6 well plates  (Thermo Fisher)


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    Name:
    Abgene 48 Well Polypropylene Storage Plate
    Description:
    Thermo Scientific Abgene polypropylene microplates and deepwell plates offer storage security for assays compound libraries or storing samples for either intermediate or long term use Abgene microtiter plates are manufactured to exacting specifications in our Class 100 000 clean room ISO 9001 conditions using high quality medical grade virgin polypropylene resins which ensure confidence in the quality and performance of the storage plates Designed to ANSI standards these microplates and deepwell plates are compatible with a variety of automated liquid handling for high throughput workflows The polypropylene storage plates are offered with spindle pyramidal U and V bottom wells along with a wide range of volumes to accommodate most applications
    Catalog Number:
    ab0988
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    Cell Counting & Viability|Cell Culture|Laboratory Plastics and Supplies|Microplates, Dishes and Flasks|Cell Counting, Viability, & Cryopreservation
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    Lab Supplies Plastics Glassware
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    Thermo Fisher 6 well plates
    Growth kinetics of recombinant H5N1 viruses in MDCK and A549 cells. Each recombinant virus was diluted to 10 5 50% chicken embryo infection dose (EID 50 )/0.1 mL, and 0.5 mL of diluents were inoculated into confluent ( a ) MDCK and ( b ) A549 cells in <t>6-well</t> plates for 1 h. After 1 h, the inoculated virus was removed, and 1 mL of fresh medium was added. During 72 h of incubation, the supernatant was harvested at 0, 24, 48, and 72 hpi, and 50% tissue culture infectious dose (TCID 50 )/0.1 mL of each time point was measured in MDCK cells. The TCID 50 /0.1 mL values are the average of three independent experiments. #, *, significant differences of rH5N1-N144S-I223V (#) and rPR8 (*) in comparison with other viruses ( p
    Thermo Scientific Abgene polypropylene microplates and deepwell plates offer storage security for assays compound libraries or storing samples for either intermediate or long term use Abgene microtiter plates are manufactured to exacting specifications in our Class 100 000 clean room ISO 9001 conditions using high quality medical grade virgin polypropylene resins which ensure confidence in the quality and performance of the storage plates Designed to ANSI standards these microplates and deepwell plates are compatible with a variety of automated liquid handling for high throughput workflows The polypropylene storage plates are offered with spindle pyramidal U and V bottom wells along with a wide range of volumes to accommodate most applications
    https://www.bioz.com/result/6 well plates/product/Thermo Fisher
    Average 99 stars, based on 937 article reviews
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    6 well plates - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Novel Mutations Evading Avian Immunity around the Receptor Binding Site of the Clade 2.3.2.1c Hemagglutinin Gene Reduce Viral Thermostability and Mammalian Pathogenicity"

    Article Title: Novel Mutations Evading Avian Immunity around the Receptor Binding Site of the Clade 2.3.2.1c Hemagglutinin Gene Reduce Viral Thermostability and Mammalian Pathogenicity

    Journal: Viruses

    doi: 10.3390/v11100923

    Growth kinetics of recombinant H5N1 viruses in MDCK and A549 cells. Each recombinant virus was diluted to 10 5 50% chicken embryo infection dose (EID 50 )/0.1 mL, and 0.5 mL of diluents were inoculated into confluent ( a ) MDCK and ( b ) A549 cells in 6-well plates for 1 h. After 1 h, the inoculated virus was removed, and 1 mL of fresh medium was added. During 72 h of incubation, the supernatant was harvested at 0, 24, 48, and 72 hpi, and 50% tissue culture infectious dose (TCID 50 )/0.1 mL of each time point was measured in MDCK cells. The TCID 50 /0.1 mL values are the average of three independent experiments. #, *, significant differences of rH5N1-N144S-I223V (#) and rPR8 (*) in comparison with other viruses ( p
    Figure Legend Snippet: Growth kinetics of recombinant H5N1 viruses in MDCK and A549 cells. Each recombinant virus was diluted to 10 5 50% chicken embryo infection dose (EID 50 )/0.1 mL, and 0.5 mL of diluents were inoculated into confluent ( a ) MDCK and ( b ) A549 cells in 6-well plates for 1 h. After 1 h, the inoculated virus was removed, and 1 mL of fresh medium was added. During 72 h of incubation, the supernatant was harvested at 0, 24, 48, and 72 hpi, and 50% tissue culture infectious dose (TCID 50 )/0.1 mL of each time point was measured in MDCK cells. The TCID 50 /0.1 mL values are the average of three independent experiments. #, *, significant differences of rH5N1-N144S-I223V (#) and rPR8 (*) in comparison with other viruses ( p

    Techniques Used: Recombinant, Infection, Incubation

    2) Product Images from "A small-molecule ligand of valosin-containing protein/p97 inhibits cancer cell–accelerated fibroblast migration"

    Article Title: A small-molecule ligand of valosin-containing protein/p97 inhibits cancer cell–accelerated fibroblast migration

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.004741

    Silencing of VCP in NIH3T3 cells suppresses the enhanced migration of NIH3T3 cells co-cultured with MCF7. A , MCF7 cells or NIH3T3 cells were treated with control ( Co ) or VCP-targeted siRNA ( Si ) and cultured for 24, 48, or 72 h. The cell lysates were analyzed for VCP expression by Western blotting. Coomassie Brilliant Blue staining of the blot was used as a loading control. MW , molecular weight. B , NIH3T3 cells or VCP presilenced NIH3T3 cells ( NIH3T3 siVCP ) were cultured alone ( top panels ) or with MCF7 or VCP presilenced MCF7 ( MCF7 siVCP ) ( bottom panels ) overnight in 6-well plates with 10% FCS. The next day, the cells were scratched and cultured in medium containing 1% FCS for 24 h, and migration was examined. The wound edge at the 0 time point is marked with red lines and overlaid with other images taken at 24 h. C , the cell migration of fibroblasts and co-cultured cells in B was quantitated using ImageJ. The graph shows the percentage of fibroblast migration in co-cultures relative to the control (fibroblast alone) at 24 h. The migration of NIH3T3 cells was significantly enhanced when co-cultured with MCF7 cells ( n = 3; ***, p
    Figure Legend Snippet: Silencing of VCP in NIH3T3 cells suppresses the enhanced migration of NIH3T3 cells co-cultured with MCF7. A , MCF7 cells or NIH3T3 cells were treated with control ( Co ) or VCP-targeted siRNA ( Si ) and cultured for 24, 48, or 72 h. The cell lysates were analyzed for VCP expression by Western blotting. Coomassie Brilliant Blue staining of the blot was used as a loading control. MW , molecular weight. B , NIH3T3 cells or VCP presilenced NIH3T3 cells ( NIH3T3 siVCP ) were cultured alone ( top panels ) or with MCF7 or VCP presilenced MCF7 ( MCF7 siVCP ) ( bottom panels ) overnight in 6-well plates with 10% FCS. The next day, the cells were scratched and cultured in medium containing 1% FCS for 24 h, and migration was examined. The wound edge at the 0 time point is marked with red lines and overlaid with other images taken at 24 h. C , the cell migration of fibroblasts and co-cultured cells in B was quantitated using ImageJ. The graph shows the percentage of fibroblast migration in co-cultures relative to the control (fibroblast alone) at 24 h. The migration of NIH3T3 cells was significantly enhanced when co-cultured with MCF7 cells ( n = 3; ***, p

    Techniques Used: Migration, Cell Culture, Expressing, Western Blot, Staining, Molecular Weight

    NPD8733 suppresses the enhanced migration of NIH3T3 cells co-cultured with MCF7 cells using a wound healing co-culture assay. A , chemical structure of NPD8733. B , chemical structure of NPD8732. C , NIH3T3 cells alone or NIH3T3 cells co-cultured with MCF7 cells were seeded overnight in 6-well plates in 10% FCS. The next day, the cells were scratched, treated with different concentrations of NPD8733, and cultured in medium containing 1% FCS. The edge of the wound at the 0 time point is marked with a red outline and was overlaid with other images taken at 24 h. D , the cell migration percentages for fibroblasts and co-cultured cells were calculated using ImageJ. The graph shows the percentage of fibroblast migration in co-cultures relative to the control (fibroblasts alone) at 24 h and was plotted against the NPD8733 concentration ( n = 3; ***, p
    Figure Legend Snippet: NPD8733 suppresses the enhanced migration of NIH3T3 cells co-cultured with MCF7 cells using a wound healing co-culture assay. A , chemical structure of NPD8733. B , chemical structure of NPD8732. C , NIH3T3 cells alone or NIH3T3 cells co-cultured with MCF7 cells were seeded overnight in 6-well plates in 10% FCS. The next day, the cells were scratched, treated with different concentrations of NPD8733, and cultured in medium containing 1% FCS. The edge of the wound at the 0 time point is marked with a red outline and was overlaid with other images taken at 24 h. D , the cell migration percentages for fibroblasts and co-cultured cells were calculated using ImageJ. The graph shows the percentage of fibroblast migration in co-cultures relative to the control (fibroblasts alone) at 24 h and was plotted against the NPD8733 concentration ( n = 3; ***, p

    Techniques Used: Migration, Cell Culture, Co-culture Assay, Concentration Assay

    3) Product Images from "Siva-1 regulates multidrug resistance of gastric cancer by targeting MDR1 and MRP1 via the NF-κB pathway"

    Article Title: Siva-1 regulates multidrug resistance of gastric cancer by targeting MDR1 and MRP1 via the NF-κB pathway

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2020.11211

    Effect of Siva-1 overexpression on KATO III/VCR cell growth. (A and B) Apoptotic rate in Siva-1 overexpressed-KATO III/VCR cells was analyzed by flow cytometry. (C and D) KATO III/VCR + LV-Siva-1 cells, KATO III/VCR + LV-NC cells and KATO III/VCR cells were plated in 6-well plates at a density of 200 cells/well, after which colony growth was observed under an optical microscope following 14 days (magnification, ×40). The surviving fraction of cells (visible colonies) was stained with gentian violet and counted manually. Data are presented as the mean ± standard deviation from 3 independent experiments. *P
    Figure Legend Snippet: Effect of Siva-1 overexpression on KATO III/VCR cell growth. (A and B) Apoptotic rate in Siva-1 overexpressed-KATO III/VCR cells was analyzed by flow cytometry. (C and D) KATO III/VCR + LV-Siva-1 cells, KATO III/VCR + LV-NC cells and KATO III/VCR cells were plated in 6-well plates at a density of 200 cells/well, after which colony growth was observed under an optical microscope following 14 days (magnification, ×40). The surviving fraction of cells (visible colonies) was stained with gentian violet and counted manually. Data are presented as the mean ± standard deviation from 3 independent experiments. *P

    Techniques Used: Over Expression, Flow Cytometry, Microscopy, Staining, Standard Deviation

    Siva-1 overexpression increases KATO III/VCR cell migration and invasion. (A and B) KATO III/VCR + LV-Siva-1 cells, KATO III/VCR + LV-NC cells and KATO III/VCR cells were cultured to confluence on 6-well plates, after which a central linear wound was created with a 200-µl sterile pipette tip. The wound was imaged over a 4-day interval (magnification, ×40). (C and D) KATO III/VCR + LV-Siva-1 cells, KATO III/VCR + LV-NC cells and KATO III/VCR cells were loaded into the upper chambers of a Matrigel-coated Transwell plate. Filtrated cells on the undersurface of the polycarbonate membranes were stained and counted under an optical microscope after 48 h (magnification, ×200). *P
    Figure Legend Snippet: Siva-1 overexpression increases KATO III/VCR cell migration and invasion. (A and B) KATO III/VCR + LV-Siva-1 cells, KATO III/VCR + LV-NC cells and KATO III/VCR cells were cultured to confluence on 6-well plates, after which a central linear wound was created with a 200-µl sterile pipette tip. The wound was imaged over a 4-day interval (magnification, ×40). (C and D) KATO III/VCR + LV-Siva-1 cells, KATO III/VCR + LV-NC cells and KATO III/VCR cells were loaded into the upper chambers of a Matrigel-coated Transwell plate. Filtrated cells on the undersurface of the polycarbonate membranes were stained and counted under an optical microscope after 48 h (magnification, ×200). *P

    Techniques Used: Over Expression, Migration, Cell Culture, Transferring, Staining, Microscopy

    4) Product Images from "CASC21, a FOXP1 induced long non-coding RNA, promotes colorectal cancer growth by regulating CDK6"

    Article Title: CASC21, a FOXP1 induced long non-coding RNA, promotes colorectal cancer growth by regulating CDK6

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.103376

    CASC21 promotes CRC cells growth in vitro . ( A ) CCK-8 assays of HCT-116 and HCT-8 cells transfected with CASC21 siRNAs. ( B ) HCT-116 and HCT-8 cells transfected with CASC21 siRNAs were seeded onto 6-well plates. The number of colonies was counted on the 14 th day after seeding. ( C ) Flow cytometric cell apoptosis assays used to assess the effect of CASC21 knockdown on cell apoptosis. All data represent mean ± SEM (n = 3-6). *P
    Figure Legend Snippet: CASC21 promotes CRC cells growth in vitro . ( A ) CCK-8 assays of HCT-116 and HCT-8 cells transfected with CASC21 siRNAs. ( B ) HCT-116 and HCT-8 cells transfected with CASC21 siRNAs were seeded onto 6-well plates. The number of colonies was counted on the 14 th day after seeding. ( C ) Flow cytometric cell apoptosis assays used to assess the effect of CASC21 knockdown on cell apoptosis. All data represent mean ± SEM (n = 3-6). *P

    Techniques Used: In Vitro, CCK-8 Assay, Transfection

    5) Product Images from "WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition"

    Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.006122

    WISP1 induced an EMT gene signature in mouse/human melanoma cells. Unless otherwise specified, all cells were plated on 6-well plates in complete growth medium for 48 h before they were harvested for RNA analysis or treated with the indicated conditioned medium or recombinant protein. A , mRNA expression, revealed by real-time quantitative RT-PCR, of select EMT marker genes and Mitf in uninvaded and invaded B16F10 cells from a Boyden transwell invasion assay. B , immunoblot analysis of WISP1 protein to confirm the disruption of Wisp1 gene in B16F10 and YUMM1.7 knockout cells. 20 μg of whole-cell lysate was loaded in each lane, and β-actin was used as an internal loading control. B16F10-KO1-mWisp1 cells, in which mouse WISP1 expression was resumed with retroviral transduction, were used as a positive control. C , immunoblot analysis of certain EMT marker proteins in B16F10 and YUMM1.7 knockout cells. 20 μg of whole-cell lysate was loaded in each lane, and all cells were compared on the same gel to reveal the relative intensity of each protein. D , comparison of EMT marker gene expression in mouse melanoma B16F10 and its two Wisp1 -knockout cells (-KO1 and -KO2). E , comparison of EMT marker gene expression in mouse melanoma YUMM1.7 and its two Wisp1 -knockout cells (-KO1 and -KO2). F , comparison of EMT marker gene expression in human melanoma RPMI-7951 and its two WISP1 -knockout cells (-KO1 and -KO2). G , stimulation of EMT marker gene expression with recombinant mouse WISP1 protein (rmWISP1). B16F10-KO1 cells were treated with rmWISP1 (final concentration 5 μg/ml) and harvested at the indicated time point for real-time quantitative RT-PCR analysis. H , stimulation of EMT marker gene expression with WISP1-overexpressed or WISP1-immunodepleted conditioned medium ( CM ). The conditioned media were pretreated with the indicated antibodies for 30 min before they were used on Wisp1 -knockout B16F10 cells (-KO1). The cells were collected for real-time qRT-PCR after 3 h of treatment. *, p
    Figure Legend Snippet: WISP1 induced an EMT gene signature in mouse/human melanoma cells. Unless otherwise specified, all cells were plated on 6-well plates in complete growth medium for 48 h before they were harvested for RNA analysis or treated with the indicated conditioned medium or recombinant protein. A , mRNA expression, revealed by real-time quantitative RT-PCR, of select EMT marker genes and Mitf in uninvaded and invaded B16F10 cells from a Boyden transwell invasion assay. B , immunoblot analysis of WISP1 protein to confirm the disruption of Wisp1 gene in B16F10 and YUMM1.7 knockout cells. 20 μg of whole-cell lysate was loaded in each lane, and β-actin was used as an internal loading control. B16F10-KO1-mWisp1 cells, in which mouse WISP1 expression was resumed with retroviral transduction, were used as a positive control. C , immunoblot analysis of certain EMT marker proteins in B16F10 and YUMM1.7 knockout cells. 20 μg of whole-cell lysate was loaded in each lane, and all cells were compared on the same gel to reveal the relative intensity of each protein. D , comparison of EMT marker gene expression in mouse melanoma B16F10 and its two Wisp1 -knockout cells (-KO1 and -KO2). E , comparison of EMT marker gene expression in mouse melanoma YUMM1.7 and its two Wisp1 -knockout cells (-KO1 and -KO2). F , comparison of EMT marker gene expression in human melanoma RPMI-7951 and its two WISP1 -knockout cells (-KO1 and -KO2). G , stimulation of EMT marker gene expression with recombinant mouse WISP1 protein (rmWISP1). B16F10-KO1 cells were treated with rmWISP1 (final concentration 5 μg/ml) and harvested at the indicated time point for real-time quantitative RT-PCR analysis. H , stimulation of EMT marker gene expression with WISP1-overexpressed or WISP1-immunodepleted conditioned medium ( CM ). The conditioned media were pretreated with the indicated antibodies for 30 min before they were used on Wisp1 -knockout B16F10 cells (-KO1). The cells were collected for real-time qRT-PCR after 3 h of treatment. *, p

    Techniques Used: Recombinant, Expressing, Quantitative RT-PCR, Marker, Transwell Invasion Assay, Knock-Out, Transduction, Positive Control, Concentration Assay

    WISP1 knockout in mouse and human melanoma cells inhibited tumor cell migration and invasion. A , 48-h 2D growth of mouse metastatic melanoma cell line B16F10 and two B16F10 Wisp1 -knockout cells (-KO1 and -KO2). B , anchorage-independent growth assay of B16F10 and the two knockout cells in soft agar. Colonies were fixed and counted after 14 days. A representative staining image for each sample is shown on the left , and colony counts are plotted on the right. C , wound healing assay of B16F10 and the two knockout cells. Scratches were created on 6-well plates in biological triplicate, and the healing rate was calculated after 24 h. D , Boyden transwell migration assay of B16F10 and the two knockout cells. A representative staining image for each sample is shown on the left , and relative migration efficiency is graphed on the right. E , Boyden transwell invasion assay of B16F10 and the two knockout cells. F , Boyden transwell invasion assay of human metastatic melanoma cell line RPMI-7951 and its two WISP1 -knockout cells (-KO1 and -KO2). G , transwell migration assay of B16F10 and its knockout cell (-KO1) using conditioned media with different concentrations of WISP1 as chemoattractant. B16F10 migrated cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative migration efficiency and compared with other cells. H , transwell invasion assay of B16F10 and the two knockout cells using conditioned media with different concentrations of WISP1 as chemoattractant. B16F10 invaded cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative invasion efficiency and compared with other cells. Statistical significance was determined by Student's t test, where p
    Figure Legend Snippet: WISP1 knockout in mouse and human melanoma cells inhibited tumor cell migration and invasion. A , 48-h 2D growth of mouse metastatic melanoma cell line B16F10 and two B16F10 Wisp1 -knockout cells (-KO1 and -KO2). B , anchorage-independent growth assay of B16F10 and the two knockout cells in soft agar. Colonies were fixed and counted after 14 days. A representative staining image for each sample is shown on the left , and colony counts are plotted on the right. C , wound healing assay of B16F10 and the two knockout cells. Scratches were created on 6-well plates in biological triplicate, and the healing rate was calculated after 24 h. D , Boyden transwell migration assay of B16F10 and the two knockout cells. A representative staining image for each sample is shown on the left , and relative migration efficiency is graphed on the right. E , Boyden transwell invasion assay of B16F10 and the two knockout cells. F , Boyden transwell invasion assay of human metastatic melanoma cell line RPMI-7951 and its two WISP1 -knockout cells (-KO1 and -KO2). G , transwell migration assay of B16F10 and its knockout cell (-KO1) using conditioned media with different concentrations of WISP1 as chemoattractant. B16F10 migrated cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative migration efficiency and compared with other cells. H , transwell invasion assay of B16F10 and the two knockout cells using conditioned media with different concentrations of WISP1 as chemoattractant. B16F10 invaded cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative invasion efficiency and compared with other cells. Statistical significance was determined by Student's t test, where p

    Techniques Used: Knock-Out, Migration, Growth Assay, Staining, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

