Lengend Snippet: The tc aptamer controlled CD20 expression. ( A ) Schematic representation of the suicide system. When treated with the therapeutic antibody rituximab (RTX), cell death was induced in cells expressing CD20. As a positive control, CD20 was stably integrated into HeLa HF1–3 cells (cell line HF1–3CD20). The aptamer-controlled exon 2 along with the neighboring introns from construct L2 was inserted into the CD20 coding region, which resulted in the cell line HF1–3C2. In the presence of tc, exon 2 was skipped, the coding sequence of CD20 was restored, and apoptosis was triggered. Without tc, exon 2 was retained, which destroyed the CD20 reading frame and resulted in cell survival. ( B, C ) Cell lines were incubated with and without 50 μM tc for 2 days. Afterwards, RT-PCR and a western blot were performed. Primers used for RT-PCR bind to the coding sequence of CD20, which spans the alternatively spliced exon 2. HSP60 was used as a loading control for western blot. ( D ) Viability assay. The cell lines were incubated with and without RTX and 50 μM tc or doxycycline (dox) for 3 days. Afterwards, cell viability was analyzed by an AlamarBlue ® .

    Article Snippet: Afterwards, 100 μl DMEM (without phenol red) containing 10% (v/v) AlamarBlue® (Life Technologies) was added.

    Techniques: Expressing, Positive Control, Stable Transfection, Construct, Sequencing, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Viability Assay

    Inhibition of JNK and ERK renders glioma cells sensitive to EGFR inhibition (a) AlamarBlue assay in established GBM cell lines exposed to erlotinib (10µM). Cells are completely resistant to the effects of EGFR inhibition. (b–c) Patient derived GBM9 or GBM39 neurospheres were exposed to erlotinib (100nM) with or without JNK inhibitor SP600125 (1µM), p38 inhibitor SB203580 (10µM), or ERK inhibitor U0126 (1µM), followed by Alamarblue Cell Survival Assay after 72h of inhibitor exposure. (b) Erlotinib vs erlotinib+SP600125: P =0.0015,t=7.75, d.f.=4; erlotinib vs erlotinib+U0126: P =0.0013, t=8.10, d.f.=4. (c) erlotinib vs erlotinib+SP600125: P =0.0057,t=5.41, d.f.=4; Erlotinib vs Erlotinib+U0126: P =0.0023, t=6.93, d.f.=4. (d–e) A similar experiment was conducted in U87EGFRwt and U87EGFRvIII cells. (d) erlotinib vs erlotinib+SP600125: P =0.0016,t=7.61, d.f.=4; Erlotinib vs Erlotinib+U0126: P =0.0017, t=7.48, d.f.=4. (e) erlotinib vs erlotinib+SP600125: P =0.0003,t=12.23, d.f.=4; erlotinib vs erlotinib+U0126: P =0.0014, t=7.87, d.f.=4. (f) siRNA knockdown of JNK1 and JNK2 in GBM9 neurospheres results in an enhanced sensitivity to erlotinib, whereas control siRNA has no effect. Erlotinib+siCtrl vs erlotinib+siJNK1/2: P =0.0002, t=13.96, d.f.=4. (g) siRNA knockdown of JNK1 and JNK2 in GBM39 neurosphere cells has a similar effect. Erlotinib+siCtrl vs Erlotinib+siJNK1/2: P =0.0003,t=11.86, d.f.=4. (h) U87EGFRwt cells results in an enhanced sensitivity to erlotinib, whereas control siRNA has no effect. Erlotinib+siCtrl vs erlotinib+siJNK1/2: P =0.0017, t=7.52, d.f.=4. (i–j) Patient derived GBM9 or GBM39 neurospheres were exposed to erlotinib (100nM) with or without Axl inhibitor R428 (1µM) followed by Alamarblue Cell Survival Assay after 72h. (i) Erlotinib vs erlotinib+R428: P =0.0025,t=6.75, d.f.=4. (i) Erlotinib vs erlotinib+R428: P =0.0023,t=6.93, d.f.=4. (k) A similar experiment was done in U87EGFRwt cells using an erlotinib concentration of 1µM. Erlotinib vs erlotinib+R428: P =0.0094, t=4.69, d.f.=4. (l–n) siRNA knockdown of Axl in GBM9 and GBM39 neurospheres or U87EGFRwt cells sensitizes cells to the effect of erlotinib but not control siRNA as determined by Alamarblue Cell Viability Assay. (l) Erlotinib+siCtrl vs erlotinib+siAxl: P =0.0004,t=11.23, d.f.=4. (m) Erlotinib+siCtrl vs erlotinib+siAxl: P =0.0003, t=12.80, d.f.=4. (n) Erlotinib+siCtrl vs erlotinib+R428: P =0.0058, t=5.38, d.f.=4. Data are presented as mean±s.e.m; ** P

