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  • 99
    Thermo Fisher 6 well plates
    WISP1 induced an EMT gene signature in mouse/human melanoma cells. Unless otherwise specified, all cells were plated on <t>6-well</t> 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
    6 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6900 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 6 well plates
    The 1, 25 dihyroxyvitamin D3 (vitamin D3) induced VDR expression and a decreased DNA damage load in HuLM cells. HuLM cells were treated with 100 nM of vitamin D3 for 3 d. Ethanol vehicle was added to the control cells. a Real-time PCR analysis of the mRNA level of the VDR gene was performed. The mRNA levels were normalized to 18 S rRNA, and normalized values were used to generate the graph. Data are presented as the mean ± SEM of triplicate measurements. Cell lysates were analyzed by Western blot analysis using ( b ) anti-VDR antibody and ( c ) anti-γ-H2AX. The intensity of each protein signal was quantified and normalized to the corresponding β-actin and presented in the graph as the mean ± SD. d HuLM cells (8 × 10 4 ) were seeded on sterile glass coverslips in <t>6-well</t> plates and treated with 100 nM vitamin D3 for 3 d. Ethanol vehicle was added to the control cells. γ-H2AX foci reflecting DNA DSB were assessed using immunofluorescence staining and confocal laser microscopy 40x imaging. Individual data points were the γ-H2AX focus corrected fluorescence intensity per nuclear area, as measured by ImageJ, and lines represent the median ± 95% CI. *** P
    6 Well Plates, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1641 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Corning Life Sciences 6 well plates
    (A) Schematic illustration of mechanical vibration experiment in this work. Vertical mechanical vibration was applied to the <t>6-well</t> plate for 10 min every 24 h after the medium change. Phase-contrast microscopic images were acquired every 12 h. Cells were passaged continuously from P3 (Cycle 1) to P5 (Cycle 3). On the 7th day, the samples treated with vibration were collected, and seeded into a new 6-well plate. At the same time, partial samples were stained to evaluate the undifferentiation marker (B) Visualization of relative distances between designed conditions in this work. Four related parameters (acceleration, frequency, amplitude, energy) are visualized by principal component analysis. The proportion of variance for each principal component (PC) is PC1 (0.56), PC2 (0.27), and PC3 (0.16). The loadings are: acceleration: frequency: amplitude: energy = PC1 (−0.04, 0.42, −0.65, −0.64), PC2 (0.89, −0.43, 0.09, 0.13), PC3 (0.46, −0.80, −0.25, −0.30). The color representation is visualized in the legend. Representative four points from tapping and closing of incubator door condition measured in the preliminary examination is visualized with smaller dots to indicate the range of vibration.
    6 Well Plates, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 1368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pierce streptavidin coated high capacity plates
    Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-GalNAc-T2 or non-transfected, control samples. Organoids were fed with DMSO (top) or 50 µM compound 11 (bottom), fixed and treated with biotin alkyne under CuAAC conditions followed by <t>Streptavidin</t> Alexa Fluor 647 staining. Data are from one representative out of two independent experiments and shown as grayscale images for each channel and a color merge image of all three channels. Scale bar, 100 µm.
    Pierce Streptavidin Coated High Capacity Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam 6 well plates
    Rabeprazole promotes topo I degradation and irinotecan resistance. A , HCT116 cells were plated in a <t>6-well</t> plate and treated with various concentrations of rabeprazole (0, 10, 20 μM) for 72 h, and then with 2.5 μM SN-38 and harvested after 90 or 180 min. Cell lysates were immunoblotted with anti-topoI and anti-β-actin. B , Genomically edited HCT116 cells with TopoI-EGFP fusion proteins were treated with 5 and 10 μM of Rabeprazole for 48 hours and topoI-GFP protein level was analyzed by confocal microscope. C . HCT116 cells were plated in a 6-well plate, treated with 40 μM rabeprazole or DMSO for 72 h, and then with 2.5 μM SN-38, and harvested after 90 or 180 min. Cell lysates were immunoblotted with anti-pDNA-PKcs and anti-β-actin. D , HCT 116 cells were treated with rabeprazole, control and treated cells were analyzed by immunofluorescence analysis with anti-phospho-DNA-PKcs-pS2056 and confocal microscopy. E . HCT116 cells were plated in a 6-well plate and treated with rabeprazole or DMSO for 72 h. Then, 50 cells were plated in each well of a 6-well plate and treated with various concentrations of SN-38 for 24 hours. Cell colonies were counted after 14 days. F . HCT116 cells were plated in a 6-well plate and treated with 40 μM rabeprazole or DMSO for 72 h. Then, cells were plated in a 96-well plate and treated with various concentrations of SN-38 for 72 h. Cell viability was determined by luminescence detection.
    6 Well Plates, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Greiner Bio 6 well plates
    Rabeprazole promotes topo I degradation and irinotecan resistance. A , HCT116 cells were plated in a <t>6-well</t> plate and treated with various concentrations of rabeprazole (0, 10, 20 μM) for 72 h, and then with 2.5 μM SN-38 and harvested after 90 or 180 min. Cell lysates were immunoblotted with anti-topoI and anti-β-actin. B , Genomically edited HCT116 cells with TopoI-EGFP fusion proteins were treated with 5 and 10 μM of Rabeprazole for 48 hours and topoI-GFP protein level was analyzed by confocal microscope. C . HCT116 cells were plated in a 6-well plate, treated with 40 μM rabeprazole or DMSO for 72 h, and then with 2.5 μM SN-38, and harvested after 90 or 180 min. Cell lysates were immunoblotted with anti-pDNA-PKcs and anti-β-actin. D , HCT 116 cells were treated with rabeprazole, control and treated cells were analyzed by immunofluorescence analysis with anti-phospho-DNA-PKcs-pS2056 and confocal microscopy. E . HCT116 cells were plated in a 6-well plate and treated with rabeprazole or DMSO for 72 h. Then, 50 cells were plated in each well of a 6-well plate and treated with various concentrations of SN-38 for 24 hours. Cell colonies were counted after 14 days. F . HCT116 cells were plated in a 6-well plate and treated with 40 μM rabeprazole or DMSO for 72 h. Then, cells were plated in a 96-well plate and treated with various concentrations of SN-38 for 72 h. Cell viability was determined by luminescence detection.