    WISP1 activated AKT and MEK/ERK signaling and promoted EMT marker gene expression in mouse melanoma cells. Unless otherwise specified, cell treatment for kinase immunoblot analysis was maintained for 30 min before cells were lysed for protein extraction, whereas cell treatment for comparison of EMT marker gene expression was maintained for 3 h before cells were harvested for RNA extraction. A , comparison of EMT marker gene expression after inhibition of AKT and/or MEK/ERK signaling in B16F10 cells. DMSO was used for control cells. Immunoblotting for phospho-AKT ( pAKT ) and phospho-ERK1/2 ( pERK1/2 ) is shown in the top right corner . Pan-AKT and total ERK1/2 were probed as loading control. B , comparison of EMT marker gene expression after inhibition of AKT and/or MEK/ERK signaling in YUMM1.7 cells. C , immunoblot analysis of AKT and ERK1/2 activation in the indicated mouse melanoma cells with treatment of recombinant mouse WISP1 protein (rmWISP1; final concentration 5 μg/ml). All cells were grown on 6-well plates in complete DMEM for 48 h and SFM for another 48 h before rmWISP1 was added. D , immunoblot analysis of AKT and ERK1/2 activation in B16F10 knockout cell (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were grown on 6-well plates in complete DMEM for 48 h (0-h point for SFM) and switched to SFM for 24 or 48 h. The indicated cells were treated with rmWISP1 at the 0-, 24-, and 48-h time points (of SFM) for 30 min before they were lysed for kinase analysis. The first lane on the gels was loaded with YUMM1.7 at the 0-h point to compare the relative kinase level between B16F10 and YUMM1.7 cells. E , immunoblot analysis of AKT and ERK1/2 activation in YUMM1.7 knockout cells (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were treated similarly as described in D . The first lane on the gels was loaded with B16F10 at the 0-h point to compare the relative kinase level between B16F10 and YUMM1.7 cells. F , comparison of SNAI11 activation and E-cadherin repression in B16F10 knockout cells (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were treated similarly as described in D except that rmWISP1 treatment at each point was maintained for 3 h. G and H , comparison of EMT marker gene expression after AKT/ERK1/2 activation in B16F10-KO1 ( G ) or YUMM1.7-KO1 ( H ) by rmWISP1 was blocked. rmWISP1 with DMSO or inhibitors was added after the indicated cells were grown on 6-well plates in complete DMEM for 48 h and in SFM for 24 h. The relative protein levels of pAKT and AKT and of pERK1/2 and ERK1/2 in C–E . *, p
    Figure Legend Snippet: WISP1 activated AKT and MEK/ERK signaling and promoted EMT marker gene expression in mouse melanoma cells. Unless otherwise specified, cell treatment for kinase immunoblot analysis was maintained for 30 min before cells were lysed for protein extraction, whereas cell treatment for comparison of EMT marker gene expression was maintained for 3 h before cells were harvested for RNA extraction. A , comparison of EMT marker gene expression after inhibition of AKT and/or MEK/ERK signaling in B16F10 cells. DMSO was used for control cells. Immunoblotting for phospho-AKT ( pAKT ) and phospho-ERK1/2 ( pERK1/2 ) is shown in the top right corner . Pan-AKT and total ERK1/2 were probed as loading control. B , comparison of EMT marker gene expression after inhibition of AKT and/or MEK/ERK signaling in YUMM1.7 cells. C , immunoblot analysis of AKT and ERK1/2 activation in the indicated mouse melanoma cells with treatment of recombinant mouse WISP1 protein (rmWISP1; final concentration 5 μg/ml). All cells were grown on 6-well plates in complete DMEM for 48 h and SFM for another 48 h before rmWISP1 was added. D , immunoblot analysis of AKT and ERK1/2 activation in B16F10 knockout cell (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were grown on 6-well plates in complete DMEM for 48 h (0-h point for SFM) and switched to SFM for 24 or 48 h. The indicated cells were treated with rmWISP1 at the 0-, 24-, and 48-h time points (of SFM) for 30 min before they were lysed for kinase analysis. The first lane on the gels was loaded with YUMM1.7 at the 0-h point to compare the relative kinase level between B16F10 and YUMM1.7 cells. E , immunoblot analysis of AKT and ERK1/2 activation in YUMM1.7 knockout cells (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were treated similarly as described in D . The first lane on the gels was loaded with B16F10 at the 0-h point to compare the relative kinase level between B16F10 and YUMM1.7 cells. F , comparison of SNAI11 activation and E-cadherin repression in B16F10 knockout cells (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were treated similarly as described in D except that rmWISP1 treatment at each point was maintained for 3 h. G and H , comparison of EMT marker gene expression after AKT/ERK1/2 activation in B16F10-KO1 ( G ) or YUMM1.7-KO1 ( H ) by rmWISP1 was blocked. rmWISP1 with DMSO or inhibitors was added after the indicated cells were grown on 6-well plates in complete DMEM for 48 h and in SFM for 24 h. The relative protein levels of pAKT and AKT and of pERK1/2 and ERK1/2 in C–E . *, p

    Techniques Used: Marker, Expressing, Protein Extraction, RNA Extraction, Inhibition, Activation Assay, Recombinant, Concentration Assay, Knock-Out

    SNAI1 overexpression in B16F10 Wisp1 -knockout cell rescued the repression of tumor invasion in vitro and metastasis in vivo . A , immunoblot analysis of WISP1 and SNAI1 using B16F10-KO1 cell that were transduced with retroviral vector control (-pBabe) or retrovirus expressing either mouse WISP1 (-mWisp1) or human SNAI1 (-hSnai1). B , comparison of EMT marker gene expression after overexpression of SNAI1 or reintroduction of WISP1 in B16F10-KO1 cells. Cells were plated on 6-well plates in complete growth medium for 48 h before they were harvested for RNA analysis. C , Boyden transwell invasion assay after overexpression of SNAI1 or reintroduction of WISP1 in B16F10-KO1 cells. A representative staining image for each sample is shown on the left , and relative invasion efficiency is graphed on the right. D , experimental metastasis assay in NSG mice using the indicated cells. Each group contained 3–4 mice. All mice were imaged 1 day before the end of the assay, and representative bioluminescence images are shown. E , representative lung and liver images from NSG mice in the experimental metastasis assay described in D . Metastatic tumor colonies on the lung surface from mice with -mWisp1 or -hSnai1 cells are indicated by arrows. F , real-time genomic qPCR for lungs and livers from the experimental metastasis assay in D . The quantitative tumor metastatic burdens were presented as tumor cell number within 10,000 mouse tissue cells. A high-resolution image for E . *, p
    Figure Legend Snippet: SNAI1 overexpression in B16F10 Wisp1 -knockout cell rescued the repression of tumor invasion in vitro and metastasis in vivo . A , immunoblot analysis of WISP1 and SNAI1 using B16F10-KO1 cell that were transduced with retroviral vector control (-pBabe) or retrovirus expressing either mouse WISP1 (-mWisp1) or human SNAI1 (-hSnai1). B , comparison of EMT marker gene expression after overexpression of SNAI1 or reintroduction of WISP1 in B16F10-KO1 cells. Cells were plated on 6-well plates in complete growth medium for 48 h before they were harvested for RNA analysis. C , Boyden transwell invasion assay after overexpression of SNAI1 or reintroduction of WISP1 in B16F10-KO1 cells. A representative staining image for each sample is shown on the left , and relative invasion efficiency is graphed on the right. D , experimental metastasis assay in NSG mice using the indicated cells. Each group contained 3–4 mice. All mice were imaged 1 day before the end of the assay, and representative bioluminescence images are shown. E , representative lung and liver images from NSG mice in the experimental metastasis assay described in D . Metastatic tumor colonies on the lung surface from mice with -mWisp1 or -hSnai1 cells are indicated by arrows. F , real-time genomic qPCR for lungs and livers from the experimental metastasis assay in D . The quantitative tumor metastatic burdens were presented as tumor cell number within 10,000 mouse tissue cells. A high-resolution image for E . *, p

    Techniques Used: Over Expression, Knock-Out, In Vitro, In Vivo, Transduction, Plasmid Preparation, Expressing, Marker, Transwell Invasion Assay, Staining, Mouse Assay, Real-time Polymerase Chain Reaction

    6) Product Images from "UBR-Box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target"

    Article Title: UBR-Box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target

    Journal: bioRxiv

    doi: 10.1101/2020.06.03.131912

    UBR5 deficient A549 cells show decreased cell viability and clonogenic potential. (a) A549 cells were infected with the lenti-viruses from multiple shRNA construct against UBR5 and were cultured for 4 days. Alamar Blue readings were recorded every 24 hours and relative cell viability of UBR5 deficient cells were compared to control cells on each day. (b) A549 cells were infected with shRNA against UBR5 and 1000 cells were cultured in 6-well plate for 10 days. Colonies were fixed in methanol and stained with crystal violet. (c) Quantitative evaluation of clonogenic assay. Representative bar graph showing number of colonies formed per 1000 cells
    Figure Legend Snippet: UBR5 deficient A549 cells show decreased cell viability and clonogenic potential. (a) A549 cells were infected with the lenti-viruses from multiple shRNA construct against UBR5 and were cultured for 4 days. Alamar Blue readings were recorded every 24 hours and relative cell viability of UBR5 deficient cells were compared to control cells on each day. (b) A549 cells were infected with shRNA against UBR5 and 1000 cells were cultured in 6-well plate for 10 days. Colonies were fixed in methanol and stained with crystal violet. (c) Quantitative evaluation of clonogenic assay. Representative bar graph showing number of colonies formed per 1000 cells

    Techniques Used: Infection, shRNA, Construct, Cell Culture, Staining, Clonogenic Assay

    7) Product Images from "Protein arginine methyltransferase 6 mediates cardiac hypertrophy by differential regulation of histone H3 arginine methylation"

    Article Title: Protein arginine methyltransferase 6 mediates cardiac hypertrophy by differential regulation of histone H3 arginine methylation

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2020.e03864

    PRMT6 silencing blunts the effect of phenylephrine. NRVM were transfected with PRMT6 siRNA (siPRMT6) or control siRNA (siCTL) as mentioned in Materials and Methods. (A) after 48 h, total cell lysates were prepared and subjected to Western blot analysis to measure PRMT6 and ANP protein expression. GAPDH is shown as loading control. (B) Independent experiments performed to measure histone marks. Cell lysates were analyzed for H3R2Me2a and H3K4Me3 by Western blot. GAPDH was used as loading control. (C) Relationship between H3R2Me2a and H3K4Me3 in cells expressing normal or low PRMT6 maintained in serum-free condition or treated with PE. (D) NRVM were cultured in 6-well plates and transfected with control siRNA or PRMT6 siRNA. After transfection, cells were maintained in serum-free condition or challenged with 100 μM PE. After 24 h, the cells were stained with TRITC-labelled phalloidin for F-actin. Cells were visualized by fluorescence microscopy. ( E ) Cell surface area was measured in at least 5 different fields using a 40x objective using Adobe acrobat CS6. Data are expressed as mean ± SD. Statistical analysis was performed by One-way ANOVA (panel E) or two-way ANOVA (panel C) followed by Tukey's multiple comparison test, with ∗p
    Figure Legend Snippet: PRMT6 silencing blunts the effect of phenylephrine. NRVM were transfected with PRMT6 siRNA (siPRMT6) or control siRNA (siCTL) as mentioned in Materials and Methods. (A) after 48 h, total cell lysates were prepared and subjected to Western blot analysis to measure PRMT6 and ANP protein expression. GAPDH is shown as loading control. (B) Independent experiments performed to measure histone marks. Cell lysates were analyzed for H3R2Me2a and H3K4Me3 by Western blot. GAPDH was used as loading control. (C) Relationship between H3R2Me2a and H3K4Me3 in cells expressing normal or low PRMT6 maintained in serum-free condition or treated with PE. (D) NRVM were cultured in 6-well plates and transfected with control siRNA or PRMT6 siRNA. After transfection, cells were maintained in serum-free condition or challenged with 100 μM PE. After 24 h, the cells were stained with TRITC-labelled phalloidin for F-actin. Cells were visualized by fluorescence microscopy. ( E ) Cell surface area was measured in at least 5 different fields using a 40x objective using Adobe acrobat CS6. Data are expressed as mean ± SD. Statistical analysis was performed by One-way ANOVA (panel E) or two-way ANOVA (panel C) followed by Tukey's multiple comparison test, with ∗p

    Techniques Used: Transfection, Western Blot, Aqueous Normal-phase Chromatography, Expressing, Cell Culture, Staining, Fluorescence, Microscopy

    8) Product Images from "FK506 induces lung lymphatic endothelial cell senescence and downregulates LYVE-1 expression, with associated decreased hyaluronan uptake"

    Article Title: FK506 induces lung lymphatic endothelial cell senescence and downregulates LYVE-1 expression, with associated decreased hyaluronan uptake

    Journal: Molecular Medicine

    doi: 10.1186/s10020-020-00204-z

    FK506 inhibits TNF-α-induced lymphangiogenesis. Lung lymphatic endothelial cells were grown in 6 well dishes and treated or not with FK506 for 48 h (15 ng/ml). Cells were then placed on Matrigel and treated as indicated with or without TNF-α (100 ng/ml) or FK506 (15 ng/ml) for 16 h. After Calcein-AM labeling, random 4X images were taken and representative images of lymphangiogenesis in different treatment groups after Calcein-AM labeling are presented ( a ) (Size bar 200 μm). Mesh area ( b ) and total segment length ( c ) were analyzed with ImageJ Angiogenesis Analyzer plugin. Results are expressed as mean ± SEM of one experiment. ***  P
    Figure Legend Snippet: FK506 inhibits TNF-α-induced lymphangiogenesis. Lung lymphatic endothelial cells were grown in 6 well dishes and treated or not with FK506 for 48 h (15 ng/ml). Cells were then placed on Matrigel and treated as indicated with or without TNF-α (100 ng/ml) or FK506 (15 ng/ml) for 16 h. After Calcein-AM labeling, random 4X images were taken and representative images of lymphangiogenesis in different treatment groups after Calcein-AM labeling are presented ( a ) (Size bar 200 μm). Mesh area ( b ) and total segment length ( c ) were analyzed with ImageJ Angiogenesis Analyzer plugin. Results are expressed as mean ± SEM of one experiment. *** P

    Techniques Used: Labeling

    FK506 reduces HA uptake in vitro and LYVE-1 expression ex-vivo in PCLS. LEC were plated in 6-well plates and treated with vehicle or FK506 (15 ng/mL) for 72 h. Cells were then incubated in media containing 1000 μg/mL of FITC-HA for 5 h. Percentage of FITC-positive cells ( a ) were analyzed by flow cytometry. Representative images of immunofluorescent staining of single and merged images of HA (green) and DIC without and with treatment with FK506 (15 ng/ml) for 72 h ( b ) (Size bar: 50 μm). Precision cut lung slices obtained from dt-Tomato Prox1 mice, treated with FK506 (15 ng/mL) or DMSO for 7 days, followed by digestion and cell isolation. Percent LYVE1+/dt-Tomato-Prox1+ cells ( c ) were analyzed by flow cytometry. Results for flow cytometry are expressed as mean ± SEM of 3 experiments. Dotted line represents each data point and solid line represents mean of 3 experiments
    Figure Legend Snippet: FK506 reduces HA uptake in vitro and LYVE-1 expression ex-vivo in PCLS. LEC were plated in 6-well plates and treated with vehicle or FK506 (15 ng/mL) for 72 h. Cells were then incubated in media containing 1000 μg/mL of FITC-HA for 5 h. Percentage of FITC-positive cells ( a ) were analyzed by flow cytometry. Representative images of immunofluorescent staining of single and merged images of HA (green) and DIC without and with treatment with FK506 (15 ng/ml) for 72 h ( b ) (Size bar: 50 μm). Precision cut lung slices obtained from dt-Tomato Prox1 mice, treated with FK506 (15 ng/mL) or DMSO for 7 days, followed by digestion and cell isolation. Percent LYVE1+/dt-Tomato-Prox1+ cells ( c ) were analyzed by flow cytometry. Results for flow cytometry are expressed as mean ± SEM of 3 experiments. Dotted line represents each data point and solid line represents mean of 3 experiments

    Techniques Used: In Vitro, Expressing, Ex Vivo, Incubation, Flow Cytometry, Staining, Mouse Assay, Cell Isolation

    9) Product Images from "The Inactive 44-kDa Processed Form of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Enhances Proteolytic Activity via Regulation of Endocytosis of Active MT1-MMP *The Inactive 44-kDa Processed Form of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Enhances Proteolytic Activity via Regulation of Endocytosis of Active MT1-MMP * S⃞"

    Article Title: The Inactive 44-kDa Processed Form of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Enhances Proteolytic Activity via Regulation of Endocytosis of Active MT1-MMP *The Inactive 44-kDa Processed Form of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Enhances Proteolytic Activity via Regulation of Endocytosis of Active MT1-MMP * S⃞

    Journal:

    doi: 10.1074/jbc.M708943200

    Recombinant 44-MT1 expression correlates with enhanced pro-MMP-2 activation. Analyses of pro-MMP-2 activation by gelatin zymography. HT1080 ( A, parental; B, pooled populations and clones), MDA-MB-231 ( F ), and U87 ( G ) cells grown in 6-well plates were
    Figure Legend Snippet: Recombinant 44-MT1 expression correlates with enhanced pro-MMP-2 activation. Analyses of pro-MMP-2 activation by gelatin zymography. HT1080 ( A, parental; B, pooled populations and clones), MDA-MB-231 ( F ), and U87 ( G ) cells grown in 6-well plates were

    Techniques Used: Recombinant, Expressing, Activation Assay, Zymography, Multiple Displacement Amplification

    10) Product Images from "The tyrosine 73 and serine 83 dephosphorylation of H1N1 swine influenza virus NS1 protein attenuates virus replication and induces high levels of beta interferon"

    Article Title: The tyrosine 73 and serine 83 dephosphorylation of H1N1 swine influenza virus NS1 protein attenuates virus replication and induces high levels of beta interferon

    Journal: Virology Journal

    doi: 10.1186/s12985-019-1255-0

    Growth kinetics of recombinant viruses with NS1 mutants lacking a phosphorylation site. a Growth kinetics of the recombinant viruses in MDCK cells. Recombinant viruses possessing wild-type NS1, NS1 Y73F and NS1 S83A were generated by reverse genetics, as described in the Materials and Methods. MDCK cells were infected with rSIV or mutated virus (rSIV NS1 Y73F and rSIV NS1 S83A) at an MOI of 0.001. Supernatants were collected at 12, 24, 48, and 72 hpi, and virus titers were determined by TCID 50 assay. The mean values from three independent experiments are shown for each sample. b Expression kinetics of NS1 protein during infection. MDCK cells on 6-well plates were infected at an MOI of 0.001 with rSIV or mutated viruses, and cell lysates were collected at the indicated time points for western blot analysis of NS1 protein levels.C. Percentage of the NS1 protein reduction level. The percentage of protein reduction (%) was calculated by the formula [(NS1 expression of rSIV infected cells - NS1 expression of mutant viruses infected cells)/ NS1 expression of rSIV infected cells] × 100%; Abscissa: time points after infection
    Figure Legend Snippet: Growth kinetics of recombinant viruses with NS1 mutants lacking a phosphorylation site. a Growth kinetics of the recombinant viruses in MDCK cells. Recombinant viruses possessing wild-type NS1, NS1 Y73F and NS1 S83A were generated by reverse genetics, as described in the Materials and Methods. MDCK cells were infected with rSIV or mutated virus (rSIV NS1 Y73F and rSIV NS1 S83A) at an MOI of 0.001. Supernatants were collected at 12, 24, 48, and 72 hpi, and virus titers were determined by TCID 50 assay. The mean values from three independent experiments are shown for each sample. b Expression kinetics of NS1 protein during infection. MDCK cells on 6-well plates were infected at an MOI of 0.001 with rSIV or mutated viruses, and cell lysates were collected at the indicated time points for western blot analysis of NS1 protein levels.C. Percentage of the NS1 protein reduction level. The percentage of protein reduction (%) was calculated by the formula [(NS1 expression of rSIV infected cells - NS1 expression of mutant viruses infected cells)/ NS1 expression of rSIV infected cells] × 100%; Abscissa: time points after infection

    Techniques Used: Recombinant, Generated, Infection, Expressing, Western Blot, Mutagenesis

    11) Product Images from "Enhancement of Endometrial Receptivity by Cnidium officinale through Expressing LIF and Integrins"

    Article Title: Enhancement of Endometrial Receptivity by Cnidium officinale through Expressing LIF and Integrins

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2019/7560631

    Effect of antagonist for LIFR and neutralization for integrins α V, β 3, and β 5 on the adhesion of JAr cells to CoM-stimulated Ishikawa cells. (a) Ishikawa cells were treated with or without LIF antagonist (hLA) (50 ng/mL) for 1 h and then CoM (50 μ g/ml) was added to hLA-pretreated Ishikawa cells for 48 h. (b) Ishikawa cells (1.5 × 10 6 cells) were cultured in 6-well plates and treated with or without CoM (50 μ g/ml) for 48 h, and the cells were then incubated in the presence of integrin α V, β 3, β 5, and IgG antibodies for 2 h. CMFDA-labeled JAr cells were added onto Ishikawa cell monolayer. The attached cells were fixed and pictured using fluorescent microscopy. Data were calculated as mean ± SEM of three independent experiments and expressed as fold of controls. ∗∗∗ p
    Figure Legend Snippet: Effect of antagonist for LIFR and neutralization for integrins α V, β 3, and β 5 on the adhesion of JAr cells to CoM-stimulated Ishikawa cells. (a) Ishikawa cells were treated with or without LIF antagonist (hLA) (50 ng/mL) for 1 h and then CoM (50 μ g/ml) was added to hLA-pretreated Ishikawa cells for 48 h. (b) Ishikawa cells (1.5 × 10 6 cells) were cultured in 6-well plates and treated with or without CoM (50 μ g/ml) for 48 h, and the cells were then incubated in the presence of integrin α V, β 3, β 5, and IgG antibodies for 2 h. CMFDA-labeled JAr cells were added onto Ishikawa cell monolayer. The attached cells were fixed and pictured using fluorescent microscopy. Data were calculated as mean ± SEM of three independent experiments and expressed as fold of controls. ∗∗∗ p