    Journal: Nature neuroscience

    Article Title: Primary resistance to EGFR inhibition in glioblastoma is mediated by a TNF-JNK-Axl-ERK signaling axis

    doi: 10.1038/nn.4584

    Figure Lengend Snippet: Inhibition of JNK and ERK renders glioma cells sensitive to EGFR inhibition (a) AlamarBlue assay in established GBM cell lines exposed to erlotinib (10µM). Cells are completely resistant to the effects of EGFR inhibition. (b–c) Patient derived GBM9 or GBM39 neurospheres were exposed to erlotinib (100nM) with or without JNK inhibitor SP600125 (1µM), p38 inhibitor SB203580 (10µM), or ERK inhibitor U0126 (1µM), followed by Alamarblue Cell Survival Assay after 72h of inhibitor exposure. (b) Erlotinib vs erlotinib+SP600125: P =0.0015,t=7.75, d.f.=4; erlotinib vs erlotinib+U0126: P =0.0013, t=8.10, d.f.=4. (c) erlotinib vs erlotinib+SP600125: P =0.0057,t=5.41, d.f.=4; Erlotinib vs Erlotinib+U0126: P =0.0023, t=6.93, d.f.=4. (d–e) A similar experiment was conducted in U87EGFRwt and U87EGFRvIII cells. (d) erlotinib vs erlotinib+SP600125: P =0.0016,t=7.61, d.f.=4; Erlotinib vs Erlotinib+U0126: P =0.0017, t=7.48, d.f.=4. (e) erlotinib vs erlotinib+SP600125: P =0.0003,t=12.23, d.f.=4; erlotinib vs erlotinib+U0126: P =0.0014, t=7.87, d.f.=4. (f) siRNA knockdown of JNK1 and JNK2 in GBM9 neurospheres results in an enhanced sensitivity to erlotinib, whereas control siRNA has no effect. Erlotinib+siCtrl vs erlotinib+siJNK1/2: P =0.0002, t=13.96, d.f.=4. (g) siRNA knockdown of JNK1 and JNK2 in GBM39 neurosphere cells has a similar effect. Erlotinib+siCtrl vs Erlotinib+siJNK1/2: P =0.0003,t=11.86, d.f.=4. (h) U87EGFRwt cells results in an enhanced sensitivity to erlotinib, whereas control siRNA has no effect. Erlotinib+siCtrl vs erlotinib+siJNK1/2: P =0.0017, t=7.52, d.f.=4. (i–j) Patient derived GBM9 or GBM39 neurospheres were exposed to erlotinib (100nM) with or without Axl inhibitor R428 (1µM) followed by Alamarblue Cell Survival Assay after 72h. (i) Erlotinib vs erlotinib+R428: P =0.0025,t=6.75, d.f.=4. (i) Erlotinib vs erlotinib+R428: P =0.0023,t=6.93, d.f.=4. (k) A similar experiment was done in U87EGFRwt cells using an erlotinib concentration of 1µM. Erlotinib vs erlotinib+R428: P =0.0094, t=4.69, d.f.=4. (l–n) siRNA knockdown of Axl in GBM9 and GBM39 neurospheres or U87EGFRwt cells sensitizes cells to the effect of erlotinib but not control siRNA as determined by Alamarblue Cell Viability Assay. (l) Erlotinib+siCtrl vs erlotinib+siAxl: P =0.0004,t=11.23, d.f.=4. (m) Erlotinib+siCtrl vs erlotinib+siAxl: P =0.0003, t=12.80, d.f.=4. (n) Erlotinib+siCtrl vs erlotinib+R428: P =0.0058, t=5.38, d.f.=4. Data are presented as mean±s.e.m; ** P

    Article Snippet: Cell viability assay Cell viability assay was conducted using AlamarBlue cell viability assay kit (ThermoFisher) following manufacturer’s protocol.