    6 Well Plates, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 92/100, based on 368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson 6 well plates
    Immunobiotic mediated regulation of adipocyte differentiation in an immune cell-adipocyte co-culture model. The mRNA expressions of PPARγ and GLUT4 resulted from various immunobiotic lactic acid bacteria (LAB) stimulation into the co-culture model. A total of 1 × 10 6 immune cells/well of <t>6-well</t> plate were cultured on adipocytes 4-days differentiated. LAB samples (5 × 10 8 cells/well) also added into the adipocyte plate and maintained for further 2 or 4 Days. All data shown are the average ±SD of 3 independent experiments performed in triplicate. The asterisk (*) indicated statistical differences when compared with control at the significance level of p
    6 Well Plates, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 952 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson collagen coated 6 well plates
    Immunobiotic mediated regulation of adipocyte differentiation in an immune cell-adipocyte co-culture model. The mRNA expressions of PPARγ and GLUT4 resulted from various immunobiotic lactic acid bacteria (LAB) stimulation into the co-culture model. A total of 1 × 10 6 immune cells/well of <t>6-well</t> plate were cultured on adipocytes 4-days differentiated. LAB samples (5 × 10 8 cells/well) also added into the adipocyte plate and maintained for further 2 or 4 Days. All data shown are the average ±SD of 3 independent experiments performed in triplicate. The asterisk (*) indicated statistical differences when compared with control at the significance level of p
    Collagen Coated 6 Well Plates, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Sarstedt 6 well plates
    Immunobiotic mediated regulation of adipocyte differentiation in an immune cell-adipocyte co-culture model. The mRNA expressions of PPARγ and GLUT4 resulted from various immunobiotic lactic acid bacteria (LAB) stimulation into the co-culture model. A total of 1 × 10 6 immune cells/well of <t>6-well</t> plate were cultured on adipocytes 4-days differentiated. LAB samples (5 × 10 8 cells/well) also added into the adipocyte plate and maintained for further 2 or 4 Days. All data shown are the average ±SD of 3 independent experiments performed in triplicate. The asterisk (*) indicated statistical differences when compared with control at the significance level of p
    6 Well Plates, supplied by Sarstedt, used in various techniques. Bioz Stars score: 93/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher collagen coated 6 well plates
    Protection of 3D spheroid-derived MSCs on hepatocyte injury  in vitro . Hepatocytes were isolated from C57BL/6 mice and cultured in collagen-coated 6-well plates. After treatment with CCl 4  for 6 h, the medium of cells was replaced with normal medium, conditioned medium of 2D cultured MSCs, or conditioned medium of 3D spheroids of MSCs for 24 h before LDH assays, apoptosis analysis, and RT-PCR. (a) LDH assays. (b) Statistics of Annexin V staining. (c) Images of Annexin V staining. Annexin V staining was performed to evaluate cell apoptosis (red), with DPAI for nucleus staining (blue). (d) RT-PCR. The mRNA levels of Bax, Bcl-1, NF κ B, and TGF- β  were tested by RT-PCR, with GAPDH as the internal control. (e) Quantitative analysis of Bax/Bcl mRNA level. (f) Quantitative analysis of NF κ B mRNA level. Band intensities of NF κ B were normalized to those of the internal control. (g) Quantitative analysis of TGF- β  mRNA level. Band intensities of TGF- β  were normalized to those of the internal control.  $$ p
    Collagen Coated 6 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eppendorf AG 6 well plates
    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in <t>6-well</t> format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.
    6 Well Plates, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 99/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Becton Dickinson 6 well plates pre coated
    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in <t>6-well</t> format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.
    6 Well Plates Pre Coated, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher nunc cell culture treated multidishes
    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in <t>6-well</t> format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.
    Nunc Cell Culture Treated Multidishes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 459 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore collagen coated 6 well plates
    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in <t>6-well</t> format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.
    Collagen Coated 6 Well Plates, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    MatTek glass bottom 6 well plates
    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in <t>6-well</t> format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.
    Glass Bottom 6 Well Plates, supplied by MatTek, used in various techniques. Bioz Stars score: 90/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Nest Biotechnology 6 well plates
    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in <t>6-well</t> format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.
    6 Well Plates, supplied by Nest Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gelatin sigma coated 6 well plates
    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in <t>6-well</t> format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.
    Gelatin Sigma Coated 6 Well Plates, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences polystyrene 6 well plates
    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in <t>6-well</t> format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.
    Polystyrene 6 Well Plates, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 88/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific 6 well plates
    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in <t>6-well</t> format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.
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    Becton Dickinson fibronectin coated 6 well plates
    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in <t>6-well</t> format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.
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    SPL Life Sciences 6 well plates
    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in <t>6-well</t> format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.
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    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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.RA118.006122