    Techniques Used: Neutralization, Cell Culture, Incubation, Labeling, Microscopy

    12) Product Images from "RNF8 Dysregulation and Down-regulation During HTLV-1 Infection Promote Genomic Instability in Adult T-Cell Leukemia"

    Article Title: RNF8 Dysregulation and Down-regulation During HTLV-1 Infection Promote Genomic Instability in Adult T-Cell Leukemia

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008618

    The DDR impairment in HTLV-1-infected HeLa cells cannot be rescued by RNF8 over-expression. (A) HTLV-1-infected HeLa cells are DDR impaired . HeLa-G: ΔN-IκBα and its progeny, HeLa-G: ΔN-IκBα:HTLV-1, that has been stably infected by HTLV-1 were treated with 12 μM bleomycin for 1 hr to induce DNA damage, washed, grown in fresh bleomycin-free media and harvested at the indicated times post-treatment (0, 1, 3, 8 and 16 hrs) and monitored by IB for γH2AX, RNF8, p19 matrix protein, and β-actin control. (B) RNF8 is crucial for the repair of DNA double-strand breaks. HeLa-G and HeLa-G ΔRNF8 were similarly treated with bleomycin as in (A) and monitored by IB for RNF8, γH2AX, H2AX and β-actin control. (C) RNF8 over-expression rescued the DDR defect in RNF8-null cells. HeLa-G ΔRNF8 cells (2X10 5 ) grown in 6-well plates were infected by Ad-GFP (odd-number lanes) or Ad-RNF8 (even-number lanes) at an MOI of 5 for 16 hrs. The infected cells were treated with bleomycin and monitored for rescue at indicated times by γH2AX, RNF8, and β-actin IB. (D) RNF8 over-expression could not rescue the DDR defect of HTLV-1-infected cells. HeLa-G: ΔN-IκBα (lanes 1 2) and HeLa-G: ΔN-IκBα:HTLV-1 (lanes 3 4) were infected by Ad-GFP or Ad-RNF8 at MOI of 5, respectively. Twenty-four hours post-infection, cells were treated with bleomycin as in (A), and grown in bleomycin-free media for another 16 hrs. Thereupon, cells were harvested and immunoblotted for the RNF8, γH2AX, Tax and GAPDH. See Supplemental Information for quantitation of representative immunoblots.
    Figure Legend Snippet: The DDR impairment in HTLV-1-infected HeLa cells cannot be rescued by RNF8 over-expression. (A) HTLV-1-infected HeLa cells are DDR impaired . HeLa-G: ΔN-IκBα and its progeny, HeLa-G: ΔN-IκBα:HTLV-1, that has been stably infected by HTLV-1 were treated with 12 μM bleomycin for 1 hr to induce DNA damage, washed, grown in fresh bleomycin-free media and harvested at the indicated times post-treatment (0, 1, 3, 8 and 16 hrs) and monitored by IB for γH2AX, RNF8, p19 matrix protein, and β-actin control. (B) RNF8 is crucial for the repair of DNA double-strand breaks. HeLa-G and HeLa-G ΔRNF8 were similarly treated with bleomycin as in (A) and monitored by IB for RNF8, γH2AX, H2AX and β-actin control. (C) RNF8 over-expression rescued the DDR defect in RNF8-null cells. HeLa-G ΔRNF8 cells (2X10 5 ) grown in 6-well plates were infected by Ad-GFP (odd-number lanes) or Ad-RNF8 (even-number lanes) at an MOI of 5 for 16 hrs. The infected cells were treated with bleomycin and monitored for rescue at indicated times by γH2AX, RNF8, and β-actin IB. (D) RNF8 over-expression could not rescue the DDR defect of HTLV-1-infected cells. HeLa-G: ΔN-IκBα (lanes 1 2) and HeLa-G: ΔN-IκBα:HTLV-1 (lanes 3 4) were infected by Ad-GFP or Ad-RNF8 at MOI of 5, respectively. Twenty-four hours post-infection, cells were treated with bleomycin as in (A), and grown in bleomycin-free media for another 16 hrs. Thereupon, cells were harvested and immunoblotted for the RNF8, γH2AX, Tax and GAPDH. See Supplemental Information for quantitation of representative immunoblots.

    Techniques Used: Infection, Over Expression, Stable Transfection, Quantitation Assay, Western Blot

    13) Product Images from "Titanium dioxide nanoparticles induce endoplasmic reticulum stress-mediated apoptotic cell death in liver cancer cells"

    Article Title: Titanium dioxide nanoparticles induce endoplasmic reticulum stress-mediated apoptotic cell death in liver cancer cells

    Journal: The Journal of International Medical Research

    doi: 10.1177/0300060520903652

    Influence of TiO 2 nanotubes on cell growth. L02 (a) and HepG2 (b) cells were incubated in TiO 2 nanotube-coated (2.5, 5.0, 7.5, and 10 µg/mL) 6-well plates for 48 hours at 60°C. Cell numbers were counted using cell counting chambers. Cells incubated in 6-well plates coated with 0 µg/mL TiO 2 nanotubes were used as a control. * P
    Figure Legend Snippet: Influence of TiO 2 nanotubes on cell growth. L02 (a) and HepG2 (b) cells were incubated in TiO 2 nanotube-coated (2.5, 5.0, 7.5, and 10 µg/mL) 6-well plates for 48 hours at 60°C. Cell numbers were counted using cell counting chambers. Cells incubated in 6-well plates coated with 0 µg/mL TiO 2 nanotubes were used as a control. * P

    Techniques Used: Incubation, Cell Counting

    L02 (a) and HepG2 (b) cells were cultured in 6-well plates for 48 hours, then apoptosis of L02 cells and HepG2 cells was detected by flow cytometry. TiO 2 increased the percentage of apoptotic liver cancer cells. Annexin V-positive cells were considered as apoptotic liver cancer cells. Cells incubated with 0 µg/mL TiO 2 were used as a control. * P
    Figure Legend Snippet: L02 (a) and HepG2 (b) cells were cultured in 6-well plates for 48 hours, then apoptosis of L02 cells and HepG2 cells was detected by flow cytometry. TiO 2 increased the percentage of apoptotic liver cancer cells. Annexin V-positive cells were considered as apoptotic liver cancer cells. Cells incubated with 0 µg/mL TiO 2 were used as a control. * P

    Techniques Used: Cell Culture, Flow Cytometry, Incubation

    14) Product Images from "Selection and validation of reference genes by RT-qPCR for murine cementoblasts in mechanical loading experiments simulating orthodontic forces in vitro"

    Article Title: Selection and validation of reference genes by RT-qPCR for murine cementoblasts in mechanical loading experiments simulating orthodontic forces in vitro

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-67449-w

    Influence of different cell confluences with and without loading compression in gene expression. ( A ) Illustration of the loading compression (LC) setup. Cell monolayer loaded with a sterile glass cylinder in 6-well plate to apply a static compression of 2 g/cm 2 . ( B ) Light microscopy photography of cells before loading (control) and before unloading (LC 6 h and LC 24 h). ×200 magnification, cropped, exposure adjustment and sharpening in Photoshop CC. ( C ) mRNA expression of selected markers of inflammation, osteoblastic and osteoclastic markers mean ± SD, n = 6 (two independent experiments in triplicate); normalization by ddC t method to Rpl22 and control 100% statistical analysis with GraphPad Prism 7, two-way-ANOVA, p
    Figure Legend Snippet: Influence of different cell confluences with and without loading compression in gene expression. ( A ) Illustration of the loading compression (LC) setup. Cell monolayer loaded with a sterile glass cylinder in 6-well plate to apply a static compression of 2 g/cm 2 . ( B ) Light microscopy photography of cells before loading (control) and before unloading (LC 6 h and LC 24 h). ×200 magnification, cropped, exposure adjustment and sharpening in Photoshop CC. ( C ) mRNA expression of selected markers of inflammation, osteoblastic and osteoclastic markers mean ± SD, n = 6 (two independent experiments in triplicate); normalization by ddC t method to Rpl22 and control 100% statistical analysis with GraphPad Prism 7, two-way-ANOVA, p

    Techniques Used: Expressing, Light Microscopy

    15) Product Images from "Double stranded-RNA-mediated activation of P21 gene induced apoptosis and cell cycle arrest in renal cell carcinoma"

    Article Title: Double stranded-RNA-mediated activation of P21 gene induced apoptosis and cell cycle arrest in renal cell carcinoma

    Journal:

    doi: 10.1002/ijc.24370

    p21 promoter-targeted RNAa inhibits cell proliferation and survival. A. Cells were plated in 6-well plate format and transfected with 50 nM dsRNA. On day 3 following transfection, cells were photographed at low power 10x ( A ) and at high power 200x ( B
    Figure Legend Snippet: p21 promoter-targeted RNAa inhibits cell proliferation and survival. A. Cells were plated in 6-well plate format and transfected with 50 nM dsRNA. On day 3 following transfection, cells were photographed at low power 10x ( A ) and at high power 200x ( B

    Techniques Used: Transfection

    dsP21 transfection is associated with a decrease in survivin expression in renal cell carcinoma A498 cell line. Cells were plated in 6-well plates and transfected with 50 nM dsRNAs for 72 hrs. mRNA was isolated for quantitative analysis ( A ). Survivin
    Figure Legend Snippet: dsP21 transfection is associated with a decrease in survivin expression in renal cell carcinoma A498 cell line. Cells were plated in 6-well plates and transfected with 50 nM dsRNAs for 72 hrs. mRNA was isolated for quantitative analysis ( A ). Survivin

    Techniques Used: Transfection, Expressing, Isolation

    A depiction of the p21 promoter and target site is shown in part A . dsP21 induces p21 expression in renal cell carcinoma A498 cell line. Cells were plated in 6-well plates and transfected with 50 nM dsRNAs for 72 hrs. mRNA was isolated for semi-quantitative
    Figure Legend Snippet: A depiction of the p21 promoter and target site is shown in part A . dsP21 induces p21 expression in renal cell carcinoma A498 cell line. Cells were plated in 6-well plates and transfected with 50 nM dsRNAs for 72 hrs. mRNA was isolated for semi-quantitative

    Techniques Used: Expressing, Transfection, Isolation

    16) Product Images from "HIV-1 Vpr induces cell cycle arrest and enhances viral gene expression by depleting CCDC137"

    Article Title: HIV-1 Vpr induces cell cycle arrest and enhances viral gene expression by depleting CCDC137

    Journal: eLife

    doi: 10.7554/eLife.55806

    Discovery and analysis of proteins labelled with Vpr-BirA (R118G). ( A ) Proteins recovered from lysates of MT-4 cells expressing Vpr-BirA (R118G) or BirA (R118G) using streptavidin beads. Biotinylated proteins were detected with IRDye 680RD-conjugated Streptavidin. Representative of two experiments. ( B ) Immunostaining/FACS analysis of endogenous Ki-67 levels in 293 T cells 28 hr after transfection (in 6-well plates) with 100 ng of a GFP expression plasmid and increasing amounts (0 ng, 50 ng, 100 ng, 200 ng, or 400 ng) of an HA-Vpr expression plasmid. Representative of two experiments. ( C ) Western blot analysis of endogenous Ki-67 and HA-Vpr expression in 293 T cells 48 hr after infection with V1/δ-Vpr or V1/HA-Vpr at an MOI of 2. Representative of two experiments. ( D ) Western blot analysis of Ki-67 and tubulin (loading control) levels in U2OS cells transduced with lentiviruses carrying no shRNA (vector) or shRNA against Ki-67 (shRNA Ki-67) after selection with puromycin. Representative of three experiments. ( E ) Analysis of DNA content by propidium iodide staining in U2OS cells transduced with lentiviruses carrying no shRNA (vector) or shRNA against Ki-67 (shRNA Ki-67) after selection with puromycin. Representative of three experiments.
    Figure Legend Snippet: Discovery and analysis of proteins labelled with Vpr-BirA (R118G). ( A ) Proteins recovered from lysates of MT-4 cells expressing Vpr-BirA (R118G) or BirA (R118G) using streptavidin beads. Biotinylated proteins were detected with IRDye 680RD-conjugated Streptavidin. Representative of two experiments. ( B ) Immunostaining/FACS analysis of endogenous Ki-67 levels in 293 T cells 28 hr after transfection (in 6-well plates) with 100 ng of a GFP expression plasmid and increasing amounts (0 ng, 50 ng, 100 ng, 200 ng, or 400 ng) of an HA-Vpr expression plasmid. Representative of two experiments. ( C ) Western blot analysis of endogenous Ki-67 and HA-Vpr expression in 293 T cells 48 hr after infection with V1/δ-Vpr or V1/HA-Vpr at an MOI of 2. Representative of two experiments. ( D ) Western blot analysis of Ki-67 and tubulin (loading control) levels in U2OS cells transduced with lentiviruses carrying no shRNA (vector) or shRNA against Ki-67 (shRNA Ki-67) after selection with puromycin. Representative of three experiments. ( E ) Analysis of DNA content by propidium iodide staining in U2OS cells transduced with lentiviruses carrying no shRNA (vector) or shRNA against Ki-67 (shRNA Ki-67) after selection with puromycin. Representative of three experiments.

    Techniques Used: Expressing, Immunostaining, FACS, Transfection, Plasmid Preparation, Western Blot, Infection, Transduction, shRNA, Selection, Staining

    17) Product Images from "Identification of key hemagglutinin residues responsible for cleavage, acid stability, and virulence of fifth-wave highly pathogenic avian influenza A(H7N9) viruses"

    Article Title: Identification of key hemagglutinin residues responsible for cleavage, acid stability, and virulence of fifth-wave highly pathogenic avian influenza A(H7N9) viruses

    Journal: Virology

    doi: 10.1016/j.virol.2019.07.012

    Replication and stability of wild-type and mutant PR8:TW/17-HA/NA viruses. (A) Vero cells grown on 6-well-plates were inoculated in triplicate with wild-type or mutant (HA2-K64E) recombinant viruses at an MOI of 0.01 and incubated at 37 °C; supernatants from the cultures were collected at 4, 24, 48, and 72 h p. i. for titration in eggs and are expressed as the mean log 10 EID 50 /ml with standard deviation (SD). (B) Mean hemagglutination titers (log 2 with SD) are presented from three replicates of the wild-type or mutant viruses after incubation at 56 °C for the indicated times. (C) Wild-type and mutant viruses (75 μl) were incubated in triplicate at 20 °C and 50% relative humidity for the indicated times. Viral titers were determined in eggs and are expressed as the mean log 10 EID 50 /mL with SD. (D) Wild-type and mutant viruses (10 μl) were deposited onto 96-well plates and were incubated in an environmental chamber set for 20 °C, 50% relative humidity for the indicated lengths of time. Six replicates for each time point were collected by reconstitution in 60 μl of PBS and viral titers were determined in eggs and are expressed as the mean log 10 EID 50 /mL with SD. Limit of detection (
    Figure Legend Snippet: Replication and stability of wild-type and mutant PR8:TW/17-HA/NA viruses. (A) Vero cells grown on 6-well-plates were inoculated in triplicate with wild-type or mutant (HA2-K64E) recombinant viruses at an MOI of 0.01 and incubated at 37 °C; supernatants from the cultures were collected at 4, 24, 48, and 72 h p. i. for titration in eggs and are expressed as the mean log 10 EID 50 /ml with standard deviation (SD). (B) Mean hemagglutination titers (log 2 with SD) are presented from three replicates of the wild-type or mutant viruses after incubation at 56 °C for the indicated times. (C) Wild-type and mutant viruses (75 μl) were incubated in triplicate at 20 °C and 50% relative humidity for the indicated times. Viral titers were determined in eggs and are expressed as the mean log 10 EID 50 /mL with SD. (D) Wild-type and mutant viruses (10 μl) were deposited onto 96-well plates and were incubated in an environmental chamber set for 20 °C, 50% relative humidity for the indicated lengths of time. Six replicates for each time point were collected by reconstitution in 60 μl of PBS and viral titers were determined in eggs and are expressed as the mean log 10 EID 50 /mL with SD. Limit of detection (

    Techniques Used: Mutagenesis, Recombinant, Incubation, Titration, Standard Deviation

    Replication kinetics of recombinant PR8:TW/17-HA/NA viruses in Calu-3 or MDCK cells. Calu-3 cells (grown on 24 mm transwells), which produce extracellular proteases, and MDCK cells (grown on 6-well plates), which do not produce extracellular proteases, were inoculated with recombinant PR8 viruses bearing wild-type or mutant HAs of TW/17 virus at an MOI of 0.01 for 1 h followed by incubation with fresh viral infection medium. The supernatants from the infected Calu-3 (A) or MDCK cells (B) were collected at various time points for subsequent viral titer determination in eggs. Viral titers were plotted as the mean with standard deviation (SD) from three independent replicates. The detection limit indicated by the dashed line was 1.5 log 10 EID 50 /ml. Two-way analysis of variance followed by Dunnett’s multiple comparisons test between the wild-type and each of the mutant viruses was performed with GraphPad Prism. *, p ≤ 0.05; ***, p ≤ 0.001; ‡, p ≤ 0.0001.
    Figure Legend Snippet: Replication kinetics of recombinant PR8:TW/17-HA/NA viruses in Calu-3 or MDCK cells. Calu-3 cells (grown on 24 mm transwells), which produce extracellular proteases, and MDCK cells (grown on 6-well plates), which do not produce extracellular proteases, were inoculated with recombinant PR8 viruses bearing wild-type or mutant HAs of TW/17 virus at an MOI of 0.01 for 1 h followed by incubation with fresh viral infection medium. The supernatants from the infected Calu-3 (A) or MDCK cells (B) were collected at various time points for subsequent viral titer determination in eggs. Viral titers were plotted as the mean with standard deviation (SD) from three independent replicates. The detection limit indicated by the dashed line was 1.5 log 10 EID 50 /ml. Two-way analysis of variance followed by Dunnett’s multiple comparisons test between the wild-type and each of the mutant viruses was performed with GraphPad Prism. *, p ≤ 0.05; ***, p ≤ 0.001; ‡, p ≤ 0.0001.

    Techniques Used: Recombinant, Mutagenesis, Incubation, Infection, Standard Deviation

    18) Product Images from "The Effect of Lithium on Inflammation-Associated Genes in Lipopolysaccharide-Activated Raw 264.7 Macrophages"

    Article Title: The Effect of Lithium on Inflammation-Associated Genes in Lipopolysaccharide-Activated Raw 264.7 Macrophages

    Journal: International Journal of Inflammation

    doi: 10.1155/2020/8340195

    Determination of the apoptosis effect of lithium on Raw 264.7 macrophage cells using fluorescent microscopy. Cells were cultured at a density of 6 × 10 5 cell/ml on coverslip in 6-well plates. Followed by treating cell with lithium 10 mM, NaCl 10 mM, actinomycin-D 0.02 mg/ml, and untreated cell were used as control for 24 hrs. Staining with Annexin-V and PI followed for 30 min at RT in the dark. Thereafter, cells were fixed and mounted on the slides and then measurement of fluorescence was accomplished by capturing pictures at 20x magnification with Nikon Ti-E inverted fluorescent microscope.
    Figure Legend Snippet: Determination of the apoptosis effect of lithium on Raw 264.7 macrophage cells using fluorescent microscopy. Cells were cultured at a density of 6 × 10 5 cell/ml on coverslip in 6-well plates. Followed by treating cell with lithium 10 mM, NaCl 10 mM, actinomycin-D 0.02 mg/ml, and untreated cell were used as control for 24 hrs. Staining with Annexin-V and PI followed for 30 min at RT in the dark. Thereafter, cells were fixed and mounted on the slides and then measurement of fluorescence was accomplished by capturing pictures at 20x magnification with Nikon Ti-E inverted fluorescent microscope.