    Techniques: Inhibition, Alamar Blue Assay, Derivative Assay, Clonogenic Cell Survival Assay, Concentration Assay, Viability Assay

    TNF inhibition sensitizes glioma cells to EGFR inhibition (a–b) AlamarBlue cell viability assay in GBM9 or GBM39 neurospheres. Enbrel (100 nM) sensitizes cells to EGFR inhibition with erlotinib. Enbrel and erlotinib were added to GBM9 or GBM39 neurospheres concurrently and AlamarBlue assay was done after 72h. (a) Erlotinib vs Erlotinib+Enbrel: P =0.0027, t=6.59, d.f.=4. (b) Erlotinib vs erlotinib+enbrel: P =0.0044, t=6.59, d.f.=4. (c) A similar experiment was performed in U87EGFRwt cells. Erlotinib vs Erlotinib+Enbrel: P =0.0056, t=5.41, d.f.=4. (d–e) TNFR1 was silenced using siRNA in GBM9 and GBM39 cells and cells were exposed to erlotinib for 72h in stem cell medium without EGF for 72h followed by Alamarblue Assay. (d) Erlotinib+siCtrl vs erlotinib+siTNFR1: P =0.0014, t=7.95, d.f.=4. (e) Erlotinib+siCtrl vs erlotinib+siTNFR1: P =0.0041, t=5.90, d.f.=4. (f) A similar experiment was done in U87EGFRwt cells. Erlotinib+siCtrl vs erlotinib+siTNFR1: P =0.0021, t=7.11, d.f.=4. (g–i) Thalidomide sensitizes GBM9 and GBM39 cells to EGFR inhibition with erlotinib. Thalidomide (1µM) and erlotinib were added to GBM9 and GBM39 neurospheres (100nM) or U87EGFRwt cells (1uM) concurrently and AlamarBlue assay was done after 72h. (g) Erlotinib vs erlotinib+thalidomide: P =0.0030, t=6.42, d.f.=4. (h) Erlotinib vs erlotinib+thalidomide: P =0.0027, t=6.59, d.f.=4. (i) Erlotinib vs erlotinib+thalidomide: P =0.0013, t=8.11, d.f.=4. (j–k) Enbrel or thalidomide block erlotinib induced activation of JNK, Axl and ERK in GBM9 and GBM39 neurospheres as shown in the Western blot. (l) A similar experiment was conducted in U87EGFRwt cells. Western blots shown in j-l are representative of at least three independent replicates. Full-length blots are presented in Supplementary Figure 14 . (m–n) show that exogenous TNF protects GBM9 and GBM39 neurospheres from erlotinib induced cell death. TNF (1ng/ml) and erlotinib (1µM) were added to cells concurrently and AlamarBlue cell viability assay was done after 72h. (m) Erlotinib vs erlotinib+TNF: P =0.0018, t=7.41, d.f.=4. (n) Erlotinib vs erlotinib+TNF: P =0.0087, t=4.79, d.f.=4. Data are presented as mean±s.e.m; ** P