    Figure Lengend 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

    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.

    Techniques: 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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.RA118.006122

    Figure Lengend 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

    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.

    Techniques: 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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.RA118.006122

    Figure Lengend 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

    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.

    Techniques: 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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.RA118.006122

    Figure Lengend 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

    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.

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

    The 1, 25 dihyroxyvitamin D3 (vitamin D3) induced VDR expression and a decreased DNA damage load in HuLM cells. HuLM cells were treated with 100 nM of vitamin D3 for 3 d. Ethanol vehicle was added to the control cells. a Real-time PCR analysis of the mRNA level of the VDR gene was performed. The mRNA levels were normalized to 18 S rRNA, and normalized values were used to generate the graph. Data are presented as the mean ± SEM of triplicate measurements. Cell lysates were analyzed by Western blot analysis using ( b ) anti-VDR antibody and ( c ) anti-γ-H2AX. The intensity of each protein signal was quantified and normalized to the corresponding β-actin and presented in the graph as the mean ± SD. d HuLM cells (8 × 10 4 ) were seeded on sterile glass coverslips in 6-well plates and treated with 100 nM vitamin D3 for 3 d. Ethanol vehicle was added to the control cells. γ-H2AX foci reflecting DNA DSB were assessed using immunofluorescence staining and confocal laser microscopy 40x imaging. Individual data points were the γ-H2AX focus corrected fluorescence intensity per nuclear area, as measured by ImageJ, and lines represent the median ± 95% CI. *** P

    Journal: Acta Pharmacologica Sinica

    Article Title: Hypovitaminosis D exacerbates the DNA damage load in human uterine fibroids, which is ameliorated by vitamin D3 treatment

    doi: 10.1038/s41401-018-0184-6

    Figure Lengend Snippet: The 1, 25 dihyroxyvitamin D3 (vitamin D3) induced VDR expression and a decreased DNA damage load in HuLM cells. HuLM cells were treated with 100 nM of vitamin D3 for 3 d. Ethanol vehicle was added to the control cells. a Real-time PCR analysis of the mRNA level of the VDR gene was performed. The mRNA levels were normalized to 18 S rRNA, and normalized values were used to generate the graph. Data are presented as the mean ± SEM of triplicate measurements. Cell lysates were analyzed by Western blot analysis using ( b ) anti-VDR antibody and ( c ) anti-γ-H2AX. The intensity of each protein signal was quantified and normalized to the corresponding β-actin and presented in the graph as the mean ± SD. d HuLM cells (8 × 10 4 ) were seeded on sterile glass coverslips in 6-well plates and treated with 100 nM vitamin D3 for 3 d. Ethanol vehicle was added to the control cells. γ-H2AX foci reflecting DNA DSB were assessed using immunofluorescence staining and confocal laser microscopy 40x imaging. Individual data points were the γ-H2AX focus corrected fluorescence intensity per nuclear area, as measured by ImageJ, and lines represent the median ± 95% CI. *** P