    Techniques Used: Microscopy, Cell Culture, Staining, Fluorescence

    19) Product Images from "Innate Biomineralization"

    Article Title: Innate Biomineralization

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21144820

    Calcium ion (Ca 2+ ), a phosphoester salt, and alkaline phosphatase (ALP) are essential for promoting biomineralization in MG-63 and Saos-2 cell lines and human blood mononuclear cells (MNCs). ( A ) Alizarin Red S assays (ARS) show mineralization within 7 days, visualized as the intensity of red staining, of Saos-2 cells in MEMα/10% FBS supplemented only with βGP or the combination of βGP/ascorbic acid (Vit. C)/dexamethasone (Dex); MG-63 cell line was inactive under similar conditions. No mineralization was observed in either cell line cultured with MEMα/10% FBS supplemented with Vit. C or Dex. In MEMα/10% FBS supplemented with βGP and ALPL, calf intestinal ALP (CIP), or shrimp ALP (SAP), MG-63 and Saos-2 cell lines were mineralized. ( B ) Similar results were iterated when αGP was used instead of βGP. ( C ) Compared to βGP and phosphoenolpyruvate monosodium (PEP), αGP was the most efficient phosphoester salt elicited biomineralization. Pamidronate (Pamidn) and glycerophosphoric acid (NSC9231) did not elicit the reaction. ( D ) Human MNCs also have an innate ability of mineralization in 7 days without the induction of cellular differentiation (top row indicates initial cell counts per well in a 6-well plate). ( E – G ) Titration assays indicated that biomineralization depended on the doses of αGP, CIP, and Ca 2+ . ( H ) In a 48-well plate, human blood MNCs were seeded (10 5 /well) and exposed to MEMα/10% FBS, the medium supplemented with αGP, with αGP and CIP, or with CIP for 7 days (the media were changed on day 4). On day 7, ARS indicates that biomineralization occurred in MNCs exposed to MEMα/10% FBS supplemented with αGP and CIP in the uncoated wells (top row) and the wells coated with Collagen Type I, rat tail (middle row). Biomineralization did not occur if αGP or CIP was missing. None of the cell-less wells coated with Collagen Type I was positive for HAP by ARS (bottom row). Each of the tests was in duplicates.
    Figure Legend Snippet: Calcium ion (Ca 2+ ), a phosphoester salt, and alkaline phosphatase (ALP) are essential for promoting biomineralization in MG-63 and Saos-2 cell lines and human blood mononuclear cells (MNCs). ( A ) Alizarin Red S assays (ARS) show mineralization within 7 days, visualized as the intensity of red staining, of Saos-2 cells in MEMα/10% FBS supplemented only with βGP or the combination of βGP/ascorbic acid (Vit. C)/dexamethasone (Dex); MG-63 cell line was inactive under similar conditions. No mineralization was observed in either cell line cultured with MEMα/10% FBS supplemented with Vit. C or Dex. In MEMα/10% FBS supplemented with βGP and ALPL, calf intestinal ALP (CIP), or shrimp ALP (SAP), MG-63 and Saos-2 cell lines were mineralized. ( B ) Similar results were iterated when αGP was used instead of βGP. ( C ) Compared to βGP and phosphoenolpyruvate monosodium (PEP), αGP was the most efficient phosphoester salt elicited biomineralization. Pamidronate (Pamidn) and glycerophosphoric acid (NSC9231) did not elicit the reaction. ( D ) Human MNCs also have an innate ability of mineralization in 7 days without the induction of cellular differentiation (top row indicates initial cell counts per well in a 6-well plate). ( E – G ) Titration assays indicated that biomineralization depended on the doses of αGP, CIP, and Ca 2+ . ( H ) In a 48-well plate, human blood MNCs were seeded (10 5 /well) and exposed to MEMα/10% FBS, the medium supplemented with αGP, with αGP and CIP, or with CIP for 7 days (the media were changed on day 4). On day 7, ARS indicates that biomineralization occurred in MNCs exposed to MEMα/10% FBS supplemented with αGP and CIP in the uncoated wells (top row) and the wells coated with Collagen Type I, rat tail (middle row). Biomineralization did not occur if αGP or CIP was missing. None of the cell-less wells coated with Collagen Type I was positive for HAP by ARS (bottom row). Each of the tests was in duplicates.

    Techniques Used: Staining, Cell Culture, Cell Differentiation, Titration

    20) Product Images from "M3 Muscarinic Acetylcholine Receptor-Mediated Signaling is Regulated by Distinct Mechanisms"

    Article Title: M3 Muscarinic Acetylcholine Receptor-Mediated Signaling is Regulated by Distinct Mechanisms

    Journal: Molecular pharmacology

    doi: 10.1124/mol.107.044750

    Characterization of the Muscarinic Acetylcholine Receptor Subtype Endogenously Expressed in HEK-293 Cells. A) HEK-293 cells loaded with the ratiometric calcium indicator Fura2/AM were incubated with 100 nM pirenzepine (orange), 1 μM p-FHHsiD (green), vehicle (red), or not pretreated (black) and stimulated with 100 μM carbachol. Changes in calcium mobilization were assayed by monitoring the change in Fura-2AM fluorescence. Shown is a representative tracing from three independent experiments. B) Following a 6 hr serum starve, HEK-293 cells were incubated with 100 nM pirenzepine, 1 μM p-FHHsiD, vehicle, or not pretreated and stimulated with 100 μM carbachol for the indicated times. Cells from a 6-well plate were harvested and equal amounts of total cellular lysate were separated by SDS-PAGE and probed for phospho-ERK1/2 as described in Materials and Methods. Shown is a representative immunoblot of three independent experiments. C) Cells were treated with Bis I (2.5 μM), Bis V (2.5 μM) or rottlerin (5 μM) for 30 min prior to stimulation with carbachol (100 μM) for 5 min or PMA (100 nM) for 15 min.
    Figure Legend Snippet: Characterization of the Muscarinic Acetylcholine Receptor Subtype Endogenously Expressed in HEK-293 Cells. A) HEK-293 cells loaded with the ratiometric calcium indicator Fura2/AM were incubated with 100 nM pirenzepine (orange), 1 μM p-FHHsiD (green), vehicle (red), or not pretreated (black) and stimulated with 100 μM carbachol. Changes in calcium mobilization were assayed by monitoring the change in Fura-2AM fluorescence. Shown is a representative tracing from three independent experiments. B) Following a 6 hr serum starve, HEK-293 cells were incubated with 100 nM pirenzepine, 1 μM p-FHHsiD, vehicle, or not pretreated and stimulated with 100 μM carbachol for the indicated times. Cells from a 6-well plate were harvested and equal amounts of total cellular lysate were separated by SDS-PAGE and probed for phospho-ERK1/2 as described in Materials and Methods. Shown is a representative immunoblot of three independent experiments. C) Cells were treated with Bis I (2.5 μM), Bis V (2.5 μM) or rottlerin (5 μM) for 30 min prior to stimulation with carbachol (100 μM) for 5 min or PMA (100 nM) for 15 min.

    Techniques Used: Incubation, Fluorescence, SDS Page

    21) Product Images from "Virus subtype-specific suppression of MAVS aggregation and activation by PB1-F2 protein of influenza A (H7N9) virus"

    Article Title: Virus subtype-specific suppression of MAVS aggregation and activation by PB1-F2 protein of influenza A (H7N9) virus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008611

    Suppression of RIG-I-MAVS antiviral signaling by H7N9 PB1-F2. ( A and B ) HEK293T cells in 12-well plates were transfected with 400 ng PB1-F2 expression constructs, 200 ng p125-Luc and 10 ng pRL-TK. After 24 hours, cells were treated with 100 hemagglutinating units/ml of Sendai virus (A) or 1 μg/mL of poly (I:C) (B) for a further 16 hours. Then, cells were harvested for dual-luciferase reporter assay. ( C-G ) HEK293T cells in 24-well plates were transfected with 200 ng PB1-F2 expression constructs, 100ng p125-Luc, 10ng pRL-TK and 50ng expression constructs for type I IFN stimulants including RIG-IN (C), MyD88 (D), MAVS in pEF-Bos vector (E), TBK1 (F) or IRF3-5D (G). After 48 hours, cells were harvested for dual-luciferase reporter assay. ( H and I ) HEK293T cells in 24-well plates were transfected with 200 ng PB1-F2 expression constructs, 100 ng kB-FLuc (H) or IRF3-FLuc (I), 10 ng pRL-TK with or without 50 ng MAVS expression construct for 48 hours before harvested for dual-luciferase reporter assay. ( J ) HEK293T cells in 24-well plates were transfected with 200 ng PB1-F2 expression constructs, 50 ng MAVS expression construct, 10 ng pRL-TK plus 100ng ISRE-FLuc. After 24 hours, cells were mock-treated or treated with 1000 U/mL human recombinant IFNβ protein for 24 hours before harvested for dual-luciferase reporter assay. ( K ) Same as in (A), except the inclusion of a group for H7N9 PB1-F2 S66 (S). ( L ) Same as in (E), except that 50 ng H7N9 PB1-F2 N66 (N) or S66 (S) was used. ( M ) HEK293T cells in 6-well plates were transfected with 1 μg expression construct for H7N9 PB1-F2 with N66 (N) or S66 (S) for 48 hours before SDS-PAGE and Western blot analysis. Anti-Flag recognized PB1-F2 protein. GAPDH served as internal loading control. All bars denote means ± SD of triplicate experiments. Unpaired Student’s t-test was performed to evaluate the statistical significance of the difference between vector control (vec) and the indicated sample. ***: P
    Figure Legend Snippet: Suppression of RIG-I-MAVS antiviral signaling by H7N9 PB1-F2. ( A and B ) HEK293T cells in 12-well plates were transfected with 400 ng PB1-F2 expression constructs, 200 ng p125-Luc and 10 ng pRL-TK. After 24 hours, cells were treated with 100 hemagglutinating units/ml of Sendai virus (A) or 1 μg/mL of poly (I:C) (B) for a further 16 hours. Then, cells were harvested for dual-luciferase reporter assay. ( C-G ) HEK293T cells in 24-well plates were transfected with 200 ng PB1-F2 expression constructs, 100ng p125-Luc, 10ng pRL-TK and 50ng expression constructs for type I IFN stimulants including RIG-IN (C), MyD88 (D), MAVS in pEF-Bos vector (E), TBK1 (F) or IRF3-5D (G). After 48 hours, cells were harvested for dual-luciferase reporter assay. ( H and I ) HEK293T cells in 24-well plates were transfected with 200 ng PB1-F2 expression constructs, 100 ng kB-FLuc (H) or IRF3-FLuc (I), 10 ng pRL-TK with or without 50 ng MAVS expression construct for 48 hours before harvested for dual-luciferase reporter assay. ( J ) HEK293T cells in 24-well plates were transfected with 200 ng PB1-F2 expression constructs, 50 ng MAVS expression construct, 10 ng pRL-TK plus 100ng ISRE-FLuc. After 24 hours, cells were mock-treated or treated with 1000 U/mL human recombinant IFNβ protein for 24 hours before harvested for dual-luciferase reporter assay. ( K ) Same as in (A), except the inclusion of a group for H7N9 PB1-F2 S66 (S). ( L ) Same as in (E), except that 50 ng H7N9 PB1-F2 N66 (N) or S66 (S) was used. ( M ) HEK293T cells in 6-well plates were transfected with 1 μg expression construct for H7N9 PB1-F2 with N66 (N) or S66 (S) for 48 hours before SDS-PAGE and Western blot analysis. Anti-Flag recognized PB1-F2 protein. GAPDH served as internal loading control. All bars denote means ± SD of triplicate experiments. Unpaired Student’s t-test was performed to evaluate the statistical significance of the difference between vector control (vec) and the indicated sample. ***: P

    Techniques Used: Transfection, Expressing, Construct, Luciferase, Reporter Assay, Plasmid Preparation, Recombinant, SDS Page, Western Blot

    Suppression of MAVS aggregation by H7N9 PB1-F2. ( A ) SDD-AGE analysis of MAVS aggregation. HEK293T cells in 6-well plates were co-transfected with 1 μg pEF-Bos-Flag-MAVS and 0.2 μg expression construct for PB1-F2 of WSN, H5N1 or H7N9 virus. After 48 hours, cells were harvested for SDD-AGE or SDS-PAGE followed by Western blot analysis with anti-MAVS. ( B ) Co-immunoprecipitation assay for PB1-F2-MAVS association. HEK293T cells in 60mm dishes were co-transfected with 1 μg pCAGEN-myc-MAVS and 0.5 μg PB1-F2-Flag expression construct. After 48 hours, cells were harvested for co-immunoprecipitation with anti-Flag (IP: Flag). Both input and immunoprecipitates were analyzed by SDS-PAGE and Western blot analysis with anti-Myc for MAVS, anti-Flag for PB1-F2 and anti-GAPDH for normalization. ( C ) Distribution of unaggregated MAVS on fissioned mitochondria in cells expressing H7N9 PB1-F2. HEK293T cells in 6-well plates were co-transfected with 0.25 μg of CAGEN-V5-MAVS and 0.25 μg of PB1-F2-Flag expression constructs. After 48 hours, cells were stained with 500 nM Mitotracker Red CMXRos for 30 min and then fixed with 4% paraformaldehyde and probed with anti-Flag for PB1-F2, anti-MAVS and DAPI. The stained cells were analyzed by confocal microscopy. Fractions of cells with unaggregated and aggregated MAVS were calculated by counting 100 cells per sample. Aggregated MAVS or PB1-F2 was visually defined as concentrated dots or structures with intense MAVS or PB1-F2 signal. Bars, 20 μm. Similar results were obtained from three independent experiments.
    Figure Legend Snippet: Suppression of MAVS aggregation by H7N9 PB1-F2. ( A ) SDD-AGE analysis of MAVS aggregation. HEK293T cells in 6-well plates were co-transfected with 1 μg pEF-Bos-Flag-MAVS and 0.2 μg expression construct for PB1-F2 of WSN, H5N1 or H7N9 virus. After 48 hours, cells were harvested for SDD-AGE or SDS-PAGE followed by Western blot analysis with anti-MAVS. ( B ) Co-immunoprecipitation assay for PB1-F2-MAVS association. HEK293T cells in 60mm dishes were co-transfected with 1 μg pCAGEN-myc-MAVS and 0.5 μg PB1-F2-Flag expression construct. After 48 hours, cells were harvested for co-immunoprecipitation with anti-Flag (IP: Flag). Both input and immunoprecipitates were analyzed by SDS-PAGE and Western blot analysis with anti-Myc for MAVS, anti-Flag for PB1-F2 and anti-GAPDH for normalization. ( C ) Distribution of unaggregated MAVS on fissioned mitochondria in cells expressing H7N9 PB1-F2. HEK293T cells in 6-well plates were co-transfected with 0.25 μg of CAGEN-V5-MAVS and 0.25 μg of PB1-F2-Flag expression constructs. After 48 hours, cells were stained with 500 nM Mitotracker Red CMXRos for 30 min and then fixed with 4% paraformaldehyde and probed with anti-Flag for PB1-F2, anti-MAVS and DAPI. The stained cells were analyzed by confocal microscopy. Fractions of cells with unaggregated and aggregated MAVS were calculated by counting 100 cells per sample. Aggregated MAVS or PB1-F2 was visually defined as concentrated dots or structures with intense MAVS or PB1-F2 signal. Bars, 20 μm. Similar results were obtained from three independent experiments.

    Techniques Used: Transfection, Expressing, Construct, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Staining, Confocal Microscopy

    Influence of H7N9 PB1-F2 expression on mitochondrial morphology and function. ( A ) Mitotracker Red CMXRos staining. HEK293T cells over coverslips in 6-well plates were transfected with 1μg of PB1-F2-Flag expression construct. After 48 hours, cells were stained with 500 nM Mitotracker Red CMXRos for 30 min and then fixed with 4% paraformaldehyde. PB1-F2 was probed with anti-Flag and nuclei were stained with DAPI. The stained cells were analyzed by confocal microscopy. Arrows indicated distinct mitochondrial clusters colocalized with PB1-F2. vec: vector control. Bars, 20 μm. ( B ) JC-1 staining. HEK293T cells in 12-well plates were transfected with 0.5 μg of PB1-F2-Flag expression construct. After 48 hours, cells were stained with JC-1 dye. For CCCP control, cells were co-treated with 50 μM CCCP and JC-1 dye. JC-1 dye-stained cells were analyzed through fluorescence microplate reader for red and green signals, which respectively indicated healthy and depolarized mitochondria. The data represent mean values ± SD of red-to-green ratio of JC-1 staining of three independent experiments. Unpaired Student’s t-test was used to assess the statistical significance of the difference between vector control (vec) and sample. ***: P
    Figure Legend Snippet: Influence of H7N9 PB1-F2 expression on mitochondrial morphology and function. ( A ) Mitotracker Red CMXRos staining. HEK293T cells over coverslips in 6-well plates were transfected with 1μg of PB1-F2-Flag expression construct. After 48 hours, cells were stained with 500 nM Mitotracker Red CMXRos for 30 min and then fixed with 4% paraformaldehyde. PB1-F2 was probed with anti-Flag and nuclei were stained with DAPI. The stained cells were analyzed by confocal microscopy. Arrows indicated distinct mitochondrial clusters colocalized with PB1-F2. vec: vector control. Bars, 20 μm. ( B ) JC-1 staining. HEK293T cells in 12-well plates were transfected with 0.5 μg of PB1-F2-Flag expression construct. After 48 hours, cells were stained with JC-1 dye. For CCCP control, cells were co-treated with 50 μM CCCP and JC-1 dye. JC-1 dye-stained cells were analyzed through fluorescence microplate reader for red and green signals, which respectively indicated healthy and depolarized mitochondria. The data represent mean values ± SD of red-to-green ratio of JC-1 staining of three independent experiments. Unpaired Student’s t-test was used to assess the statistical significance of the difference between vector control (vec) and sample. ***: P

    Techniques Used: Expressing, Staining, Transfection, Construct, Confocal Microscopy, Plasmid Preparation, Fluorescence

    Facilitation of MAVS degradation by H7N9 PB1-F2 in immune-stimulated cells. ( A and B ) Impact of H7N9 PB1-F2 on MAVS protein level. THP-1 cells were infected with H7-2 WT and H7-2ΔF viruses at MOI = 1. At 6, 12, and 24 hpi, infected cells were harvested for total protein extraction, SDS-PAGE and Western blot analysis with anti-MAVS and anti-H7N9 PB1-F2 (A). PA and GAPDH served as normalization controls for viral infection and loading. Harvested cells were also subjected to RT-qPCR analysis of MAVS mRNA level relative to that of GAPDH mRNA using comparative Ct method (B). ( C and D ) Impact of WSN PB1-F2 on MAVS protein level. THP-1 cells were infected with WSN WT and WSN ΔF viruses at MOI = 1. MAVS protein and mRNA were analyzed as above. ( E ) HEK293T cells in 6-well plates were co-transfected with 1 μg pEF-Bos-Flag-MAVS and 0.2 μg PB1-F2 expression constructs. After 24 hours, 100 μg/mL cycloheximide (CHX) was added. Cells were harvested for total protein extraction at 0, 6, 12 and 24 hours after drug treatment for SDS-PAGE followed by Western blot analysis with anti-Flag. Relative band intensity of MAVS over GAPDH was plotted in the lower panel. ( F ) HEK293T cells were transfected with 0.5 μg pEF-Bos-Flag-MAVS together with increasing dosage of expression construct for PB1-F2-Flag of WSN and H7N9 (0, 0.25, 0.5, 1, 1.5 and 2 μg). After 48 hours, cells were harvested for total protein extraction followed by SDS-PAGE and Western blot analysis with anti-Flag for detection of both MAVS (70 kDa) and PB1-F2 (15–20 kDa) normalized to GAPDH. ( G and H ) HEK293T cells in 6-well plates were transfected with 1.5 μg PB1-F2 expression constructs. After 24 hours, cells were either mock-transfected or transfected with 1 μg/mL poly (I:C) for 18 hours before harvested for total protein extraction followed by SDS-PAGE and Western blot analysis with anti-MAVS and anti-Flag (G) or for RT-qPCR analysis of MAVS mRNA (H). ( I and J ) HEK293T cells in 6-well plates were transfected with 1.5 μg PB1-F2 expression constru cts. After 24 hours, cells were either mock-infected or infected with Sendai virus (100 hemagglutinating units/ml) for 18 hours before harvested for total protein extraction followed by SDS-PAGE and Western blot analysis with anti-MAVS and anti-Flag (I) or for RT-qPCR analysis of MAVS mRNA relative to HPRT mRNA by comparative Ct method (J). Bars represent means ± SD of triplicate experiments.
    Figure Legend Snippet: Facilitation of MAVS degradation by H7N9 PB1-F2 in immune-stimulated cells. ( A and B ) Impact of H7N9 PB1-F2 on MAVS protein level. THP-1 cells were infected with H7-2 WT and H7-2ΔF viruses at MOI = 1. At 6, 12, and 24 hpi, infected cells were harvested for total protein extraction, SDS-PAGE and Western blot analysis with anti-MAVS and anti-H7N9 PB1-F2 (A). PA and GAPDH served as normalization controls for viral infection and loading. Harvested cells were also subjected to RT-qPCR analysis of MAVS mRNA level relative to that of GAPDH mRNA using comparative Ct method (B). ( C and D ) Impact of WSN PB1-F2 on MAVS protein level. THP-1 cells were infected with WSN WT and WSN ΔF viruses at MOI = 1. MAVS protein and mRNA were analyzed as above. ( E ) HEK293T cells in 6-well plates were co-transfected with 1 μg pEF-Bos-Flag-MAVS and 0.2 μg PB1-F2 expression constructs. After 24 hours, 100 μg/mL cycloheximide (CHX) was added. Cells were harvested for total protein extraction at 0, 6, 12 and 24 hours after drug treatment for SDS-PAGE followed by Western blot analysis with anti-Flag. Relative band intensity of MAVS over GAPDH was plotted in the lower panel. ( F ) HEK293T cells were transfected with 0.5 μg pEF-Bos-Flag-MAVS together with increasing dosage of expression construct for PB1-F2-Flag of WSN and H7N9 (0, 0.25, 0.5, 1, 1.5 and 2 μg). After 48 hours, cells were harvested for total protein extraction followed by SDS-PAGE and Western blot analysis with anti-Flag for detection of both MAVS (70 kDa) and PB1-F2 (15–20 kDa) normalized to GAPDH. ( G and H ) HEK293T cells in 6-well plates were transfected with 1.5 μg PB1-F2 expression constructs. After 24 hours, cells were either mock-transfected or transfected with 1 μg/mL poly (I:C) for 18 hours before harvested for total protein extraction followed by SDS-PAGE and Western blot analysis with anti-MAVS and anti-Flag (G) or for RT-qPCR analysis of MAVS mRNA (H). ( I and J ) HEK293T cells in 6-well plates were transfected with 1.5 μg PB1-F2 expression constru cts. After 24 hours, cells were either mock-infected or infected with Sendai virus (100 hemagglutinating units/ml) for 18 hours before harvested for total protein extraction followed by SDS-PAGE and Western blot analysis with anti-MAVS and anti-Flag (I) or for RT-qPCR analysis of MAVS mRNA relative to HPRT mRNA by comparative Ct method (J). Bars represent means ± SD of triplicate experiments.