    Journal: Nature neuroscience

    Article Title: Primary resistance to EGFR inhibition in glioblastoma is mediated by a TNF-JNK-Axl-ERK signaling axis

    doi: 10.1038/nn.4584

    Figure Lengend Snippet: TNF inhibition sensitizes glioma cells to EGFR inhibition (a–b) AlamarBlue cell viability assay in GBM9 or GBM39 neurospheres. Enbrel (100 nM) sensitizes cells to EGFR inhibition with erlotinib. Enbrel and erlotinib were added to GBM9 or GBM39 neurospheres concurrently and AlamarBlue assay was done after 72h. (a) Erlotinib vs Erlotinib+Enbrel: P =0.0027, t=6.59, d.f.=4. (b) Erlotinib vs erlotinib+enbrel: P =0.0044, t=6.59, d.f.=4. (c) A similar experiment was performed in U87EGFRwt cells. Erlotinib vs Erlotinib+Enbrel: P =0.0056, t=5.41, d.f.=4. (d–e) TNFR1 was silenced using siRNA in GBM9 and GBM39 cells and cells were exposed to erlotinib for 72h in stem cell medium without EGF for 72h followed by Alamarblue Assay. (d) Erlotinib+siCtrl vs erlotinib+siTNFR1: P =0.0014, t=7.95, d.f.=4. (e) Erlotinib+siCtrl vs erlotinib+siTNFR1: P =0.0041, t=5.90, d.f.=4. (f) A similar experiment was done in U87EGFRwt cells. Erlotinib+siCtrl vs erlotinib+siTNFR1: P =0.0021, t=7.11, d.f.=4. (g–i) Thalidomide sensitizes GBM9 and GBM39 cells to EGFR inhibition with erlotinib. Thalidomide (1µM) and erlotinib were added to GBM9 and GBM39 neurospheres (100nM) or U87EGFRwt cells (1uM) concurrently and AlamarBlue assay was done after 72h. (g) Erlotinib vs erlotinib+thalidomide: P =0.0030, t=6.42, d.f.=4. (h) Erlotinib vs erlotinib+thalidomide: P =0.0027, t=6.59, d.f.=4. (i) Erlotinib vs erlotinib+thalidomide: P =0.0013, t=8.11, d.f.=4. (j–k) Enbrel or thalidomide block erlotinib induced activation of JNK, Axl and ERK in GBM9 and GBM39 neurospheres as shown in the Western blot. (l) A similar experiment was conducted in U87EGFRwt cells. Western blots shown in j-l are representative of at least three independent replicates. Full-length blots are presented in Supplementary Figure 14 . (m–n) show that exogenous TNF protects GBM9 and GBM39 neurospheres from erlotinib induced cell death. TNF (1ng/ml) and erlotinib (1µM) were added to cells concurrently and AlamarBlue cell viability assay was done after 72h. (m) Erlotinib vs erlotinib+TNF: P =0.0018, t=7.41, d.f.=4. (n) Erlotinib vs erlotinib+TNF: P =0.0087, t=4.79, d.f.=4. Data are presented as mean±s.e.m; ** P

    Article Snippet: Cell viability assay Cell viability assay was conducted using AlamarBlue cell viability assay kit (ThermoFisher) following manufacturer’s protocol.

    Techniques: Inhibition, Viability Assay, Alamar Blue Assay, Blocking Assay, Activation Assay, Western Blot

    PCS inhibits cell viability of HASMC in a time- and dose-dependent manner. ( A ) HASMCs were treated with various concentrations of PCS (0, 62.5, 125, 250, and 500 μM) for 24 h. ( B ) HASMCs were treated with various concentrations of PCS (0, 62.5, 125, 250, and 500 μM) for 48 h. Cell viability was determined using the colorimetric Alamar Blue assay. Data were expressed as mean ± standard deviation. Each experiment was replicated for three times. Data were analyzed with ANOVA (Dunnett’s multiple comparisons test). ** p

    Journal: Toxins

    Article Title: Scavenging Intracellular ROS Attenuates p-Cresyl Sulfate-Triggered Osteogenesis through MAPK Signaling Pathway and NF-κB Activation in Human Arterial Smooth Muscle Cells

    doi: 10.3390/toxins12080472

    Figure Lengend Snippet: PCS inhibits cell viability of HASMC in a time- and dose-dependent manner. ( A ) HASMCs were treated with various concentrations of PCS (0, 62.5, 125, 250, and 500 μM) for 24 h. ( B ) HASMCs were treated with various concentrations of PCS (0, 62.5, 125, 250, and 500 μM) for 48 h. Cell viability was determined using the colorimetric Alamar Blue assay. Data were expressed as mean ± standard deviation. Each experiment was replicated for three times. Data were analyzed with ANOVA (Dunnett’s multiple comparisons test). ** p