    Article Snippet: Immunofluorescence and laser confocal microscopy HuLM cells (8 × 104) were cultured on sterile glass coverslips in 6-well plates and were serum-starved overnight when they reached 60% confluence and treated with vitamin D3 100 nM for 3 d. Ethanol vehicle was added to the control cells.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Microscopy, Imaging, Fluorescence

    (A) Schematic illustration of mechanical vibration experiment in this work. Vertical mechanical vibration was applied to the 6-well plate for 10 min every 24 h after the medium change. Phase-contrast microscopic images were acquired every 12 h. Cells were passaged continuously from P3 (Cycle 1) to P5 (Cycle 3). On the 7th day, the samples treated with vibration were collected, and seeded into a new 6-well plate. At the same time, partial samples were stained to evaluate the undifferentiation marker (B) Visualization of relative distances between designed conditions in this work. Four related parameters (acceleration, frequency, amplitude, energy) are visualized by principal component analysis. The proportion of variance for each principal component (PC) is PC1 (0.56), PC2 (0.27), and PC3 (0.16). The loadings are: acceleration: frequency: amplitude: energy = PC1 (−0.04, 0.42, −0.65, −0.64), PC2 (0.89, −0.43, 0.09, 0.13), PC3 (0.46, −0.80, −0.25, −0.30). The color representation is visualized in the legend. Representative four points from tapping and closing of incubator door condition measured in the preliminary examination is visualized with smaller dots to indicate the range of vibration.

    Journal: Regenerative Therapy

    Article Title: Effect of mechanical vibration stress in cell culture on human induced pluripotent stem cells

    doi: 10.1016/j.reth.2019.05.002

    Figure Lengend Snippet: (A) Schematic illustration of mechanical vibration experiment in this work. Vertical mechanical vibration was applied to the 6-well plate for 10 min every 24 h after the medium change. Phase-contrast microscopic images were acquired every 12 h. Cells were passaged continuously from P3 (Cycle 1) to P5 (Cycle 3). On the 7th day, the samples treated with vibration were collected, and seeded into a new 6-well plate. At the same time, partial samples were stained to evaluate the undifferentiation marker (B) Visualization of relative distances between designed conditions in this work. Four related parameters (acceleration, frequency, amplitude, energy) are visualized by principal component analysis. The proportion of variance for each principal component (PC) is PC1 (0.56), PC2 (0.27), and PC3 (0.16). The loadings are: acceleration: frequency: amplitude: energy = PC1 (−0.04, 0.42, −0.65, −0.64), PC2 (0.89, −0.43, 0.09, 0.13), PC3 (0.46, −0.80, −0.25, −0.30). The color representation is visualized in the legend. Representative four points from tapping and closing of incubator door condition measured in the preliminary examination is visualized with smaller dots to indicate the range of vibration.

    Article Snippet: Cells dissociated into single cells were seeded at the concentration of 5000 cells/well in 6-well plates (Corning Life Sciences, Corning, NY, USA).

    Techniques: Staining, Marker

    Clonogenic and self-renewal potential of MDA-MB-231 cells cultured on a 2D surface or within Col-I gels. (A–D) Evaluation of the clonogenic potential of MDA-MB-231 cells previously cultured on a 2D surface or within Col-I gels. Cells were collected and processed to obtain individualized cell suspension. After plating (500 cells/35 mm plate), cells were cultured for 14 days and then washed, fixed and stained with crystal violet. The images were acquired with a transilluminator and represent technical replicates of one independent experiment. (E) Bar graphs represent mean ± SD of five independent experiments performed in technical quadruplicates. (F–I) Evaluation of mammosphere (MS)-formation efficiency by MDA-MB-231 cells previously cultured on a 2D surface or within Col-I gels. Cells were collected, processed to obtain individualized cell suspension and grown in ultra-low attachment 6-well plates (5,000 cells/well) with culture medium enriched for MS formation. After 10 days, images of at least 25 fields per well were acquired. Dashed squares outline zoomed-in MS, which have a minimum diameter of 50 μm (scale) and compact shape. Bar graphs (J) and scatter plots (K–M) represent mean ± SD of five independent experiments, performed in technical triplicate.