    Techniques Used: Infection, Protein Extraction, SDS Page, Western Blot, Quantitative RT-PCR, Transfection, Expressing, Construct

    H7N9 PB1-F2-induced destabilization of MAVS protein aggregate for proteasomal and lysosomal degradation. ( A ) Treatment with proteasome and lysosome inhibitors. HEK293T cells in 6-well plates were co-transfected with 1 μg PB1-F2-Flag expression construct and 1 μg pEF-Bos-Flag-MAVS. After 24 hours, 20 μM MG132, 100 nM bafilomycin A1 (BaA1) or their combination was added for 6 or 16 hours before cells were harvested for total protein extraction and SDS-PAGE Western blot analysis against anti-Flag. Relative band intensity of MAVS over GAPDH was plotted in the right panel. ( B ) Analysis of aggregation-defective mutants of MAVS. HEK293T cells were co-transfected with 0.5 μg PB1-F2 expression construct and 0.5 μg pEF-Bos-Flag-MAVS WT, E26A, W56R or R64, 65A. After 48 hours, total protein was extracted from the cells and subjected to SDS-PAGE followed by Western blot analysis with anti-Flag for detection of MAVS at 70 kDa and PB1-F2 at 15-20kDa. Relative band intensity of MAVS over GAPDH was plotted in the lower panel. Three independent experiments were performed with similar results.
    Figure Legend Snippet: H7N9 PB1-F2-induced destabilization of MAVS protein aggregate for proteasomal and lysosomal degradation. ( A ) Treatment with proteasome and lysosome inhibitors. HEK293T cells in 6-well plates were co-transfected with 1 μg PB1-F2-Flag expression construct and 1 μg pEF-Bos-Flag-MAVS. After 24 hours, 20 μM MG132, 100 nM bafilomycin A1 (BaA1) or their combination was added for 6 or 16 hours before cells were harvested for total protein extraction and SDS-PAGE Western blot analysis against anti-Flag. Relative band intensity of MAVS over GAPDH was plotted in the right panel. ( B ) Analysis of aggregation-defective mutants of MAVS. HEK293T cells were co-transfected with 0.5 μg PB1-F2 expression construct and 0.5 μg pEF-Bos-Flag-MAVS WT, E26A, W56R or R64, 65A. After 48 hours, total protein was extracted from the cells and subjected to SDS-PAGE followed by Western blot analysis with anti-Flag for detection of MAVS at 70 kDa and PB1-F2 at 15-20kDa. Relative band intensity of MAVS over GAPDH was plotted in the lower panel. Three independent experiments were performed with similar results.

    Techniques Used: Transfection, Expressing, Construct, Protein Extraction, SDS Page, Western Blot

    High aggregation potential of H7N9 PB1-F2. (A) Sequence alignment of PB1-F2 proteins from WSN, H5N1 and H7N9 viruses. Amino acid sequences of PB1-F2 proteins from human WSN, H5N1 and H7N9 viruses were aligned by the Clustal Omega program ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). Identical, similar and weakly similar residues are indicated by *,: and., respectively. ( B ) Protein expression. HEK293T and DF-1 cells in 6-well plates were transfected with expression construct (1 μg) for PB1-F2-Flag from WSN (W), H5N1 (H5) and H7N9 (H7) viruses. After 42 hours, cells were either mock treated with DMSO or treated with 10 μM MG132 for 6 more hours and then subjected to total protein extraction and Western blot analysis with anti-Flag. α-tubulin served as an internal control for protein loading. Slight differences in the actual molecular masses of PB1-F2 proteins from WSN, H5N1 and H7N9 viruses on the 12% polyacrylamide gel might be explained at least partially by the differences in their calculated molecular masses. ( C ) Bioinformatic prediction of cross-β sheet structure. Amino acid sequences of PB1-F2 from WSN, H5N1 and H7N9 viruses were analyzed for putative cross-β sheet structure by PASTA 2.0 ( http://protein.bio.unipd.it/pasta2/ ). The top panels were the free-energy profile plotting free energy of cross β sheet pairing against residue number k. The bottom panels represented probability matrix that showed probability of pairing of residues m and k of two self-aligned PB1-F2 amino acid sequences. Probability calculation was as described [ 29 ]. Dot intensity was proportional to probability of pairing. ( D and E ) Aggregation and solubility assays. HEK293T cells in 6-well plates were transfected with PB1-F2-Flag expression construct (1 μg). After 42 hours, cells were either mock treated with DMSO or treated with 10 μM MG132 for 6 more hours and subjected to SDD-AGE and SDS-PAGE analysis (D) as well as protein solubility assay with RIPA lysis buffer (E). GAPDH or β-actin served as internal control for protein loading. Similar results were obtained from three independent experiments.
    Figure Legend Snippet: High aggregation potential of H7N9 PB1-F2. (A) Sequence alignment of PB1-F2 proteins from WSN, H5N1 and H7N9 viruses. Amino acid sequences of PB1-F2 proteins from human WSN, H5N1 and H7N9 viruses were aligned by the Clustal Omega program ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). Identical, similar and weakly similar residues are indicated by *,: and., respectively. ( B ) Protein expression. HEK293T and DF-1 cells in 6-well plates were transfected with expression construct (1 μg) for PB1-F2-Flag from WSN (W), H5N1 (H5) and H7N9 (H7) viruses. After 42 hours, cells were either mock treated with DMSO or treated with 10 μM MG132 for 6 more hours and then subjected to total protein extraction and Western blot analysis with anti-Flag. α-tubulin served as an internal control for protein loading. Slight differences in the actual molecular masses of PB1-F2 proteins from WSN, H5N1 and H7N9 viruses on the 12% polyacrylamide gel might be explained at least partially by the differences in their calculated molecular masses. ( C ) Bioinformatic prediction of cross-β sheet structure. Amino acid sequences of PB1-F2 from WSN, H5N1 and H7N9 viruses were analyzed for putative cross-β sheet structure by PASTA 2.0 ( http://protein.bio.unipd.it/pasta2/ ). The top panels were the free-energy profile plotting free energy of cross β sheet pairing against residue number k. The bottom panels represented probability matrix that showed probability of pairing of residues m and k of two self-aligned PB1-F2 amino acid sequences. Probability calculation was as described [ 29 ]. Dot intensity was proportional to probability of pairing. ( D and E ) Aggregation and solubility assays. HEK293T cells in 6-well plates were transfected with PB1-F2-Flag expression construct (1 μg). After 42 hours, cells were either mock treated with DMSO or treated with 10 μM MG132 for 6 more hours and subjected to SDD-AGE and SDS-PAGE analysis (D) as well as protein solubility assay with RIPA lysis buffer (E). GAPDH or β-actin served as internal control for protein loading. Similar results were obtained from three independent experiments.

    Techniques Used: Sequencing, Expressing, Transfection, Construct, Protein Extraction, Western Blot, Solubility, SDS Page, Lysis

    22) Product Images from "MiR-1587 Regulates DNA Damage Repair and the Radiosensitivity of CRC Cells via Targeting LIG4"

    Article Title: MiR-1587 Regulates DNA Damage Repair and the Radiosensitivity of CRC Cells via Targeting LIG4

    Journal: Dose-Response

    doi: 10.1177/1559325820936906

    Overexpression of miR-1587 induces CRC cells DSBs and induces G1 phase arrest. A, MiR-1587 enhances γ-H2AX expression. CRC cells were transfected with miR-1587 or miR-NC mimics for 48 hours and the expression of γ-H2AX was detected by Western blot analysis (left). The protein was quantified by gray-scale statistics (right). B, Overexpression of miR-1587 triggers the formation of γ-H2AX foci. CRC cells were seeded onto coverslips in 6-well plates and transfected with miR-1587 or miR-NC mimics for 48 hours. γ-H2AX foci were detected by indirect immunofluorescence staining from a minimum of 200 randomly selected cells in each group. Scale bar: 20 μm. t test, mean ± SD, n = 200. C, MiR-1587 overexpression induces G1 phase arrest. CRC cells were transfected with miR-1587 or miR-NC mimics for 48 hours and the cell cycle analysis was performed via flow cytometry. A total of 10 000 cells in each sample were scored. t test, mean ± SD, n = 3. * P
    Figure Legend Snippet: Overexpression of miR-1587 induces CRC cells DSBs and induces G1 phase arrest. A, MiR-1587 enhances γ-H2AX expression. CRC cells were transfected with miR-1587 or miR-NC mimics for 48 hours and the expression of γ-H2AX was detected by Western blot analysis (left). The protein was quantified by gray-scale statistics (right). B, Overexpression of miR-1587 triggers the formation of γ-H2AX foci. CRC cells were seeded onto coverslips in 6-well plates and transfected with miR-1587 or miR-NC mimics for 48 hours. γ-H2AX foci were detected by indirect immunofluorescence staining from a minimum of 200 randomly selected cells in each group. Scale bar: 20 μm. t test, mean ± SD, n = 200. C, MiR-1587 overexpression induces G1 phase arrest. CRC cells were transfected with miR-1587 or miR-NC mimics for 48 hours and the cell cycle analysis was performed via flow cytometry. A total of 10 000 cells in each sample were scored. t test, mean ± SD, n = 3. * P

    Techniques Used: Over Expression, Expressing, Transfection, Western Blot, Immunofluorescence, Staining, Cell Cycle Assay, Flow Cytometry

    Overexpression of miR-1587 enhances the radiosensitivity of HT29 cells. MiR-1587 overexpression enhances the radiosensitivity of HT29 cells. After transfection with miR-1587 or miR-NC mimics for 48 hours, HT29 cells were collected and reseeded into 6-well plates, and colony-formation assays were performed following irradiation (0, 1, 2, and 4 Gy) for 2 weeks. t test, mean ± SD, n = 3. * P
    Figure Legend Snippet: Overexpression of miR-1587 enhances the radiosensitivity of HT29 cells. MiR-1587 overexpression enhances the radiosensitivity of HT29 cells. After transfection with miR-1587 or miR-NC mimics for 48 hours, HT29 cells were collected and reseeded into 6-well plates, and colony-formation assays were performed following irradiation (0, 1, 2, and 4 Gy) for 2 weeks. t test, mean ± SD, n = 3. * P

    Techniques Used: Over Expression, Transfection, Irradiation

    Overexpression of miR-1587 inhibits proliferation and promotes apoptosis of CRC cells. A, Identification of miR-1587 overexpression. Cells were transfected with miR-1587 or miR-NC mimics. After 48 hours, miR-1587 expression was detected by RT-qPCR. U6 was used as an internal control. t test, mean ± SD, n = 3. B, MiR-1587 inhibits the proliferation of CRC cells. Cells were seeded into 96-well plates and transfected with miR-1587 or miR-NC mimics. Cell proliferation was measured via CCK-8 assays at the indicated time points. Cells morphologies were assessed via microscopy. Scale bar: 300 μm. t test, mean ± SD, n = 5. C, MiR-1587 overexpression regulates CRC clone formation. CRC cells transfected with miR-1587 or miR-NC mimics for 48 hours and reseeded at low density into 6-well plates, and colony-formation assays were performed after 14 days. t test, mean ± SD, n = 3. D, MiR-1587 overexpression promotes CRC cells apoptosis. Cells were transfected with miR-1587 and miR-NC mimics for 48 hours and apoptosis was assessed by flow cytometry. * P
    Figure Legend Snippet: Overexpression of miR-1587 inhibits proliferation and promotes apoptosis of CRC cells. A, Identification of miR-1587 overexpression. Cells were transfected with miR-1587 or miR-NC mimics. After 48 hours, miR-1587 expression was detected by RT-qPCR. U6 was used as an internal control. t test, mean ± SD, n = 3. B, MiR-1587 inhibits the proliferation of CRC cells. Cells were seeded into 96-well plates and transfected with miR-1587 or miR-NC mimics. Cell proliferation was measured via CCK-8 assays at the indicated time points. Cells morphologies were assessed via microscopy. Scale bar: 300 μm. t test, mean ± SD, n = 5. C, MiR-1587 overexpression regulates CRC clone formation. CRC cells transfected with miR-1587 or miR-NC mimics for 48 hours and reseeded at low density into 6-well plates, and colony-formation assays were performed after 14 days. t test, mean ± SD, n = 3. D, MiR-1587 overexpression promotes CRC cells apoptosis. Cells were transfected with miR-1587 and miR-NC mimics for 48 hours and apoptosis was assessed by flow cytometry. * P

    Techniques Used: Over Expression, Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Microscopy, Flow Cytometry

    23) Product Images from "Wnt10b-overexpressing umbilical cord mesenchymal stem cells promote critical size rat calvarial defect healing by enhanced osteogenesis and VEGF-mediated angiogenesis"

    Article Title: Wnt10b-overexpressing umbilical cord mesenchymal stem cells promote critical size rat calvarial defect healing by enhanced osteogenesis and VEGF-mediated angiogenesis

    Journal: Journal of Orthopaedic Translation

    doi: 10.1016/j.jot.2020.02.009

    HUCMSCs Wnt10b had robust self-renewal ability and osteogenic differentiation ability but decreased adipogenic differentiation ability . (A) The self-renewal ability of HUCMSCs Emp and HUCMSCs Wnt10b was assessed by CFU-F assays. HUCMSCs Emp and HUCMSCs Wnt10b were seeded in 6-well plates at 1000 cells/well. Seven days later, the cells were stained with crystal violet (scale bar = 0.5 mm). (B) Colonies, which contain more than 50 cells, were quantified ( n = 3, mean ± SEM, ∗∗: p
    Figure Legend Snippet: HUCMSCs Wnt10b had robust self-renewal ability and osteogenic differentiation ability but decreased adipogenic differentiation ability . (A) The self-renewal ability of HUCMSCs Emp and HUCMSCs Wnt10b was assessed by CFU-F assays. HUCMSCs Emp and HUCMSCs Wnt10b were seeded in 6-well plates at 1000 cells/well. Seven days later, the cells were stained with crystal violet (scale bar = 0.5 mm). (B) Colonies, which contain more than 50 cells, were quantified ( n = 3, mean ± SEM, ∗∗: p

    Techniques Used: Staining

    24) Product Images from "Anti-Cancer Potential of Oxialis obtriangulata in Pancreatic Cancer Cell through Regulation of the ERK/Src/STAT3-Mediated Pathway"

    Article Title: Anti-Cancer Potential of Oxialis obtriangulata in Pancreatic Cancer Cell through Regulation of the ERK/Src/STAT3-Mediated Pathway

    Journal: Molecules

    doi: 10.3390/molecules25102301

    Effects of OOE on proliferation of BxPC3 cells. ( A , B ) BxPC3 cells were seeded in 6-well plates and incubated for 14 days in media with or without OOE (50, 100, and 200 μg/mL), which was changed every three days. Using crystal violet solution, the effect of OOE on long-term cell proliferation was detected. Formed colonies were dissolved in DMSO and shown as a bar graph compared to the control. ( C , D ) BxPC3 cells were seeded with or without OOE (0 or 200 μg/mL) in 4-well plates and incubated for 24 h. Cell were fixed and stained with the anti-Ki67 antibody. Stained cells were observed by a microscope (magnification: 200×, scale bar: 100 μm). Colony formation graph and fluorescence intensity are expressed as the mean ± S.D. *** p
    Figure Legend Snippet: Effects of OOE on proliferation of BxPC3 cells. ( A , B ) BxPC3 cells were seeded in 6-well plates and incubated for 14 days in media with or without OOE (50, 100, and 200 μg/mL), which was changed every three days. Using crystal violet solution, the effect of OOE on long-term cell proliferation was detected. Formed colonies were dissolved in DMSO and shown as a bar graph compared to the control. ( C , D ) BxPC3 cells were seeded with or without OOE (0 or 200 μg/mL) in 4-well plates and incubated for 24 h. Cell were fixed and stained with the anti-Ki67 antibody. Stained cells were observed by a microscope (magnification: 200×, scale bar: 100 μm). Colony formation graph and fluorescence intensity are expressed as the mean ± S.D. *** p

    Techniques Used: Incubation, Staining, Microscopy, Fluorescence

    Effect of OOE on apoptosis and cell cycle arrest of BxPC3 cells. BxPC3 cells were seeded in 6-well plates and incubated with 0, 100, and 200 μg/mL OOE for 24 h. ( A ) Propidium iodide staining analysis of BxPC3 cells was performed by FACS. ( B ) Annexin V analysis was used to confirm the apoptotic effect of OOE on BxPC3 cells. ( C ) Data are presented as the mean ± SEM of three separate experiments. ( D ) The percentages of Annexin-V+/PI− (lower right quadrant) and Annexin-V+/PI+ (upper right quadrant) cells were calculated and shown as a bar graph.* p
    Figure Legend Snippet: Effect of OOE on apoptosis and cell cycle arrest of BxPC3 cells. BxPC3 cells were seeded in 6-well plates and incubated with 0, 100, and 200 μg/mL OOE for 24 h. ( A ) Propidium iodide staining analysis of BxPC3 cells was performed by FACS. ( B ) Annexin V analysis was used to confirm the apoptotic effect of OOE on BxPC3 cells. ( C ) Data are presented as the mean ± SEM of three separate experiments. ( D ) The percentages of Annexin-V+/PI− (lower right quadrant) and Annexin-V+/PI+ (upper right quadrant) cells were calculated and shown as a bar graph.* p

    Techniques Used: Incubation, Staining, FACS

    25) Product Images from "Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression"

    Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008792

    Full-length WT p53 displays pro-proliferation properties in 38HK. (A and B) 38HK were seeded into 6-well plates and transfected with Scramble or CRISPR-p53 plasmid for p53 knockdown. (A) Protein extracts were processed for IB 48 h and 72 h after transfection. Images shown are representative examples of 2 independent experiments. (B) Cells were also collected at 24, 48, and 72 h after transfection, stained with trypan blue, and counted. Error bars represent the standard deviations of 2 independent experiments performed in duplicate. *, p
    Figure Legend Snippet: Full-length WT p53 displays pro-proliferation properties in 38HK. (A and B) 38HK were seeded into 6-well plates and transfected with Scramble or CRISPR-p53 plasmid for p53 knockdown. (A) Protein extracts were processed for IB 48 h and 72 h after transfection. Images shown are representative examples of 2 independent experiments. (B) Cells were also collected at 24, 48, and 72 h after transfection, stained with trypan blue, and counted. Error bars represent the standard deviations of 2 independent experiments performed in duplicate. *, p

    Techniques Used: Transfection, CRISPR, Plasmid Preparation, Staining

    26) Product Images from "Pathophysiological role of microRNA-29 in pancreatic cancer stroma"

    Article Title: Pathophysiological role of microRNA-29 in pancreatic cancer stroma

    Journal: Scientific Reports

    doi: 10.1038/srep11450

    Ectopic expression of miR-29 in PSCs reduces cancer cells viability and cancer colony growth in co-culture. ( a ) Effect of miR-29 overexpression in PSCs on cancer colony growth in direct co-cultures. mPSCs transfected with 20 nM mimic control, mimic-29a or 29b were plated simultaneously with 100 pancreatic cancer cells (Panc-1) in a 6-well plate. Co-cultures were allowed to grow for 10 days, fixed, and stained with crystal violet fixing solution to stain cancer cells as previously described 64 with few modifications. Cancer colonies greater than 50 cells were counted under phase contrast microscopy. Representative images of co-cultures stained with crystal violet are shown. ( b ) Conditioned media of PSCs expressing miR-29 show decreased effect on pancreatic cell viability. Conditioned media from mPSCs transfected with control, miR-29a, or miR-29b mimics was applied to pancreatic cancer cells (Panc-1) in a 96-well plate and viability was measured 24 and 48 hours post-treatment using the Cell Counting Kit-8 assay according to manufacture protocol. Data is normalized to pancreatic cancer cells (Panc-1) treated with non-conditioned media. All experiments were repeated 3-4 times and representative data is presented. Data represents mean + SEM. Statistics generated by t-test, *p
    Figure Legend Snippet: Ectopic expression of miR-29 in PSCs reduces cancer cells viability and cancer colony growth in co-culture. ( a ) Effect of miR-29 overexpression in PSCs on cancer colony growth in direct co-cultures. mPSCs transfected with 20 nM mimic control, mimic-29a or 29b were plated simultaneously with 100 pancreatic cancer cells (Panc-1) in a 6-well plate. Co-cultures were allowed to grow for 10 days, fixed, and stained with crystal violet fixing solution to stain cancer cells as previously described 64 with few modifications. Cancer colonies greater than 50 cells were counted under phase contrast microscopy. Representative images of co-cultures stained with crystal violet are shown. ( b ) Conditioned media of PSCs expressing miR-29 show decreased effect on pancreatic cell viability. Conditioned media from mPSCs transfected with control, miR-29a, or miR-29b mimics was applied to pancreatic cancer cells (Panc-1) in a 96-well plate and viability was measured 24 and 48 hours post-treatment using the Cell Counting Kit-8 assay according to manufacture protocol. Data is normalized to pancreatic cancer cells (Panc-1) treated with non-conditioned media. All experiments were repeated 3-4 times and representative data is presented. Data represents mean + SEM. Statistics generated by t-test, *p