    Article Snippet: Cell Viability Assay Cell viability was assessed by Alamar Blue assay (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Alamar Blue Assay, Standard Deviation

    Domain swapping analysis of ZNT2 and ZNT3 on the ZNT3 backbone. ( A ) Expression of ZNT3 (ZNT2Nter) , ZNT3 (ZNT2Loop) , or ZNT3 (ZNT2Cter) in znt1 −/− mt −/− znt4 −/− cells did not result in zinc resistance. ( B ) Cells expressing ZNT3 (ZNT2Nter , ZNT2Loop) or ZNT3 (ZNT2Nter , ZNT2Cter) , with two grafted cytosolic segments, showed almost no resistance to high zinc concentrations. In ( A , B ), alamarBlue assay was performed as shown in Fig. 2 . Stable expression of WT and mutant ZNT2 in znt1 −/− mt −/− znt4 −/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control.

    Journal: Scientific Reports

    Article Title: Evaluation of the roles of the cytosolic N-terminus and His-rich loop of ZNT proteins using ZNT2 and ZNT3 chimeric mutants

    doi: 10.1038/s41598-018-32372-8

    Figure Lengend Snippet: Domain swapping analysis of ZNT2 and ZNT3 on the ZNT3 backbone. ( A ) Expression of ZNT3 (ZNT2Nter) , ZNT3 (ZNT2Loop) , or ZNT3 (ZNT2Cter) in znt1 −/− mt −/− znt4 −/− cells did not result in zinc resistance. ( B ) Cells expressing ZNT3 (ZNT2Nter , ZNT2Loop) or ZNT3 (ZNT2Nter , ZNT2Cter) , with two grafted cytosolic segments, showed almost no resistance to high zinc concentrations. In ( A , B ), alamarBlue assay was performed as shown in Fig. 2 . Stable expression of WT and mutant ZNT2 in znt1 −/− mt −/− znt4 −/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control.

    Article Snippet: The alamarBlue reagent (AbD Serotec, Ltd., Oxford, UK) was added to the culture media and the cells were incubated for 4 h. Absorbance was determined at 570 and 600 nm using PowerScan 4 (DS Pharma Biomedical, Osaka, Japan).

    Techniques: Expressing, Alamar Blue Assay, Mutagenesis

    His residues of the cytosolic ZNT2 His-rich loop are not essential for zinc transport. ( A ) Zinc transport activity of znt1 −/− mt −/− znt4 −/− cells expressing H197R or H205D ZNT2 SNP mutants (ZNT2 (H197R) , ZNT2 (H205D) ) was comparable to that of WT ZNT2-expressing cells. ( B ) Expressing ZNT2 mutants with single His to Ala substitution in the His-rich loop (ZNT2 (H197A) , ZNT2 (H201A) , ZNT2 (H203A) , and ZNT2 (H205A) ) in znt1 −/− mt −/− znt4 −/− cells resulted in zinc transport activity comparable to that of WT ZNT2. Similar observations were made with the double substitution mutants (ZNT2 (H201AH203A) , ZNT2 (H201AH205A) , and ZNT2 (H203AH205A) ) ( C ), whereas expression of ZNT2 mutants with three or four Ala-substituted His loop residues (ZNT2 (Loop3H-3A) and ZNT2 (Loop4H-4A) ), moderately decreased zinc resistance ( D ). ( E ) Expression of ZNT2 mutant with the deleted cytosolic His-rich loop (ZNT2 (Δ201-205) ) in znt1 −/− mt −/− znt4 −/− cells did not confer zinc resistance. In experiments depicted in panels ( A – E ), cells were grown in the presence of indicated concentrations of ZnSO 4 and the number of surviving cells was estimated using the alamarBlue assay in triplicate (representative results shown). Expression of WT or mutant ZNT2 proteins in znt1 −/− mt −/− znt4 −/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control. ( F ) Comparison of the stability of ZNT2 (Loop4H-4A) and ZNT2 (Δ201-205) mutants with those of WT ZNT2 or ZNT2 (G280R) mutant. The expression levels of each protein at each time point are shown, with representative results of immunoblotting depicted in the lower panel. The results of ZNT2 (G280R) is shown as a control for the destabilized ZNT2 mutant 42 . Data show mean ± SEM of triplicate experiments (lower sub-panels). Tubulin was used as the loading control.