    Journal: PeerJ

    Article Title: High type I collagen density fails to increase breast cancer stem cell phenotype

    doi: 10.7717/peerj.9153

    Figure Lengend Snippet: Clonogenic and self-renewal potential of MDA-MB-231 cells cultured on a 2D surface or within Col-I gels. (A–D) Evaluation of the clonogenic potential of MDA-MB-231 cells previously cultured on a 2D surface or within Col-I gels. Cells were collected and processed to obtain individualized cell suspension. After plating (500 cells/35 mm plate), cells were cultured for 14 days and then washed, fixed and stained with crystal violet. The images were acquired with a transilluminator and represent technical replicates of one independent experiment. (E) Bar graphs represent mean ± SD of five independent experiments performed in technical quadruplicates. (F–I) Evaluation of mammosphere (MS)-formation efficiency by MDA-MB-231 cells previously cultured on a 2D surface or within Col-I gels. Cells were collected, processed to obtain individualized cell suspension and grown in ultra-low attachment 6-well plates (5,000 cells/well) with culture medium enriched for MS formation. After 10 days, images of at least 25 fields per well were acquired. Dashed squares outline zoomed-in MS, which have a minimum diameter of 50 μm (scale) and compact shape. Bar graphs (J) and scatter plots (K–M) represent mean ± SD of five independent experiments, performed in technical triplicate.

    Article Snippet: Analysis of self-renewal potential Cells previously cultured on 2D surface (5 × 103 cells/plate) or within Col-I gels (25 × 103 cells/gel) for 7 days were collected, dissociated, and cell suspension was plated in ultra-low attachment 6-well plates (Corning Inc., Corning, NY, USA) at low cell density (5 × 103 cells/well) in DMEM-F12 containing 20 ng/ml bFGF (Sigma, Ronkonkoma, NY, USA), 20 ng/ml EGF (Sigma, Ronkonkoma, NY, USA) and 1X B-27 supplement (Gibco, Langley, OK, USA).

    Techniques: Multiple Displacement Amplification, Cell Culture, Staining

    Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-GalNAc-T2 or non-transfected, control samples. Organoids were fed with DMSO (top) or 50 µM compound 11 (bottom), fixed and treated with biotin alkyne under CuAAC conditions followed by Streptavidin Alexa Fluor 647 staining. Data are from one representative out of two independent experiments and shown as grayscale images for each channel and a color merge image of all three channels. Scale bar, 100 µm.

    Journal: bioRxiv

    Article Title: Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

    doi: 10.1101/2020.04.23.057208

    Figure Lengend Snippet: Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-GalNAc-T2 or non-transfected, control samples. Organoids were fed with DMSO (top) or 50 µM compound 11 (bottom), fixed and treated with biotin alkyne under CuAAC conditions followed by Streptavidin Alexa Fluor 647 staining. Data are from one representative out of two independent experiments and shown as grayscale images for each channel and a color merge image of all three channels. Scale bar, 100 µm.

    Article Snippet: To amplify the amount of viral stock (P1 to P2); 30 mL of Sf21 cells (9×105 cells/mL) were seeded at 6-well plates, incubated overnight and transferred to 30 mL Sf21 cell suspension for incubation at 125 rpm, 27 °C for 3 days.

    Techniques: Transfection, Staining

    UDP-GalNAzMe 5 recognition by GalNAc-Ts and delivery to the living cell. A, in vitro peptide glycosylation by purified GalNAc-Ts. Data are biological duplicates as average of technical duplicates. B , lysate protein glycosylation by GalNAc-T1 and GalNAc-T2. A membrane preparation was used as a lysate protein source, probed with soluble GalNAc-Ts and azide-tagged UDP-sugars, and subjected to CuAAC with clickable biotin. Streptavidin blot was used to visualize glycosylation. Data are from one representative out of three independent experiments. C , Biosynthesis of UDP-GalNAzMe in K-562 cells stably transfected with WT- or mut-AGX1, as assessed by HPAEC-PAD. Standards include UDP-GalNAzMe ( 5 ) and its C4-epimer UDP-GlcNAzMe ( SI-7 ).

    Journal: bioRxiv

    Article Title: Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

    doi: 10.1101/2020.04.23.057208

    Figure Lengend Snippet: UDP-GalNAzMe 5 recognition by GalNAc-Ts and delivery to the living cell. A, in vitro peptide glycosylation by purified GalNAc-Ts. Data are biological duplicates as average of technical duplicates. B , lysate protein glycosylation by GalNAc-T1 and GalNAc-T2. A membrane preparation was used as a lysate protein source, probed with soluble GalNAc-Ts and azide-tagged UDP-sugars, and subjected to CuAAC with clickable biotin. Streptavidin blot was used to visualize glycosylation. Data are from one representative out of three independent experiments. C , Biosynthesis of UDP-GalNAzMe in K-562 cells stably transfected with WT- or mut-AGX1, as assessed by HPAEC-PAD. Standards include UDP-GalNAzMe ( 5 ) and its C4-epimer UDP-GlcNAzMe ( SI-7 ).

    Article Snippet: To amplify the amount of viral stock (P1 to P2); 30 mL of Sf21 cells (9×105 cells/mL) were seeded at 6-well plates, incubated overnight and transferred to 30 mL Sf21 cell suspension for incubation at 125 rpm, 27 °C for 3 days.