    Techniques Used: Expressing, Co-Culture Assay, Over Expression, Transfection, Staining, Microscopy, Cell Counting, Generated

    27) Product Images from "FAM83H and SCRIB stabilize β-catenin and stimulate progression of gastric carcinoma"

    Article Title: FAM83H and SCRIB stabilize β-catenin and stimulate progression of gastric carcinoma

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.103351

    The combined effect of overexpression of FAM83H and knock-down of SCRIB on the proliferation and tumor formation of gastric cancer cells. ( A , B ) The effect of overexpression of FAM83H and/or knock-down of SCRIB on the proliferation of MKN45 and NCI-N87 gastric cancer cells were evaluated with an MTT assay ( A ) and a colony-forming assay ( B ). The MTT assay was performed after seeding 3,000 cells per well of a 96-well plate. The colony-forming assay was performed by plating 1,000 cells per well in a 6-well plate for ten days. The number of colonies was determined with GeneTools analysis software. ( C , D ) In vivo tumor growth was evaluated by subcutaneously implanting 2x10 6 NCI-N87 cells with overexpression of FAM83H and/or knock-down of SCRIB. The tumor volume was measured every week after tumor implantation by the equation V = LxWxHx0.52 mm 3 ( C ). At five weeks after tumor inoculation, the mice were euthanized and tumor weight was measured ( D ). ( E ) The mice were evaluated for metastasis and histologic findings of the lung. The arrows indicate metastatic NCI-N87 cells in lung. *; P
    Figure Legend Snippet: The combined effect of overexpression of FAM83H and knock-down of SCRIB on the proliferation and tumor formation of gastric cancer cells. ( A , B ) The effect of overexpression of FAM83H and/or knock-down of SCRIB on the proliferation of MKN45 and NCI-N87 gastric cancer cells were evaluated with an MTT assay ( A ) and a colony-forming assay ( B ). The MTT assay was performed after seeding 3,000 cells per well of a 96-well plate. The colony-forming assay was performed by plating 1,000 cells per well in a 6-well plate for ten days. The number of colonies was determined with GeneTools analysis software. ( C , D ) In vivo tumor growth was evaluated by subcutaneously implanting 2x10 6 NCI-N87 cells with overexpression of FAM83H and/or knock-down of SCRIB. The tumor volume was measured every week after tumor implantation by the equation V = LxWxHx0.52 mm 3 ( C ). At five weeks after tumor inoculation, the mice were euthanized and tumor weight was measured ( D ). ( E ) The mice were evaluated for metastasis and histologic findings of the lung. The arrows indicate metastatic NCI-N87 cells in lung. *; P

    Techniques Used: Over Expression, MTT Assay, Software, In Vivo, Tumor Implantation, Mouse Assay

    The effect of knock-down or overexpression of FAM83H on the proliferation and invasiveness of gastric cancer cells. ( A ) The effect of knock-down or overexpression of FAM83H on proliferation were evaluated with an MTT assay, cell counting, and a colony-forming assay in MKN-45 and NCI-N87 cells. The MTT assay was performed after seeding 3,000 cells per well of a 96-well plate. Cell counting was performed after seeding 3,000 cells per well of a 6-well plate. The colony-forming assay was performed by plating 1,000 cells per well in a 6-well plate for ten days. The knock-down or overexpression of FAM83H was assessed via western blot for FAM83H. ( B ) The migration assay was performed by seeding 1x10 5 MKN-45 or 1x10 5 NCI-N87 cells in the upper chamber for 48 hours. ( C ) The invasion assay was performed by seeding 2x10 5 MKN-45 or 2x10 5 NCI-N87 cells in the upper chamber with Matrigel for 48 hours. The chambers were stained with DIFF-Quik staining solution, and the number of migrated or invaded cells were counted in five x200 microscopic fields in each well. *; P
    Figure Legend Snippet: The effect of knock-down or overexpression of FAM83H on the proliferation and invasiveness of gastric cancer cells. ( A ) The effect of knock-down or overexpression of FAM83H on proliferation were evaluated with an MTT assay, cell counting, and a colony-forming assay in MKN-45 and NCI-N87 cells. The MTT assay was performed after seeding 3,000 cells per well of a 96-well plate. Cell counting was performed after seeding 3,000 cells per well of a 6-well plate. The colony-forming assay was performed by plating 1,000 cells per well in a 6-well plate for ten days. The knock-down or overexpression of FAM83H was assessed via western blot for FAM83H. ( B ) The migration assay was performed by seeding 1x10 5 MKN-45 or 1x10 5 NCI-N87 cells in the upper chamber for 48 hours. ( C ) The invasion assay was performed by seeding 2x10 5 MKN-45 or 2x10 5 NCI-N87 cells in the upper chamber with Matrigel for 48 hours. The chambers were stained with DIFF-Quik staining solution, and the number of migrated or invaded cells were counted in five x200 microscopic fields in each well. *; P

    Techniques Used: Over Expression, MTT Assay, Cell Counting, Western Blot, Migration, Invasion Assay, Staining, Diff-Quik

    The effect of knock-down or overexpression of SCRIB on the proliferation and invasiveness of gastric cancer cells. ( A , B ) The effect of knock-down or overexpression of SCRIB on proliferation was evaluated with an MTT assay and colony-forming assay in MKN-45 ( A ) and NCI-N87 cells ( B ). The MTT assay was performed after seeding 3,000 cells per well of a 96-well plate. The colony-forming assay was performed by plating 1,000 cells per well in a 6-well plate for ten days. The knock-down or overexpression of SCRIB was assessed via western blot for SCRIB. ( C ) The migration assay was performed by seeding 1x10 5 MKN-45 or 1x10 5 NCI-N87 cells in the upper chamber for 48 hours. ( D ) The invasion assay was performed by seeding 2x10 5 MKN-45 or 2x10 5 NCI-N87 cells in the upper chamber with Matrigel for 48 hours. The chambers were stained with DIFF-Quik staining solution, and the number of migrated or invaded cells were counted in five x200 microscopic fields in each well. **; P
    Figure Legend Snippet: The effect of knock-down or overexpression of SCRIB on the proliferation and invasiveness of gastric cancer cells. ( A , B ) The effect of knock-down or overexpression of SCRIB on proliferation was evaluated with an MTT assay and colony-forming assay in MKN-45 ( A ) and NCI-N87 cells ( B ). The MTT assay was performed after seeding 3,000 cells per well of a 96-well plate. The colony-forming assay was performed by plating 1,000 cells per well in a 6-well plate for ten days. The knock-down or overexpression of SCRIB was assessed via western blot for SCRIB. ( C ) The migration assay was performed by seeding 1x10 5 MKN-45 or 1x10 5 NCI-N87 cells in the upper chamber for 48 hours. ( D ) The invasion assay was performed by seeding 2x10 5 MKN-45 or 2x10 5 NCI-N87 cells in the upper chamber with Matrigel for 48 hours. The chambers were stained with DIFF-Quik staining solution, and the number of migrated or invaded cells were counted in five x200 microscopic fields in each well. **; P

    Techniques Used: Over Expression, MTT Assay, Western Blot, Migration, Invasion Assay, Staining, Diff-Quik

    28) Product Images from "Alterations in TGF-β signaling leads to high HMGA2 levels potentially through modulation of PJA1/SMAD3 in HCC cells"

    Article Title: Alterations in TGF-β signaling leads to high HMGA2 levels potentially through modulation of PJA1/SMAD3 in HCC cells

    Journal: Genes & Cancer

    doi: 10.18632/genesandcancer.199

    Confocal microscopic analysis showed that PJA1 colocalize with HMGA2 in the nuclei of HepG2 and Huh7 cells and TGF-β treatment enhances the colocalization. HepG2 and Huh7 cells were transfected with T7-PJA1 on 6 well plates with cover slips and incubated in serum-free medium after one day. After 24 hours, the cells were treated with TGF-β1 for 3 hours and fixed with 4% paraformaldehyde for 20 min. The fixed cells were premetallized with 0.1% Triton X-100, blocked with 10% normal goat serum for 30 min and incubated with T7 and HMGA2 antibodies for one hour. Then, the cells were labelled with goat anti-IgG antibodies (Green: Alexa Fluor 488, Red: Alexa Fluor 555) and counterstained with DAPI.
    Figure Legend Snippet: Confocal microscopic analysis showed that PJA1 colocalize with HMGA2 in the nuclei of HepG2 and Huh7 cells and TGF-β treatment enhances the colocalization. HepG2 and Huh7 cells were transfected with T7-PJA1 on 6 well plates with cover slips and incubated in serum-free medium after one day. After 24 hours, the cells were treated with TGF-β1 for 3 hours and fixed with 4% paraformaldehyde for 20 min. The fixed cells were premetallized with 0.1% Triton X-100, blocked with 10% normal goat serum for 30 min and incubated with T7 and HMGA2 antibodies for one hour. Then, the cells were labelled with goat anti-IgG antibodies (Green: Alexa Fluor 488, Red: Alexa Fluor 555) and counterstained with DAPI.

    Techniques Used: Transfection, Incubation

    29) Product Images from "HIV-1 Vpr induces cell cycle arrest and enhances viral gene expression by depleting CCDC137"

    Article Title: HIV-1 Vpr induces cell cycle arrest and enhances viral gene expression by depleting CCDC137

    Journal: eLife

    doi: 10.7554/eLife.55806

    Discovery and analysis of proteins labelled with Vpr-BirA (R118G). ( A ) Proteins recovered from lysates of MT-4 cells expressing Vpr-BirA (R118G) or BirA (R118G) using streptavidin beads. Biotinylated proteins were detected with IRDye 680RD-conjugated Streptavidin. Representative of two experiments. ( B ) Immunostaining/FACS analysis of endogenous Ki-67 levels in 293 T cells 28 hr after transfection (in 6-well plates) with 100 ng of a GFP expression plasmid and increasing amounts (0 ng, 50 ng, 100 ng, 200 ng, or 400 ng) of an HA-Vpr expression plasmid. Representative of two experiments. ( C ) Western blot analysis of endogenous Ki-67 and HA-Vpr expression in 293 T cells 48 hr after infection with V1/δ-Vpr or V1/HA-Vpr at an MOI of 2. Representative of two experiments. ( D ) Western blot analysis of Ki-67 and tubulin (loading control) levels in U2OS cells transduced with lentiviruses carrying no shRNA (vector) or shRNA against Ki-67 (shRNA Ki-67) after selection with puromycin. Representative of three experiments. ( E ) Analysis of DNA content by propidium iodide staining in U2OS cells transduced with lentiviruses carrying no shRNA (vector) or shRNA against Ki-67 (shRNA Ki-67) after selection with puromycin. Representative of three experiments.
    Figure Legend Snippet: Discovery and analysis of proteins labelled with Vpr-BirA (R118G). ( A ) Proteins recovered from lysates of MT-4 cells expressing Vpr-BirA (R118G) or BirA (R118G) using streptavidin beads. Biotinylated proteins were detected with IRDye 680RD-conjugated Streptavidin. Representative of two experiments. ( B ) Immunostaining/FACS analysis of endogenous Ki-67 levels in 293 T cells 28 hr after transfection (in 6-well plates) with 100 ng of a GFP expression plasmid and increasing amounts (0 ng, 50 ng, 100 ng, 200 ng, or 400 ng) of an HA-Vpr expression plasmid. Representative of two experiments. ( C ) Western blot analysis of endogenous Ki-67 and HA-Vpr expression in 293 T cells 48 hr after infection with V1/δ-Vpr or V1/HA-Vpr at an MOI of 2. Representative of two experiments. ( D ) Western blot analysis of Ki-67 and tubulin (loading control) levels in U2OS cells transduced with lentiviruses carrying no shRNA (vector) or shRNA against Ki-67 (shRNA Ki-67) after selection with puromycin. Representative of three experiments. ( E ) Analysis of DNA content by propidium iodide staining in U2OS cells transduced with lentiviruses carrying no shRNA (vector) or shRNA against Ki-67 (shRNA Ki-67) after selection with puromycin. Representative of three experiments.

    Techniques Used: Expressing, Immunostaining, FACS, Transfection, Plasmid Preparation, Western Blot, Infection, Transduction, shRNA, Selection, Staining

    30) Product Images from "Pathophysiological role of microRNA-29 in pancreatic cancer stroma"

    Article Title: Pathophysiological role of microRNA-29 in pancreatic cancer stroma

    Journal: Scientific Reports

    doi: 10.1038/srep11450

    Ectopic expression of miR-29 in PSCs reduces cancer cells viability and cancer colony growth in co-culture. ( a ) Effect of miR-29 overexpression in PSCs on cancer colony growth in direct co-cultures. mPSCs transfected with 20 nM mimic control, mimic-29a or 29b were plated simultaneously with 100 pancreatic cancer cells (Panc-1) in a 6-well plate. Co-cultures were allowed to grow for 10 days, fixed, and stained with crystal violet fixing solution to stain cancer cells as previously described 64 with few modifications. Cancer colonies greater than 50 cells were counted under phase contrast microscopy. Representative images of co-cultures stained with crystal violet are shown. ( b ) Conditioned media of PSCs expressing miR-29 show decreased effect on pancreatic cell viability. Conditioned media from mPSCs transfected with control, miR-29a, or miR-29b mimics was applied to pancreatic cancer cells (Panc-1) in a 96-well plate and viability was measured 24 and 48 hours post-treatment using the Cell Counting Kit-8 assay according to manufacture protocol. Data is normalized to pancreatic cancer cells (Panc-1) treated with non-conditioned media. All experiments were repeated 3-4 times and representative data is presented. Data represents mean + SEM. Statistics generated by t-test, *p
    Figure Legend Snippet: Ectopic expression of miR-29 in PSCs reduces cancer cells viability and cancer colony growth in co-culture. ( a ) Effect of miR-29 overexpression in PSCs on cancer colony growth in direct co-cultures. mPSCs transfected with 20 nM mimic control, mimic-29a or 29b were plated simultaneously with 100 pancreatic cancer cells (Panc-1) in a 6-well plate. Co-cultures were allowed to grow for 10 days, fixed, and stained with crystal violet fixing solution to stain cancer cells as previously described 64 with few modifications. Cancer colonies greater than 50 cells were counted under phase contrast microscopy. Representative images of co-cultures stained with crystal violet are shown. ( b ) Conditioned media of PSCs expressing miR-29 show decreased effect on pancreatic cell viability. Conditioned media from mPSCs transfected with control, miR-29a, or miR-29b mimics was applied to pancreatic cancer cells (Panc-1) in a 96-well plate and viability was measured 24 and 48 hours post-treatment using the Cell Counting Kit-8 assay according to manufacture protocol. Data is normalized to pancreatic cancer cells (Panc-1) treated with non-conditioned media. All experiments were repeated 3-4 times and representative data is presented. Data represents mean + SEM. Statistics generated by t-test, *p

    Techniques Used: Expressing, Co-Culture Assay, Over Expression, Transfection, Staining, Microscopy, Cell Counting, Generated

    31) Product Images from "Risuteganib Protects against Hydroquinone–induced Injury in Human RPE Cells"

    Article Title: Risuteganib Protects against Hydroquinone–induced Injury in Human RPE Cells

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.61.10.35

    RSG cotreatment upregulated HQ-induced HO-1 expression. ( A , B ) RPE cells were treated for 4 hours with HQ (250 µM) in the presence or absence of RSG. RNA was extracted and gene expression was analyzed via RNA-seq ( A ) and quantitative RT-PCR ( B ). RSG cotreatment upregulated HQ-induced HMOX-1 gene expression. ( C-D ) RPE cells in triplicate wells of a 6-well plate were treated for 8 hours with HQ (170 µM) in the presence or absence of RSG. Total proteins (30 µg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis for Western blot ( C ). ( D ) The quantity of HO-1 relative to GAPDH indicated that HQ-elevated HO-1 protein level was further upregulated by RSG cotreatment. Data are representative of two separate experiments with similar results.
    Figure Legend Snippet: RSG cotreatment upregulated HQ-induced HO-1 expression. ( A , B ) RPE cells were treated for 4 hours with HQ (250 µM) in the presence or absence of RSG. RNA was extracted and gene expression was analyzed via RNA-seq ( A ) and quantitative RT-PCR ( B ). RSG cotreatment upregulated HQ-induced HMOX-1 gene expression. ( C-D ) RPE cells in triplicate wells of a 6-well plate were treated for 8 hours with HQ (170 µM) in the presence or absence of RSG. Total proteins (30 µg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis for Western blot ( C ). ( D ) The quantity of HO-1 relative to GAPDH indicated that HQ-elevated HO-1 protein level was further upregulated by RSG cotreatment. Data are representative of two separate experiments with similar results.

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Polyacrylamide Gel Electrophoresis, Western Blot

    HQ and RSG cotreatment-initiated gene expression changes. RPE cells in sextuplicate wells of 6-well plate were treated for 4 hours with HQ at 250 µM or 300 µM in the presence or absence of RSG. Poly-A–enriched messenger RNAs were isolated and sequenced using RNA-seq. ( A ) The six treatment conditions. Orange arrows indicate the differential expression comparisons performed. ( B ) A principal component analysis was used to visualize the samples on the first two dimensions that captured the largest amount of variance (shown in axis label) in the dataset. First two dimensions showed clear separation between control, HQ-treated, and RSG cotreated cells. ( C ) The edgeR software was used to identify genes that are DE between selected conditions. The number of genes that passed the false discovery rate and log2-fold change thresholds are shown. The number of genes that were strongly downregulated, downregulated, upregulated, or strongly upregulated are colored in dark blue , blue , red , or dark red , respectively. Few DE genes were found after RSG treatment alone, whereas HQ treatment and RSG cotreatment induced large transcriptome changes.
    Figure Legend Snippet: HQ and RSG cotreatment-initiated gene expression changes. RPE cells in sextuplicate wells of 6-well plate were treated for 4 hours with HQ at 250 µM or 300 µM in the presence or absence of RSG. Poly-A–enriched messenger RNAs were isolated and sequenced using RNA-seq. ( A ) The six treatment conditions. Orange arrows indicate the differential expression comparisons performed. ( B ) A principal component analysis was used to visualize the samples on the first two dimensions that captured the largest amount of variance (shown in axis label) in the dataset. First two dimensions showed clear separation between control, HQ-treated, and RSG cotreated cells. ( C ) The edgeR software was used to identify genes that are DE between selected conditions. The number of genes that passed the false discovery rate and log2-fold change thresholds are shown. The number of genes that were strongly downregulated, downregulated, upregulated, or strongly upregulated are colored in dark blue , blue , red , or dark red , respectively. Few DE genes were found after RSG treatment alone, whereas HQ treatment and RSG cotreatment induced large transcriptome changes.

    Techniques Used: Expressing, Isolation, RNA Sequencing Assay, Software

    RSG cotreatment protected against HQ-mediated RPE cell death. RPE cells in triplicate wells of a 6-well plate were treated for 12 hours with HQ (150 µM) in the presence or absence of RSG. ( A ) Morphology of cells was captured before cell harvest. A large number of the HQ-treated RPE cells appeared less adherent ( black arrow ) and other cells seemed to be shriveled ( red arrow ). ( B – D ) Cells were then harvested and stained with Annexin V and 7-AAD. Cell death was analyzed with flow cytometry. HQ primarily induced RPE cell necrosis and to a lesser extent, apoptosis. RSG cotreatment significantly protected against both HQ-induced necrosis and apoptosis. Data were averaged from two separate experiments ( n = 6 per condition).
    Figure Legend Snippet: RSG cotreatment protected against HQ-mediated RPE cell death. RPE cells in triplicate wells of a 6-well plate were treated for 12 hours with HQ (150 µM) in the presence or absence of RSG. ( A ) Morphology of cells was captured before cell harvest. A large number of the HQ-treated RPE cells appeared less adherent ( black arrow ) and other cells seemed to be shriveled ( red arrow ). ( B – D ) Cells were then harvested and stained with Annexin V and 7-AAD. Cell death was analyzed with flow cytometry. HQ primarily induced RPE cell necrosis and to a lesser extent, apoptosis. RSG cotreatment significantly protected against both HQ-induced necrosis and apoptosis. Data were averaged from two separate experiments ( n = 6 per condition).