    Journal: Scientific Reports

    Article Title: Evaluation of the roles of the cytosolic N-terminus and His-rich loop of ZNT proteins using ZNT2 and ZNT3 chimeric mutants

    doi: 10.1038/s41598-018-32372-8

    Figure Lengend Snippet: His residues of the cytosolic ZNT2 His-rich loop are not essential for zinc transport. ( A ) Zinc transport activity of znt1 −/− mt −/− znt4 −/− cells expressing H197R or H205D ZNT2 SNP mutants (ZNT2 (H197R) , ZNT2 (H205D) ) was comparable to that of WT ZNT2-expressing cells. ( B ) Expressing ZNT2 mutants with single His to Ala substitution in the His-rich loop (ZNT2 (H197A) , ZNT2 (H201A) , ZNT2 (H203A) , and ZNT2 (H205A) ) in znt1 −/− mt −/− znt4 −/− cells resulted in zinc transport activity comparable to that of WT ZNT2. Similar observations were made with the double substitution mutants (ZNT2 (H201AH203A) , ZNT2 (H201AH205A) , and ZNT2 (H203AH205A) ) ( C ), whereas expression of ZNT2 mutants with three or four Ala-substituted His loop residues (ZNT2 (Loop3H-3A) and ZNT2 (Loop4H-4A) ), moderately decreased zinc resistance ( D ). ( E ) Expression of ZNT2 mutant with the deleted cytosolic His-rich loop (ZNT2 (Δ201-205) ) in znt1 −/− mt −/− znt4 −/− cells did not confer zinc resistance. In experiments depicted in panels ( A – E ), cells were grown in the presence of indicated concentrations of ZnSO 4 and the number of surviving cells was estimated using the alamarBlue assay in triplicate (representative results shown). Expression of WT or mutant ZNT2 proteins in znt1 −/− mt −/− znt4 −/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control. ( F ) Comparison of the stability of ZNT2 (Loop4H-4A) and ZNT2 (Δ201-205) mutants with those of WT ZNT2 or ZNT2 (G280R) mutant. The expression levels of each protein at each time point are shown, with representative results of immunoblotting depicted in the lower panel. The results of ZNT2 (G280R) is shown as a control for the destabilized ZNT2 mutant 42 . Data show mean ± SEM of triplicate experiments (lower sub-panels). Tubulin was used as the loading control.

    Article Snippet: The alamarBlue reagent (AbD Serotec, Ltd., Oxford, UK) was added to the culture media and the cells were incubated for 4 h. Absorbance was determined at 570 and 600 nm using PowerScan 4 (DS Pharma Biomedical, Osaka, Japan).

    Techniques: Activity Assay, Expressing, Mutagenesis, Alamar Blue Assay

    Domain swapping analysis of ZNT2 and ZNT3 on the ZNT2 backbone. ( A ) Expression of ZNT3 in znt1 −/− mt −/− znt4 −/− cells did not confer significant resistance to high zinc concentrations, compared to the zinc resistance of cells expressing ZNT2. ( B–D ) Expression of ZNT2 (ZNT3Nter) in znt1 −/− mt −/− znt4 −/− cells did not confer zinc resistance, whereas expression of ZNT2 (ZNT3Loop) and ZNT2 (ZNT3Cter) conferred zinc resistance, which was similar to that of WT ZNT2-expressing cells. In ( D ) *indicates the position of non-specific band. ( E ) Expression of ZNT2 (ZNT3Nter , ZNT3Loop) conferred zinc resistance in znt1 −/− mt −/− znt4 −/− cells. ( F ) Stabilities of ZNT2 (ZNT3Loop) , ZNT2 (ZNT3Nter) , and ZNT2 (ZNT3Nter , ZNT3Loop) were evaluated as described in Fig. 2F . Data show mean ± SEM of triplicate experiments (lower sub-panels). Tubulin was used as the loading control. ( G ) Cells expressing ZNT2 (ZNT2-3Nter) showed zinc resistance similar to that of ZNT2-expressing cells, whereas cells expressing ZNT2 (ZNT3-2Nter) showed moderate resistance. In ( A − E ) and ( G ), the alamarBlue assay was performed as shown in Fig. 2 . Stable expression of WT and mutant ZNT2 in znt1 −/− mt −/− znt4 −/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control.