    Techniques: In Vitro, Purification, Stable Transfection, Transfection

    An engineered BH-T2 double mutant enhances GalNAzMe labeling. A, in vitro glycosylation using WT- or BH-T2 as enzyme sources. UDP-GalNAz 2 and UDP-GalNAzMe 5 were used as substrates, and UDP-GalNAc 1 was used as a competitor at different concentrations. Azide-labeled glycoproteins were visualized as in Fig. 2B . Data are from one representative out of two independent replicates. B , live cell surface glycosylation by K-562 cells stably transfected with mut-AGX1 and WT- or BH-T2 and fed with DMSO, 50 µM compound 11 , or 3 µM Ac 4 GalNAz. Data are from one representative out of two independent replicates. C , Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-T2. Organoids were fed with 50 µM compound 11 or 1.5 µM Ac 4 GalNAz, fixed and treated with biotin alkyne under CuAAC conditions followed by Streptavidin Alexa Fluor 647 staining.

    Journal: bioRxiv

    Article Title: Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

    doi: 10.1101/2020.04.23.057208

    Figure Lengend Snippet: An engineered BH-T2 double mutant enhances GalNAzMe labeling. A, in vitro glycosylation using WT- or BH-T2 as enzyme sources. UDP-GalNAz 2 and UDP-GalNAzMe 5 were used as substrates, and UDP-GalNAc 1 was used as a competitor at different concentrations. Azide-labeled glycoproteins were visualized as in Fig. 2B . Data are from one representative out of two independent replicates. B , live cell surface glycosylation by K-562 cells stably transfected with mut-AGX1 and WT- or BH-T2 and fed with DMSO, 50 µM compound 11 , or 3 µM Ac 4 GalNAz. Data are from one representative out of two independent replicates. C , Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-T2. Organoids were fed with 50 µM compound 11 or 1.5 µM Ac 4 GalNAz, fixed and treated with biotin alkyne under CuAAC conditions followed by Streptavidin Alexa Fluor 647 staining.

    Article Snippet: To amplify the amount of viral stock (P1 to P2); 30 mL of Sf21 cells (9×105 cells/mL) were seeded at 6-well plates, incubated overnight and transferred to 30 mL Sf21 cell suspension for incubation at 125 rpm, 27 °C for 3 days.

    Techniques: Mutagenesis, Labeling, In Vitro, Stable Transfection, Transfection, Staining

    Rabeprazole promotes topo I degradation and irinotecan resistance. A , HCT116 cells were plated in a 6-well plate and treated with various concentrations of rabeprazole (0, 10, 20 μM) for 72 h, and then with 2.5 μM SN-38 and harvested after 90 or 180 min. Cell lysates were immunoblotted with anti-topoI and anti-β-actin. B , Genomically edited HCT116 cells with TopoI-EGFP fusion proteins were treated with 5 and 10 μM of Rabeprazole for 48 hours and topoI-GFP protein level was analyzed by confocal microscope. C . HCT116 cells were plated in a 6-well plate, treated with 40 μM rabeprazole or DMSO for 72 h, and then with 2.5 μM SN-38, and harvested after 90 or 180 min. Cell lysates were immunoblotted with anti-pDNA-PKcs and anti-β-actin. D , HCT 116 cells were treated with rabeprazole, control and treated cells were analyzed by immunofluorescence analysis with anti-phospho-DNA-PKcs-pS2056 and confocal microscopy. E . HCT116 cells were plated in a 6-well plate and treated with rabeprazole or DMSO for 72 h. Then, 50 cells were plated in each well of a 6-well plate and treated with various concentrations of SN-38 for 24 hours. Cell colonies were counted after 14 days. F . HCT116 cells were plated in a 6-well plate and treated with 40 μM rabeprazole or DMSO for 72 h. Then, cells were plated in a 96-well plate and treated with various concentrations of SN-38 for 72 h. Cell viability was determined by luminescence detection.