    Techniques Used: Staining, Flow Cytometry

    32) Product Images from "Distal Interleukin-1β (IL-1β) Response Element of Human Matrix Metalloproteinase-13 (MMP-13) Binds Activator Protein 1 (AP-1) Transcription Factors and Regulates Gene Expression *"

    Article Title: Distal Interleukin-1β (IL-1β) Response Element of Human Matrix Metalloproteinase-13 (MMP-13) Binds Activator Protein 1 (AP-1) Transcription Factors and Regulates Gene Expression *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.264077

    A luciferase reporter driven by the MMP-13 proximal promoter combined with the distal response element is responsive to IL-1β and mimics the response of endogenous MMP-13. SW-1353 cells were seeded in triplicate in 6-well plates and transfected
    Figure Legend Snippet: A luciferase reporter driven by the MMP-13 proximal promoter combined with the distal response element is responsive to IL-1β and mimics the response of endogenous MMP-13. SW-1353 cells were seeded in triplicate in 6-well plates and transfected

    Techniques Used: Luciferase, Transfection

    33) Product Images from "Nucleocapsid protein of porcine reproductive and respiratory syndrome virus antagonizes the antiviral activity of TRIM25 by interfering with TRIM25-mediated RIG-I ubiquitination"

    Article Title: Nucleocapsid protein of porcine reproductive and respiratory syndrome virus antagonizes the antiviral activity of TRIM25 by interfering with TRIM25-mediated RIG-I ubiquitination

    Journal: Veterinary Microbiology

    doi: 10.1016/j.vetmic.2019.05.003

    The PRRSV N protein inhibits TRIM25-mediated RIG-I activation and IFN production. (A and B) HEK293 T cells seeded in 24-well plates were co-transfected using the firefly luciferase reporter plasmid IFN- β -Luc and the Renilla luciferase control reporter plasmid pRL-TK. For the experiment, pCAGGS-RIG-I-Flag (0.25 μg), or pCAGGS -2 CARD (0.25 μg), pCAGGS-N-HA were co-transfected. (C) pCAGGS -2 CARD-Flag (0.25 μg), pCAGGS-N-Falg (0.25 μg) and pCAGGS-TRIM25-Myc (0.5 μg) plasmids were cotransfected. The luciferase activity in cell lysates was analyzed using a dual luciferase reporter assay system. (D) HEK293 T cells grown in 6-well plates were co-transfected with plasmids encoding ubiquitin-HA (0.5 μg), Flag-2CARD (0.5 μg), N-Myc (1.0 μg), or TRIM25-Myc (1.0 μg). For the experiment, 24 hpt, the cells were infected with SEV, and 16 hpi, whole-cell lysates were analyzed by immunoprecipitation using the indicated antibodies to detect the ubiquitination of RIG-I-CARD. The data are presented as the mean ± SD from three experiments. The statistical significance of differences was determined using Student’s t -test (* p
    Figure Legend Snippet: The PRRSV N protein inhibits TRIM25-mediated RIG-I activation and IFN production. (A and B) HEK293 T cells seeded in 24-well plates were co-transfected using the firefly luciferase reporter plasmid IFN- β -Luc and the Renilla luciferase control reporter plasmid pRL-TK. For the experiment, pCAGGS-RIG-I-Flag (0.25 μg), or pCAGGS -2 CARD (0.25 μg), pCAGGS-N-HA were co-transfected. (C) pCAGGS -2 CARD-Flag (0.25 μg), pCAGGS-N-Falg (0.25 μg) and pCAGGS-TRIM25-Myc (0.5 μg) plasmids were cotransfected. The luciferase activity in cell lysates was analyzed using a dual luciferase reporter assay system. (D) HEK293 T cells grown in 6-well plates were co-transfected with plasmids encoding ubiquitin-HA (0.5 μg), Flag-2CARD (0.5 μg), N-Myc (1.0 μg), or TRIM25-Myc (1.0 μg). For the experiment, 24 hpt, the cells were infected with SEV, and 16 hpi, whole-cell lysates were analyzed by immunoprecipitation using the indicated antibodies to detect the ubiquitination of RIG-I-CARD. The data are presented as the mean ± SD from three experiments. The statistical significance of differences was determined using Student’s t -test (* p

    Techniques Used: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Reporter Assay, Infection, Immunoprecipitation

    34) Product Images from "Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria"

    Article Title: Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16889-z

    IFNγ priming is required for LPS-induced caspase-4 activation in human epithelial cells. a – c Intracellular bacterial fold-replication ( a ) and release of LDH ( b , c ) in naive or IFNγ-primed wild-type, CASP4 –/– or GSDMD –/– HeLa cells, at 1 or 6-h post-infection (p.i.) with Salmonella . Cells in 96-well plates were infected for 30 min, washed and gentamicin was added to kill extracellular bacteria. At the indicated time points supernatant was collected to determine the release of LDH, and then cells were lysed and the number of viable intracellular bacteria was determined by counting colony forming units (CFUs). The bacterial fold-replication was calculated relative to 1 h p.i. d Percentage of CHQ-resistant cytosolic Salmonella in naive or IFNγ-primed HeLa at 1.5 h p.i. Cells were infected for 30 min as in ( a ) and then treated with gentamicin ± CHQ for an additional 1 h before cells were lysed and bacteria counted by CFUs. The percentage of cytosolic bacteria was calculated as the ratio of (CHQ + gentamicin resistant / gentamicin resistant ). e – g Release of LDH from naive or IFNγ-primed cells, 5 h after transfection with LPS (2.5 µg/50,000 cells) or 3–4 h after electroporation with LPS (300 ng/50,0000 cells). h Western blot analysis of full length (p43) and cleaved (p32) caspase-4 in the supernatants and cell lysates from naive or IFNγ-primed HaCaT cells, upon transfection with E. coli LPS LPS (2.5 µg/50,000 cells). i Streptavidin pull-down assay of the binding of biotin-conjugated LPS to endogenous caspase-4 from the lysates of naive or IFNγ-primed HBEC3-KT. Cells in 6-well plates were transfected with LPS-biotin (10 µg) or left untransfected, and biotinylated substrate was pulled down using equal amounts of streptavidin magnetic beads, which were then eluted in equal volumes of SDS-PAGE reducing sample buffer. Streptavidin-bound and -unbound fractions were analyzed by immunoblotting for caspase-4. Graphs show the mean ± SD, and data are pooled from two to six independent experiments performed in triplicate ( a – g ) or representative of two ( h , i ) independent experiments. *** P
    Figure Legend Snippet: IFNγ priming is required for LPS-induced caspase-4 activation in human epithelial cells. a – c Intracellular bacterial fold-replication ( a ) and release of LDH ( b , c ) in naive or IFNγ-primed wild-type, CASP4 –/– or GSDMD –/– HeLa cells, at 1 or 6-h post-infection (p.i.) with Salmonella . Cells in 96-well plates were infected for 30 min, washed and gentamicin was added to kill extracellular bacteria. At the indicated time points supernatant was collected to determine the release of LDH, and then cells were lysed and the number of viable intracellular bacteria was determined by counting colony forming units (CFUs). The bacterial fold-replication was calculated relative to 1 h p.i. d Percentage of CHQ-resistant cytosolic Salmonella in naive or IFNγ-primed HeLa at 1.5 h p.i. Cells were infected for 30 min as in ( a ) and then treated with gentamicin ± CHQ for an additional 1 h before cells were lysed and bacteria counted by CFUs. The percentage of cytosolic bacteria was calculated as the ratio of (CHQ + gentamicin resistant / gentamicin resistant ). e – g Release of LDH from naive or IFNγ-primed cells, 5 h after transfection with LPS (2.5 µg/50,000 cells) or 3–4 h after electroporation with LPS (300 ng/50,0000 cells). h Western blot analysis of full length (p43) and cleaved (p32) caspase-4 in the supernatants and cell lysates from naive or IFNγ-primed HaCaT cells, upon transfection with E. coli LPS LPS (2.5 µg/50,000 cells). i Streptavidin pull-down assay of the binding of biotin-conjugated LPS to endogenous caspase-4 from the lysates of naive or IFNγ-primed HBEC3-KT. Cells in 6-well plates were transfected with LPS-biotin (10 µg) or left untransfected, and biotinylated substrate was pulled down using equal amounts of streptavidin magnetic beads, which were then eluted in equal volumes of SDS-PAGE reducing sample buffer. Streptavidin-bound and -unbound fractions were analyzed by immunoblotting for caspase-4. Graphs show the mean ± SD, and data are pooled from two to six independent experiments performed in triplicate ( a – g ) or representative of two ( h , i ) independent experiments. *** P

    Techniques Used: Activation Assay, Infection, Transfection, Electroporation, Western Blot, Pull Down Assay, Binding Assay, Magnetic Beads, SDS Page

    35) Product Images from "Smoothened-dependent and -independent pathways in mammalian noncanonical Hedgehog signaling"

    Article Title: Smoothened-dependent and -independent pathways in mammalian noncanonical Hedgehog signaling

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.007956

    Effects of Hedgehog stimulation on cellular signaling as determined by kinome profiling. A , murine fibroblasts were stimulated with 2 μg/ml Shh. Subsequently, cells were lysed, and the resulting lysates were used to phosphorylate arrays of different kinase substrates employing [γ- 33 P]ATP, and radioactivity incorporated in the different substrates was determined. Peptide substrates were allotted to elective signal transduction elements. The figure depicts the number of peptides significantly phosphorylated (which means the number of peptides that received a Markov “on” call; see “Experimental procedures”) for each element. A darker color reflects more kinase activity toward substrate elements, and the results reveal the effects of Hedgehog stimulation on cellular signal transduction; thus, black means that all peptides were significantly phosphorylated, whereas white means that no peptides allotted to this signal transduction in this experimental condition were phosphorylated. Results were first statistically tested by a dichotomal analysis based on the number of Markov “on” calls observed in vehicle- and Shh-stimulated cultures. If statistically significant differences were noted, the signal transduction category is highlighted with a red border , and the level of significance observed is indicated in red . For signal transduction elements in which this very robust analysis failed to detect a statistically significant difference, a parametric test was performed. If this proved significant, the category is highlighted in orange , and the corresponding level of significance is depicted as well. The results provide a wealth of data on the effects of Hedgehog stimulation on cellular signaling. B , MEFs were grown in 6-well plates. To simulate Smo- and Ptc-dependent signaling, cells were treated with Shh (2 μg/ml) for 10 min and compared with unstimulated cells. Cells were lysed, and proteins were resolved by SDS-PAGE followed by blotting to PVDF and incubation of membrane with antibodies against the indicated phosphorylated proteins. Blots were reprobed with antibodies against β-actin to confirm equal loading.
    Figure Legend Snippet: Effects of Hedgehog stimulation on cellular signaling as determined by kinome profiling. A , murine fibroblasts were stimulated with 2 μg/ml Shh. Subsequently, cells were lysed, and the resulting lysates were used to phosphorylate arrays of different kinase substrates employing [γ- 33 P]ATP, and radioactivity incorporated in the different substrates was determined. Peptide substrates were allotted to elective signal transduction elements. The figure depicts the number of peptides significantly phosphorylated (which means the number of peptides that received a Markov “on” call; see “Experimental procedures”) for each element. A darker color reflects more kinase activity toward substrate elements, and the results reveal the effects of Hedgehog stimulation on cellular signal transduction; thus, black means that all peptides were significantly phosphorylated, whereas white means that no peptides allotted to this signal transduction in this experimental condition were phosphorylated. Results were first statistically tested by a dichotomal analysis based on the number of Markov “on” calls observed in vehicle- and Shh-stimulated cultures. If statistically significant differences were noted, the signal transduction category is highlighted with a red border , and the level of significance observed is indicated in red . For signal transduction elements in which this very robust analysis failed to detect a statistically significant difference, a parametric test was performed. If this proved significant, the category is highlighted in orange , and the corresponding level of significance is depicted as well. The results provide a wealth of data on the effects of Hedgehog stimulation on cellular signaling. B , MEFs were grown in 6-well plates. To simulate Smo- and Ptc-dependent signaling, cells were treated with Shh (2 μg/ml) for 10 min and compared with unstimulated cells. Cells were lysed, and proteins were resolved by SDS-PAGE followed by blotting to PVDF and incubation of membrane with antibodies against the indicated phosphorylated proteins. Blots were reprobed with antibodies against β-actin to confirm equal loading.

    Techniques Used: Radioactivity, Transduction, Activity Assay, SDS Page, Incubation

    36) Product Images from "A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein"

    Article Title: A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008941

    The c655 mutation disrupts TG transfer activity, but not PL transfer activity of the zebrafish Mtp complex. (A, B) COS-7 cells were first transfected with an expression vector for human APOB48 (5 μg), distributed equally in 6-well plates, and subsequently transfected with plasmids expressing either wild-type zebrafish mttp -FLAG, mttp stl -FLAG, mttp c655 -FLAG, or empty vector (pcDNA3) (3 μg). After 72 h, APOB48 was measured via ELISA in media (A) or in the cell (B). Data are representative of 7 independent experiments (each data point is the mean of three technical replicates), mean +/- SD, One-Way ANOVA with Bonferroni post-hoc tests, * p
    Figure Legend Snippet: The c655 mutation disrupts TG transfer activity, but not PL transfer activity of the zebrafish Mtp complex. (A, B) COS-7 cells were first transfected with an expression vector for human APOB48 (5 μg), distributed equally in 6-well plates, and subsequently transfected with plasmids expressing either wild-type zebrafish mttp -FLAG, mttp stl -FLAG, mttp c655 -FLAG, or empty vector (pcDNA3) (3 μg). After 72 h, APOB48 was measured via ELISA in media (A) or in the cell (B). Data are representative of 7 independent experiments (each data point is the mean of three technical replicates), mean +/- SD, One-Way ANOVA with Bonferroni post-hoc tests, * p

    Techniques Used: Mutagenesis, Activity Assay, Transfection, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    37) Product Images from "UBR-Box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target"

    Article Title: UBR-Box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target

    Journal: bioRxiv

    doi: 10.1101/2020.06.03.131912

    UBR5 deficient A549 cells show decreased cell viability and clonogenic potential. (a) A549 cells were infected with the lenti-viruses from multiple shRNA construct against UBR5 and were cultured for 4 days. Alamar Blue readings were recorded every 24 hours and relative cell viability of UBR5 deficient cells were compared to control cells on each day. (b) A549 cells were infected with shRNA against UBR5 and 1000 cells were cultured in 6-well plate for 10 days. Colonies were fixed in methanol and stained with crystal violet. (c) Quantitative evaluation of clonogenic assay. Representative bar graph showing number of colonies formed per 1000 cells
    Figure Legend Snippet: UBR5 deficient A549 cells show decreased cell viability and clonogenic potential. (a) A549 cells were infected with the lenti-viruses from multiple shRNA construct against UBR5 and were cultured for 4 days. Alamar Blue readings were recorded every 24 hours and relative cell viability of UBR5 deficient cells were compared to control cells on each day. (b) A549 cells were infected with shRNA against UBR5 and 1000 cells were cultured in 6-well plate for 10 days. Colonies were fixed in methanol and stained with crystal violet. (c) Quantitative evaluation of clonogenic assay. Representative bar graph showing number of colonies formed per 1000 cells

    Techniques Used: Infection, shRNA, Construct, Cell Culture, Staining, Clonogenic Assay

    38) Product Images from "UBR-box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target"

    Article Title: UBR-box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target

    Journal: BMC Cancer

    doi: 10.1186/s12885-020-07322-1

    UBR5 deficient A549 cells show decreased cell viability and clonogenic potential. a A549 cells were infected with the lenti-viruses from multiple shRNA construct against UBR5 and were cultured for 4 days. Alamar Blue readings were recorded every 24 h and relative cell viability of UBR5 deficient cells were compared to control cells on each day. b A549 cells were infected with shRNA against UBR5 and 1000 cells were cultured in 6-well plate for 10 days. Colonies were fixed in methanol and stained with crystal violet. c Quantitative evaluation of clonogenic assay. Representative bar graph showing number of colonies formed per 1000 cells
    Figure Legend Snippet: UBR5 deficient A549 cells show decreased cell viability and clonogenic potential. a A549 cells were infected with the lenti-viruses from multiple shRNA construct against UBR5 and were cultured for 4 days. Alamar Blue readings were recorded every 24 h and relative cell viability of UBR5 deficient cells were compared to control cells on each day. b A549 cells were infected with shRNA against UBR5 and 1000 cells were cultured in 6-well plate for 10 days. Colonies were fixed in methanol and stained with crystal violet. c Quantitative evaluation of clonogenic assay. Representative bar graph showing number of colonies formed per 1000 cells

    Techniques Used: Infection, shRNA, Construct, Cell Culture, Staining, Clonogenic Assay

    39) Product Images from "Icariin Improves Age-Related Testicular Dysfunction by Alleviating Sertoli Cell Injury via Upregulation of the ERα/Nrf2-Signaling Pathway"

    Article Title: Icariin Improves Age-Related Testicular Dysfunction by Alleviating Sertoli Cell Injury via Upregulation of the ERα/Nrf2-Signaling Pathway

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2020.00677

    Icariin alleviates TM4 cells injury induced by D-galactose (D-gal). (A) Cellular viability was detected using MTT assay. TM4 cells at a concentration of 3 × 10 4 cells/well in 96-well plates were pretreated with icariin (0.01–8 μM) or DMSO (0.1%) for 20 h and then incubated with 100 mM D-gal for 60 h. (B–D) TM4 cells at 5 × 10 5 /well in 6-well plates were pretreated with icariin (0.5–1 μM) for 20 h and then incubated with 100 mM of D-gal for 60 h. (B) Representative images of SA- β -gal staining of cells (scale bar = 50 μm). (C) The percentages of SA- β -gal-positive cells from a total of 500 cells. (D) The relative protein expression levels of GDNF, PLZF, BMP4, and SCF were measured using western blotting analysis. All values are expressed as means ± SEM of three independent experiments. ## P
    Figure Legend Snippet: Icariin alleviates TM4 cells injury induced by D-galactose (D-gal). (A) Cellular viability was detected using MTT assay. TM4 cells at a concentration of 3 × 10 4 cells/well in 96-well plates were pretreated with icariin (0.01–8 μM) or DMSO (0.1%) for 20 h and then incubated with 100 mM D-gal for 60 h. (B–D) TM4 cells at 5 × 10 5 /well in 6-well plates were pretreated with icariin (0.5–1 μM) for 20 h and then incubated with 100 mM of D-gal for 60 h. (B) Representative images of SA- β -gal staining of cells (scale bar = 50 μm). (C) The percentages of SA- β -gal-positive cells from a total of 500 cells. (D) The relative protein expression levels of GDNF, PLZF, BMP4, and SCF were measured using western blotting analysis. All values are expressed as means ± SEM of three independent experiments. ## P

    Techniques Used: MTT Assay, Concentration Assay, Incubation, Staining, Expressing, Western Blot

    Icariin protects against TM4 cells injury via the ER α /Nrf2 signaling pathway. TM4 cells at 1 × 10 6 /well in 6-well plates transferred with or without ER α siRNA were incubated with icariin (1 μM) or DMSO (0.1%) for 12 h, followed by treatment with 100 mM D-gal for 60 h. (A) The relative protein expression levels of ERα, Nrf2, HO-1, and NQO-1 in TM4 cells as described above were measured by western blotting analysis. All values are expressed as means ± SEM of three independent experiments. # P
    Figure Legend Snippet: Icariin protects against TM4 cells injury via the ER α /Nrf2 signaling pathway. TM4 cells at 1 × 10 6 /well in 6-well plates transferred with or without ER α siRNA were incubated with icariin (1 μM) or DMSO (0.1%) for 12 h, followed by treatment with 100 mM D-gal for 60 h. (A) The relative protein expression levels of ERα, Nrf2, HO-1, and NQO-1 in TM4 cells as described above were measured by western blotting analysis. All values are expressed as means ± SEM of three independent experiments. # P

    Techniques Used: Incubation, Expressing, Western Blot

    40) Product Images from "Exosomal MiRNA Transfer between Retinal Microglia and RPE"

    Article Title: Exosomal MiRNA Transfer between Retinal Microglia and RPE

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21103541

    Transferred miR-21 between cultured RPE and microglia. ( A ) Microglial uptake of purified EVs from RPE. EVs were labeled with the lipophilic dye DiI (red). Immunostaining of LAMP2 was performed (green), along with DAPI staining of nuclei. ( B ) and ( C ) miR-21 transferred via RPE-conditioned medium. ( B ) Fluorescein-labeled miR-21 mimics (green) were transfected into cultured mouse RPE cells. ( C ) One day after transfection, the conditioned medium was collected and used to treat cultured microglia. Red: LysoTracker Red. ( D ) Schematic model of RPE and microglia co-culture. The posterior eyecups containing RPE/choroid tissues were ex vivo cultured on 6-well plate, and microglia were grown on transwell insert. ( E ) Quantitative RT-PCR analyses of miR-21 levels in microglia after being co-cultured with RPE/choroid from either young or aged mice, compared to that of the control (without RPE co-culture). Data presented are the average of 3 independent experiments (mean ± sem; * p
    Figure Legend Snippet: Transferred miR-21 between cultured RPE and microglia. ( A ) Microglial uptake of purified EVs from RPE. EVs were labeled with the lipophilic dye DiI (red). Immunostaining of LAMP2 was performed (green), along with DAPI staining of nuclei. ( B ) and ( C ) miR-21 transferred via RPE-conditioned medium. ( B ) Fluorescein-labeled miR-21 mimics (green) were transfected into cultured mouse RPE cells. ( C ) One day after transfection, the conditioned medium was collected and used to treat cultured microglia. Red: LysoTracker Red. ( D ) Schematic model of RPE and microglia co-culture. The posterior eyecups containing RPE/choroid tissues were ex vivo cultured on 6-well plate, and microglia were grown on transwell insert. ( E ) Quantitative RT-PCR analyses of miR-21 levels in microglia after being co-cultured with RPE/choroid from either young or aged mice, compared to that of the control (without RPE co-culture). Data presented are the average of 3 independent experiments (mean ± sem; * p

    Techniques Used: Cell Culture, Purification, Labeling, Immunostaining, Staining, Transfection, Co-Culture Assay, Ex Vivo, Quantitative RT-PCR, Mouse Assay

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    Article Snippet: .. Unless otherwise specified, all cells were plated on 6-well plates in complete growth medium for 48 h before they were harvested for gene expression analysis. ..

    other:

    Article Title: The Effect of Lithium on Inflammation-Associated Genes in Lipopolysaccharide-Activated Raw 264.7 Macrophages
    Article Snippet: In summary, cells were seeded at a density of 6 × 106 cell/ml on coverslips in 6-well plates overnight.