    Journal: Scientific Reports

    Article Title: Evaluation of the roles of the cytosolic N-terminus and His-rich loop of ZNT proteins using ZNT2 and ZNT3 chimeric mutants

    doi: 10.1038/s41598-018-32372-8

    Figure Lengend Snippet: Domain swapping analysis of ZNT2 and ZNT3 on the ZNT2 backbone. ( A ) Expression of ZNT3 in znt1 −/− mt −/− znt4 −/− cells did not confer significant resistance to high zinc concentrations, compared to the zinc resistance of cells expressing ZNT2. ( B–D ) Expression of ZNT2 (ZNT3Nter) in znt1 −/− mt −/− znt4 −/− cells did not confer zinc resistance, whereas expression of ZNT2 (ZNT3Loop) and ZNT2 (ZNT3Cter) conferred zinc resistance, which was similar to that of WT ZNT2-expressing cells. In ( D ) *indicates the position of non-specific band. ( E ) Expression of ZNT2 (ZNT3Nter , ZNT3Loop) conferred zinc resistance in znt1 −/− mt −/− znt4 −/− cells. ( F ) Stabilities of ZNT2 (ZNT3Loop) , ZNT2 (ZNT3Nter) , and ZNT2 (ZNT3Nter , ZNT3Loop) were evaluated as described in Fig. 2F . Data show mean ± SEM of triplicate experiments (lower sub-panels). Tubulin was used as the loading control. ( G ) Cells expressing ZNT2 (ZNT2-3Nter) showed zinc resistance similar to that of ZNT2-expressing cells, whereas cells expressing ZNT2 (ZNT3-2Nter) showed moderate resistance. In ( A − E ) and ( G ), the alamarBlue assay was performed as shown in Fig. 2 . Stable expression of WT and mutant ZNT2 in znt1 −/− mt −/− znt4 −/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control.

    Article Snippet: The alamarBlue reagent (AbD Serotec, Ltd., Oxford, UK) was added to the culture media and the cells were incubated for 4 h. Absorbance was determined at 570 and 600 nm using PowerScan 4 (DS Pharma Biomedical, Osaka, Japan).

    Techniques: Expressing, Alamar Blue Assay, Mutagenesis

    Deletion of the ZNT2 cytosolic N-terminus did not result in loss of zinc transport function. ( A ) Expression of ZNT2 (Nter-del) and ZNT2 (Nter-del , ZNT3Loop) in znt1 −/− mt −/− znt4 −/− cells conferred zinc resistance, similar to (with minor loss) the effect of ZNT2 (Nter-del , Loop3H-3A) expression ( B) . In ( A , B ), the alamarBlue assay was performed as shown in Fig. 2 . Stable expression of WT and mutant ZNT2 in znt1 −/− mt −/− znt4 −/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control. ( C ) The stabilities of ZNT2 (Nter-del) , ZNT2 (Nter-del , ZNT3Loop) , and ZNT2 (Nter-del , Loop3H-3A) were evaluated as described in Fig. 2F . Data show mean ± SEM of triplicate experiments (lower sub-panels). Tubulin was used as the loading control. Asterisk (*) denotes a significant difference between WT ZNT2, and ZNT2 (Nter-del) mutant protein levels ( P