    Journal: PLoS ONE

    Article Title: CTDSP1 inhibitor rabeprazole regulates DNA-PKcs dependent topoisomerase I degradation and irinotecan drug resistance in colorectal cancer

    doi: 10.1371/journal.pone.0228002

    Figure Lengend Snippet: Rabeprazole promotes topo I degradation and irinotecan resistance. A , HCT116 cells were plated in a 6-well plate and treated with various concentrations of rabeprazole (0, 10, 20 μM) for 72 h, and then with 2.5 μM SN-38 and harvested after 90 or 180 min. Cell lysates were immunoblotted with anti-topoI and anti-β-actin. B , Genomically edited HCT116 cells with TopoI-EGFP fusion proteins were treated with 5 and 10 μM of Rabeprazole for 48 hours and topoI-GFP protein level was analyzed by confocal microscope. C . HCT116 cells were plated in a 6-well plate, treated with 40 μM rabeprazole or DMSO for 72 h, and then with 2.5 μM SN-38, and harvested after 90 or 180 min. Cell lysates were immunoblotted with anti-pDNA-PKcs and anti-β-actin. D , HCT 116 cells were treated with rabeprazole, control and treated cells were analyzed by immunofluorescence analysis with anti-phospho-DNA-PKcs-pS2056 and confocal microscopy. E . HCT116 cells were plated in a 6-well plate and treated with rabeprazole or DMSO for 72 h. Then, 50 cells were plated in each well of a 6-well plate and treated with various concentrations of SN-38 for 24 hours. Cell colonies were counted after 14 days. F . HCT116 cells were plated in a 6-well plate and treated with 40 μM rabeprazole or DMSO for 72 h. Then, cells were plated in a 96-well plate and treated with various concentrations of SN-38 for 72 h. Cell viability was determined by luminescence detection.

    Article Snippet: ImmunoblottingCells cultured in 6-well plates were scraped into an ice-cold RIPA buffer.

    Techniques: Microscopy, Immunofluorescence, Confocal Microscopy

    Silencing of CTDSP1 enhances topoI degradation and irinotecan resistance. A , Cells transfected with CTDSP1 or control siRNA were lyzed and the cells’ lysates were immunoblotted with anti-CTDSP1 and anti-β-actin antibodies. B , HCT116-siRNA CTDSP1 or control siRNA, treated with 2.5 μM SN-38 were harvested after 90 and 180 min. Cell lysates were immunoblotted with anti-topoI and anti-β-actin antibodies. Cells’ lysates were immunoblotted with anti-topoI and anti-β-actin antibodies. C , EGFP was integrated with topoI in HCT116 cells using CRISPR/Cas9 system and CTDSP1 was knocked down in this cell line by siRNA. Cells were treated with 2.5uM SN-38 for 60 min and the topoI-EGFP signal was imaged by Leica SP5 confocal microscope. D , HCT116-siRNA-CTDSP1 or control siRNA were plated in a 96-well plate and treated with various concentrations of SN-38 for 72 h. Cell viability was determined by detecting the luminescence. E , HCT116-siRNA-CTDSP1 or control siRNA cells were plated in a 6-well plate and treated with various concentrations of SN-38 for 24 h. Then, 50 cells per well were plated in a 6-well plate. After 14 days, when colonies were apparent (right panel), colonies were counted and the relative number of colonies was determined (left panel).

    Journal: PLoS ONE

    Article Title: CTDSP1 inhibitor rabeprazole regulates DNA-PKcs dependent topoisomerase I degradation and irinotecan drug resistance in colorectal cancer

    doi: 10.1371/journal.pone.0228002

    Figure Lengend Snippet: Silencing of CTDSP1 enhances topoI degradation and irinotecan resistance. A , Cells transfected with CTDSP1 or control siRNA were lyzed and the cells’ lysates were immunoblotted with anti-CTDSP1 and anti-β-actin antibodies. B , HCT116-siRNA CTDSP1 or control siRNA, treated with 2.5 μM SN-38 were harvested after 90 and 180 min. Cell lysates were immunoblotted with anti-topoI and anti-β-actin antibodies. Cells’ lysates were immunoblotted with anti-topoI and anti-β-actin antibodies. C , EGFP was integrated with topoI in HCT116 cells using CRISPR/Cas9 system and CTDSP1 was knocked down in this cell line by siRNA. Cells were treated with 2.5uM SN-38 for 60 min and the topoI-EGFP signal was imaged by Leica SP5 confocal microscope. D , HCT116-siRNA-CTDSP1 or control siRNA were plated in a 96-well plate and treated with various concentrations of SN-38 for 72 h. Cell viability was determined by detecting the luminescence. E , HCT116-siRNA-CTDSP1 or control siRNA cells were plated in a 6-well plate and treated with various concentrations of SN-38 for 24 h. Then, 50 cells per well were plated in a 6-well plate. After 14 days, when colonies were apparent (right panel), colonies were counted and the relative number of colonies was determined (left panel).

    Article Snippet: ImmunoblottingCells cultured in 6-well plates were scraped into an ice-cold RIPA buffer.