    Article Title: FK506 induces lung lymphatic endothelial cell senescence and downregulates LYVE-1 expression, with associated decreased hyaluronan uptake
    Article Snippet: Briefly, LEC were seeded in 6-well plates at a density of 50,000 cells per well.

    Article Title: UBR-Box containing protein, UBR5, is over-expressed in human lung adenocarcinoma and is a potential therapeutic target
    Article Snippet: Cells were allowed to grow on 6-well plates for 10 days and supplemented with fresh media every two days.

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    Article Snippet: Briefly, monolayer cells in 6-well plates were washed with PBS and lysed directly adding 1 ml Trizol.

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    Article Snippet: Cells were seeded the day before in 6-well plates to achieve ∼80% confluence the following day.

    Immunofluorescence:

    Article Title: Pathophysiological role of microRNA-29 in pancreatic cancer stroma
    Article Snippet: .. Immunofluorescence 1 × 105 mouse PSCs, human PSCs or primary human fibroblasts were plated on collagen coated cover slips in 6-well plates and grown to ~80% confluency. .. Cells were washed with PBS, fixed in 4% paraformaldehyde at 37 °C and then permeabilized with 0.25% Tween-20 in PBS (PBST).

    Infection:

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    Thermo Fisher j82
    ( A ) In four human UC cell lines, western blot analysis (WB) revealed DAB2 protein expression in UM-UC-3, <t>J82,</t> and T24 cells, whereas MGH-U-3 cells did not express DAB2 protein. The RT-qPCR analysis also revealed mRNA expression of DAB2 corresponded to the results of WB. ( B ) In three cell lines knocked-down with DAB2 siRNA transfection, DAB2 protein reduction was confirmed by WB. Epithelial-to-mesenchymal transition-related markers, proteins involved in the activation of intracellular signaling pathways, were quantified by western blot analysis. Actin was used as a loading control. The numbers below the bands are the results of densitometric quantification. NT: No treatment; NC: Negative control; SI: DAB2 -si RNA.
    J82, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of repeated viable cell estimation on growth of EPC, MSC and HUVEC cultured on <t>24-well</t> plate. Growth curves of EPC ( A ), MSC ( D ) and HUVEC ( G ), which were cultured in 24-well plate. The control group and the test group were monitored by MTT assay and resazurin assay, respectively. Each point was the mean of absorbance readings of four scaffolds. The representative standard curves of resazurin assay of EPC ( B ), MSC ( E ) and HUVEC ( H ). The representative standard curves of MTT assay of EPC ( C ), MSC ( F ) and HUVEC ( I )
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    Thermo Fisher alisertib
    TPI 287 and <t>alisertib</t> synergistically induce apoptosis in glioblastoma neurosphere cells. GB30 cells were stained with H E (A-C) after treatment with alisertib (32 nM, approximate IC 50 ) (D-F), TPI 287 (0.70 nM, approximate IC 50 ) (G-I), or both drugs (J-L) for a period of 3, 5, and 7 days. Arrows indicate large mononucleated cells; arrowheads indicate multinucleated cells. Magnification (600x) is identical in all panels. Apoptotic cells were counted, and average values from 2 experiments are shown (M). Similarly treated neurospheres were dissociated with accutase and counted and stained with an Alexafluor 594 annexin V conjugate and analyzed using a Countess II FL equipped with a Texas Red fluorescent light cube. N. Combined effects of alisertib (32 nM) and TPI 287 (0.70 nM) on apoptosis as measured by fold increase in annexin V binding. O. Combined effects of alisertib (32 nM) and TPI 287 (0.35 nM, 0.5x approximate IC 50 ) on apoptosis. GB30 neurosphere cells showed greatly increased annexin V labeling when treated with TPI 287 and alisertib. This experiment was performed twice and showed similar results. A representative example is shown
    Alisertib, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cxcr4 tango plasmid
    <t>CXCR4</t> activation with ubiquitin and CXCL12 activates similar signaling pathways in primary human monocytes Pharmacological inhibition of CXCL12 and ubiquitin (Ub) induced chemotaxis in human monocytes. Cells were incubated with vehicle or AMD3100 (10 μM, PBMNCs: n = 7, ( A ); monocytes: n= 4 ( C )) or with vehicle and the TM2 peptide (100 μM, PBMNCs: n = 3 ( B ); monocytes: n = 4, (D) ) and migration towards 10 nM of CXCL12 or Ub tested. CI (% ctrl.): chemotactic index in % of vehicle treated cells (= 100%). *: p
    Cxcr4 Tango Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( A ) In four human UC cell lines, western blot analysis (WB) revealed DAB2 protein expression in UM-UC-3, J82, and T24 cells, whereas MGH-U-3 cells did not express DAB2 protein. The RT-qPCR analysis also revealed mRNA expression of DAB2 corresponded to the results of WB. ( B ) In three cell lines knocked-down with DAB2 siRNA transfection, DAB2 protein reduction was confirmed by WB. Epithelial-to-mesenchymal transition-related markers, proteins involved in the activation of intracellular signaling pathways, were quantified by western blot analysis. Actin was used as a loading control. The numbers below the bands are the results of densitometric quantification. NT: No treatment; NC: Negative control; SI: DAB2 -si RNA.

    Journal: Diagnostics

    Article Title: Disabled Homolog 2 (DAB2) Protein in Tumor Microenvironment Correlates with Aggressive Phenotype in Human Urothelial Carcinoma of the Bladder

    doi: 10.3390/diagnostics10010054

    Figure Lengend Snippet: ( A ) In four human UC cell lines, western blot analysis (WB) revealed DAB2 protein expression in UM-UC-3, J82, and T24 cells, whereas MGH-U-3 cells did not express DAB2 protein. The RT-qPCR analysis also revealed mRNA expression of DAB2 corresponded to the results of WB. ( B ) In three cell lines knocked-down with DAB2 siRNA transfection, DAB2 protein reduction was confirmed by WB. Epithelial-to-mesenchymal transition-related markers, proteins involved in the activation of intracellular signaling pathways, were quantified by western blot analysis. Actin was used as a loading control. The numbers below the bands are the results of densitometric quantification. NT: No treatment; NC: Negative control; SI: DAB2 -si RNA.

    Article Snippet: In a 6-well plate, UM-UC-3, J82, and T24 cells were transfected for 48 h with 25 pmol of siRNA and 3.75 μL of Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions.

    Techniques: Western Blot, Expressing, Quantitative RT-PCR, Transfection, Activation Assay, Negative Control

    ( A ) In cell proliferation assays, the proliferation ability of UM-UC-3 NT tended to be higher than that of NC and SI compared to T24. But there were no significant differences in the proliferative ability for all three cell lines. ( B ) In migration assays, there was significant decreased migration ability of si-RNA transfected cells than negative control cells in UM-UC-3 and T24. ( C ) In the Matrigel invasion assay, there was significant decreased invasion ability of si-RNA transfected cells than negative controls in J82 and T24. NT: No treatment; NC: Negative control; SI: DAB2 -si RNA.

    Journal: Diagnostics

    Article Title: Disabled Homolog 2 (DAB2) Protein in Tumor Microenvironment Correlates with Aggressive Phenotype in Human Urothelial Carcinoma of the Bladder

    doi: 10.3390/diagnostics10010054

    Figure Lengend Snippet: ( A ) In cell proliferation assays, the proliferation ability of UM-UC-3 NT tended to be higher than that of NC and SI compared to T24. But there were no significant differences in the proliferative ability for all three cell lines. ( B ) In migration assays, there was significant decreased migration ability of si-RNA transfected cells than negative control cells in UM-UC-3 and T24. ( C ) In the Matrigel invasion assay, there was significant decreased invasion ability of si-RNA transfected cells than negative controls in J82 and T24. NT: No treatment; NC: Negative control; SI: DAB2 -si RNA.

    Article Snippet: In a 6-well plate, UM-UC-3, J82, and T24 cells were transfected for 48 h with 25 pmol of siRNA and 3.75 μL of Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions.

    Techniques: Migration, Transfection, Negative Control, Invasion Assay

    Effects of repeated viable cell estimation on growth of EPC, MSC and HUVEC cultured on 24-well plate. Growth curves of EPC ( A ), MSC ( D ) and HUVEC ( G ), which were cultured in 24-well plate. The control group and the test group were monitored by MTT assay and resazurin assay, respectively. Each point was the mean of absorbance readings of four scaffolds. The representative standard curves of resazurin assay of EPC ( B ), MSC ( E ) and HUVEC ( H ). The representative standard curves of MTT assay of EPC ( C ), MSC ( F ) and HUVEC ( I )

    Journal: Regenerative Biomaterials

    Article Title: A resazurin-based, nondestructive assay for monitoring cell proliferation during a scaffold-based 3D culture process

    doi: 10.1093/rb/rbaa002

    Figure Lengend Snippet: Effects of repeated viable cell estimation on growth of EPC, MSC and HUVEC cultured on 24-well plate. Growth curves of EPC ( A ), MSC ( D ) and HUVEC ( G ), which were cultured in 24-well plate. The control group and the test group were monitored by MTT assay and resazurin assay, respectively. Each point was the mean of absorbance readings of four scaffolds. The representative standard curves of resazurin assay of EPC ( B ), MSC ( E ) and HUVEC ( H ). The representative standard curves of MTT assay of EPC ( C ), MSC ( F ) and HUVEC ( I )

    Article Snippet: The cell-seeded scaffolds were cultured in 6-well plate for 24 h allowing cell adherence and then were incubated in 24-well plates with 1-ml culture media per well.

    Techniques: Cell Culture, MTT Assay, Resazurin Assay

    TPI 287 and alisertib synergistically induce apoptosis in glioblastoma neurosphere cells. GB30 cells were stained with H E (A-C) after treatment with alisertib (32 nM, approximate IC 50 ) (D-F), TPI 287 (0.70 nM, approximate IC 50 ) (G-I), or both drugs (J-L) for a period of 3, 5, and 7 days. Arrows indicate large mononucleated cells; arrowheads indicate multinucleated cells. Magnification (600x) is identical in all panels. Apoptotic cells were counted, and average values from 2 experiments are shown (M). Similarly treated neurospheres were dissociated with accutase and counted and stained with an Alexafluor 594 annexin V conjugate and analyzed using a Countess II FL equipped with a Texas Red fluorescent light cube. N. Combined effects of alisertib (32 nM) and TPI 287 (0.70 nM) on apoptosis as measured by fold increase in annexin V binding. O. Combined effects of alisertib (32 nM) and TPI 287 (0.35 nM, 0.5x approximate IC 50 ) on apoptosis. GB30 neurosphere cells showed greatly increased annexin V labeling when treated with TPI 287 and alisertib. This experiment was performed twice and showed similar results. A representative example is shown

    Journal: Journal of neuro-oncology

    Article Title: The CNS penetrating taxane TPI 287 and the AURKA inhibitor alisertib induce synergistic apoptosis in glioblastoma cells

    doi: 10.1007/s11060-018-2755-2

    Figure Lengend Snippet: TPI 287 and alisertib synergistically induce apoptosis in glioblastoma neurosphere cells. GB30 cells were stained with H E (A-C) after treatment with alisertib (32 nM, approximate IC 50 ) (D-F), TPI 287 (0.70 nM, approximate IC 50 ) (G-I), or both drugs (J-L) for a period of 3, 5, and 7 days. Arrows indicate large mononucleated cells; arrowheads indicate multinucleated cells. Magnification (600x) is identical in all panels. Apoptotic cells were counted, and average values from 2 experiments are shown (M). Similarly treated neurospheres were dissociated with accutase and counted and stained with an Alexafluor 594 annexin V conjugate and analyzed using a Countess II FL equipped with a Texas Red fluorescent light cube. N. Combined effects of alisertib (32 nM) and TPI 287 (0.70 nM) on apoptosis as measured by fold increase in annexin V binding. O. Combined effects of alisertib (32 nM) and TPI 287 (0.35 nM, 0.5x approximate IC 50 ) on apoptosis. GB30 neurosphere cells showed greatly increased annexin V labeling when treated with TPI 287 and alisertib. This experiment was performed twice and showed similar results. A representative example is shown

    Article Snippet: GB30 cells were seeded at 2×105 cells/well into 6-well plates and treated with TPI 287, alisertib, or both for 3, 5, and 7 d and stained with DRAQ5 and CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher).

    Techniques: Staining, Binding Assay, Labeling

    Cell cycle progression of GB30 cells treated with alisertib, TPI 287 alone and in combination. GB30 tumor stem cells were treated with alisertib (32 nM), TPI 287 (0.70 nM) or both for 3, 5, and 7 days, stained with DRAQ5 and CellEvent Caspase-3/7 Green Detection Reagent and analyzed by flow cytometry. Black lines represent DNA content of all single cells analyzed, while green lines represent DNA content of cells positive for caspase 3/7 activity. Counts were normalized to the mode. Numbers in the top right corners of histograms indicate the percentage of cells showing active caspase 3/7 activity. Arrows indicate G 2 /M tetraploid populations, and asterisks mark sub G 1 /G 0 populations. A replicate experiment yielded similar results. A representative example is shown

    Journal: Journal of neuro-oncology

    Article Title: The CNS penetrating taxane TPI 287 and the AURKA inhibitor alisertib induce synergistic apoptosis in glioblastoma cells

    doi: 10.1007/s11060-018-2755-2

    Figure Lengend Snippet: Cell cycle progression of GB30 cells treated with alisertib, TPI 287 alone and in combination. GB30 tumor stem cells were treated with alisertib (32 nM), TPI 287 (0.70 nM) or both for 3, 5, and 7 days, stained with DRAQ5 and CellEvent Caspase-3/7 Green Detection Reagent and analyzed by flow cytometry. Black lines represent DNA content of all single cells analyzed, while green lines represent DNA content of cells positive for caspase 3/7 activity. Counts were normalized to the mode. Numbers in the top right corners of histograms indicate the percentage of cells showing active caspase 3/7 activity. Arrows indicate G 2 /M tetraploid populations, and asterisks mark sub G 1 /G 0 populations. A replicate experiment yielded similar results. A representative example is shown

    Article Snippet: GB30 cells were seeded at 2×105 cells/well into 6-well plates and treated with TPI 287, alisertib, or both for 3, 5, and 7 d and stained with DRAQ5 and CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher).

    Techniques: Staining, Flow Cytometry, Cytometry, Activity Assay

    CXCR4 activation with ubiquitin and CXCL12 activates similar signaling pathways in primary human monocytes Pharmacological inhibition of CXCL12 and ubiquitin (Ub) induced chemotaxis in human monocytes. Cells were incubated with vehicle or AMD3100 (10 μM, PBMNCs: n = 7, ( A ); monocytes: n= 4 ( C )) or with vehicle and the TM2 peptide (100 μM, PBMNCs: n = 3 ( B ); monocytes: n = 4, (D) ) and migration towards 10 nM of CXCL12 or Ub tested. CI (% ctrl.): chemotactic index in % of vehicle treated cells (= 100%). *: p

    Journal: Molecular and cellular biochemistry

    Article Title: Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists

    doi: 10.1007/s11010-017-3044-7

    Figure Lengend Snippet: CXCR4 activation with ubiquitin and CXCL12 activates similar signaling pathways in primary human monocytes Pharmacological inhibition of CXCL12 and ubiquitin (Ub) induced chemotaxis in human monocytes. Cells were incubated with vehicle or AMD3100 (10 μM, PBMNCs: n = 7, ( A ); monocytes: n= 4 ( C )) or with vehicle and the TM2 peptide (100 μM, PBMNCs: n = 3 ( B ); monocytes: n = 4, (D) ) and migration towards 10 nM of CXCL12 or Ub tested. CI (% ctrl.): chemotactic index in % of vehicle treated cells (= 100%). *: p

    Article Snippet: HTLA cells (2.5×105 /well) were seeded in a 6-well plate and transfected with 1500 ng of the CXCR4-Tango plasmid using Lipofectamine 3000 (ThermoScientific).

    Techniques: Activation Assay, Inhibition, Chemotaxis Assay, Incubation, Migration

    CXCR4 activation with ubiquitin and CXCL12 induces chemotaxis and activates similar signaling pathways in primary human cells of non-hematopoietic origin A : Migration of human primary aortic smooth muscle cells (hVSMC, n = 6) towards CXCL12 (circles) and ubiquitin (Ub, squares). B/C : hVSMC were incubated with vehicle or AMD3100 (10 μM, n = 4, ( B )) or with vehicle and the TM2 peptide (100 μM, n = 4, ( C )) and migration towards 10 nM of CXCL12 or Ub tested. CI (% ctrl.): chemotactic index in % of vehicle treated cells (= 100%). *: p

    Journal: Molecular and cellular biochemistry

    Article Title: Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists

    doi: 10.1007/s11010-017-3044-7

    Figure Lengend Snippet: CXCR4 activation with ubiquitin and CXCL12 induces chemotaxis and activates similar signaling pathways in primary human cells of non-hematopoietic origin A : Migration of human primary aortic smooth muscle cells (hVSMC, n = 6) towards CXCL12 (circles) and ubiquitin (Ub, squares). B/C : hVSMC were incubated with vehicle or AMD3100 (10 μM, n = 4, ( B )) or with vehicle and the TM2 peptide (100 μM, n = 4, ( C )) and migration towards 10 nM of CXCL12 or Ub tested. CI (% ctrl.): chemotactic index in % of vehicle treated cells (= 100%). *: p

    Article Snippet: HTLA cells (2.5×105 /well) were seeded in a 6-well plate and transfected with 1500 ng of the CXCR4-Tango plasmid using Lipofectamine 3000 (ThermoScientific).

    Techniques: Activation Assay, Chemotaxis Assay, Migration, Incubation

    Ubiquitin induces chemical shift changes in the NMR spectrum of CXCR4 in membranes but fails to recruit β-arrestin 2 to CXCR4 A. β-arrestin 2 recruitment assay (PRESTO-Tango) for CXCR4. Ub: ubiquitin. Ub + 6bK: Ub + 1 μM 6bK. Alb: albumin. RLU (%): relative luminescence units in % of the RLU after treatment with 1 μM CXCL12 (= 100%). N= 3. B. β-arrestin 2 recruitment assay (PRESTO-Tango) for CXCR4. Ub was tested in the presence of 10 nM CXCL12. RLU (%): relative luminescence units in % of the RLU after treatment with 1 μM CXCL12 (= 100%). N= 3. C–F : 1 H- 13 C HSQC spectra of reductively methylated CXCR4 membrane preparations were recorded without (blue, ( C )) and with (red) 1 μM CXCL12 ( D ), ubiquitin ( E ) or CXCL11 ( F ). Signal 1 represents the N-terminus of CXCR4, signal 2 and signal 3 represent lysine residues that have been successfully modified with two 13 C labeled methyl groups. Black arrows indicate the difference in chemical shifts or broadening (loss) of the signal. Chemical shift perturbations (CSPs) of the N-terminus of reductively methylated CXCR4 were 0.0064, 0.0014 and 0.0024 in the presence of CXCL12, ubiquitin and CXCL11, respectively; CSPs greater 0.0058 were considered significant.

    Journal: Molecular and cellular biochemistry

    Article Title: Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists

    doi: 10.1007/s11010-017-3044-7

    Figure Lengend Snippet: Ubiquitin induces chemical shift changes in the NMR spectrum of CXCR4 in membranes but fails to recruit β-arrestin 2 to CXCR4 A. β-arrestin 2 recruitment assay (PRESTO-Tango) for CXCR4. Ub: ubiquitin. Ub + 6bK: Ub + 1 μM 6bK. Alb: albumin. RLU (%): relative luminescence units in % of the RLU after treatment with 1 μM CXCL12 (= 100%). N= 3. B. β-arrestin 2 recruitment assay (PRESTO-Tango) for CXCR4. Ub was tested in the presence of 10 nM CXCL12. RLU (%): relative luminescence units in % of the RLU after treatment with 1 μM CXCL12 (= 100%). N= 3. C–F : 1 H- 13 C HSQC spectra of reductively methylated CXCR4 membrane preparations were recorded without (blue, ( C )) and with (red) 1 μM CXCL12 ( D ), ubiquitin ( E ) or CXCL11 ( F ). Signal 1 represents the N-terminus of CXCR4, signal 2 and signal 3 represent lysine residues that have been successfully modified with two 13 C labeled methyl groups. Black arrows indicate the difference in chemical shifts or broadening (loss) of the signal. Chemical shift perturbations (CSPs) of the N-terminus of reductively methylated CXCR4 were 0.0064, 0.0014 and 0.0024 in the presence of CXCL12, ubiquitin and CXCL11, respectively; CSPs greater 0.0058 were considered significant.

    Article Snippet: HTLA cells (2.5×105 /well) were seeded in a 6-well plate and transfected with 1500 ng of the CXCR4-Tango plasmid using Lipofectamine 3000 (ThermoScientific).

    Techniques: Nuclear Magnetic Resonance, Methylation, Modification, Labeling