    Journal: Scientific Reports

    Article Title: Evaluation of the roles of the cytosolic N-terminus and His-rich loop of ZNT proteins using ZNT2 and ZNT3 chimeric mutants

    doi: 10.1038/s41598-018-32372-8

    Figure Lengend Snippet: Deletion of the ZNT2 cytosolic N-terminus did not result in loss of zinc transport function. ( A ) Expression of ZNT2 (Nter-del) and ZNT2 (Nter-del , ZNT3Loop) in znt1 −/− mt −/− znt4 −/− cells conferred zinc resistance, similar to (with minor loss) the effect of ZNT2 (Nter-del , Loop3H-3A) expression ( B) . In ( A , B ), the alamarBlue assay was performed as shown in Fig. 2 . Stable expression of WT and mutant ZNT2 in znt1 −/− mt −/− znt4 −/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control. ( C ) The stabilities of ZNT2 (Nter-del) , ZNT2 (Nter-del , ZNT3Loop) , and ZNT2 (Nter-del , Loop3H-3A) were evaluated as described in Fig. 2F . Data show mean ± SEM of triplicate experiments (lower sub-panels). Tubulin was used as the loading control. Asterisk (*) denotes a significant difference between WT ZNT2, and ZNT2 (Nter-del) mutant protein levels ( P

    Article Snippet: The alamarBlue reagent (AbD Serotec, Ltd., Oxford, UK) was added to the culture media and the cells were incubated for 4 h. Absorbance was determined at 570 and 600 nm using PowerScan 4 (DS Pharma Biomedical, Osaka, Japan).

    Techniques: Expressing, Alamar Blue Assay, Mutagenesis

    DNA methylation directly suppresses SE activity and affects ES cell state. (A) Experimental setup for assessing the causal role of SE DNA methylation suppresses H3K27ac. FACS (DNA methylation), RT-qPCR ( Mir290–295 ) and ChIP-qPCR (H3K27ac) were co-assessed from the same pool of cells from each sample. (B) Loss of DNA methylation in MIR290-SE-TG T−G− cells 8 days post Dnmt1 and Uhrf1 sgRNA transfection as compared to controls. (C) H3K27ac ChIP-qPCR at the Mir290 SE from the experimental groups in (B), respectively. Data are represented as mean ± SD. (D) . Mir290–295 pri-miRNA level from the experimental groups in (B). Data are represented as mean ± SD. (E) Summary of the dynamic regulation and functional impact of allelic SE methylation. (F) Colony formation assays in “2i” starting from 100 sorted cells. Data are represented as mean ± SD . (G) Growth curves measured by AlamarBlue Cell Viability Reagent. Data are represented as mean ± SD . (H) .

    Journal: Molecular cell

    Article Title: Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs

    doi: 10.1016/j.molcel.2019.06.045

    Figure Lengend Snippet: DNA methylation directly suppresses SE activity and affects ES cell state. (A) Experimental setup for assessing the causal role of SE DNA methylation suppresses H3K27ac. FACS (DNA methylation), RT-qPCR ( Mir290–295 ) and ChIP-qPCR (H3K27ac) were co-assessed from the same pool of cells from each sample. (B) Loss of DNA methylation in MIR290-SE-TG T−G− cells 8 days post Dnmt1 and Uhrf1 sgRNA transfection as compared to controls. (C) H3K27ac ChIP-qPCR at the Mir290 SE from the experimental groups in (B), respectively. Data are represented as mean ± SD. (D) . Mir290–295 pri-miRNA level from the experimental groups in (B). Data are represented as mean ± SD. (E) Summary of the dynamic regulation and functional impact of allelic SE methylation. (F) Colony formation assays in “2i” starting from 100 sorted cells. Data are represented as mean ± SD . (G) Growth curves measured by AlamarBlue Cell Viability Reagent. Data are represented as mean ± SD . (H) .

    Article Snippet: For measuring cell proliferation, AlamarBlue Cell Viability Reagent (Bio-Rad, BUF012A) was added to cell culture and incubated at 37°C with 5% CO2 and emission at 590nm was monitored every 50hrs.

    Techniques: DNA Methylation Assay, Activity Assay, FACS, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Functional Assay, Methylation