    Techniques: Transfection, CRISPR, Microscopy

    Immunobiotic mediated regulation of adipocyte differentiation in an immune cell-adipocyte co-culture model. The mRNA expressions of PPARγ and GLUT4 resulted from various immunobiotic lactic acid bacteria (LAB) stimulation into the co-culture model. A total of 1 × 10 6 immune cells/well of 6-well plate were cultured on adipocytes 4-days differentiated. LAB samples (5 × 10 8 cells/well) also added into the adipocyte plate and maintained for further 2 or 4 Days. All data shown are the average ±SD of 3 independent experiments performed in triplicate. The asterisk (*) indicated statistical differences when compared with control at the significance level of p

    Journal: Cells

    Article Title: Evaluation of Fat Accumulation and Adipokine Production during the Long-Term Adipogenic Differentiation of Porcine Intramuscular Preadipocytes and Study of the Influence of Immunobiotics

    doi: 10.3390/cells9071715

    Figure Lengend Snippet: Immunobiotic mediated regulation of adipocyte differentiation in an immune cell-adipocyte co-culture model. The mRNA expressions of PPARγ and GLUT4 resulted from various immunobiotic lactic acid bacteria (LAB) stimulation into the co-culture model. A total of 1 × 10 6 immune cells/well of 6-well plate were cultured on adipocytes 4-days differentiated. LAB samples (5 × 10 8 cells/well) also added into the adipocyte plate and maintained for further 2 or 4 Days. All data shown are the average ±SD of 3 independent experiments performed in triplicate. The asterisk (*) indicated statistical differences when compared with control at the significance level of p

    Article Snippet: Induction of Fat Accumulation by SFAs Stimulation Adipocytes were cultured at a density of 2.5 × 104 /cm2 in 6-well plates (BD Falcon, Tokyo, Japan).

    Techniques: Co-Culture Assay, Cell Culture

    Immunobiotic mediated inflammatory responses in the antigen presenting cells (APCs) of Payer’s patches. The APCs were seeded at 1 × 10 6 cells/well of 6-well plate and cultured with each bacterial strain (5 × 10 8 cells/well) separately for 24 h and the mRNA expressions of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-β and IFN-γ) were analyzed with RT-qPCR. Data shown are the mean ±SD of 3 independent experiments performed in triplicates. The asterisks: (*) and (**) indicated statistical differences with significant levels of p

    Journal: Cells

    Article Title: Evaluation of Fat Accumulation and Adipokine Production during the Long-Term Adipogenic Differentiation of Porcine Intramuscular Preadipocytes and Study of the Influence of Immunobiotics

    doi: 10.3390/cells9071715

    Figure Lengend Snippet: Immunobiotic mediated inflammatory responses in the antigen presenting cells (APCs) of Payer’s patches. The APCs were seeded at 1 × 10 6 cells/well of 6-well plate and cultured with each bacterial strain (5 × 10 8 cells/well) separately for 24 h and the mRNA expressions of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-β and IFN-γ) were analyzed with RT-qPCR. Data shown are the mean ±SD of 3 independent experiments performed in triplicates. The asterisks: (*) and (**) indicated statistical differences with significant levels of p

    Article Snippet: Induction of Fat Accumulation by SFAs Stimulation Adipocytes were cultured at a density of 2.5 × 104 /cm2 in 6-well plates (BD Falcon, Tokyo, Japan).

    Techniques: Cell Culture, Quantitative RT-PCR

    Immunobiotic mediated regulation of fat accumulation and inflammatory responses in an immune cell-adipocyte co-culture model. A total of 1 × 10 6 immune cells/well of a 6-well plate were cultured on adipocytes 4-days differentiated. Bacterial samples (5 × 10 8 cells/well) were also added and cultured for 2 or 4 days. Adipocyte cells were stained with Oil red O stained and fat accumulation in the adipocytes was quantitatively analyzed by image J software. The ELISA-based concentration of CCL2 protein in supernatant of adipocyte-culture stimulated by immune cells and lactic acid bacteria (LAB). All data shown are the mean ± SD of three independent experiments performed in triplicate. The asterisks: ‘*’ and ‘**’ indicated statistical differences either between bars of panel-a and control or between bars of panel-b and IMU, with significant levels of p

    Journal: Cells

    Article Title: Evaluation of Fat Accumulation and Adipokine Production during the Long-Term Adipogenic Differentiation of Porcine Intramuscular Preadipocytes and Study of the Influence of Immunobiotics

    doi: 10.3390/cells9071715

    Figure Lengend Snippet: Immunobiotic mediated regulation of fat accumulation and inflammatory responses in an immune cell-adipocyte co-culture model. A total of 1 × 10 6 immune cells/well of a 6-well plate were cultured on adipocytes 4-days differentiated. Bacterial samples (5 × 10 8 cells/well) were also added and cultured for 2 or 4 days. Adipocyte cells were stained with Oil red O stained and fat accumulation in the adipocytes was quantitatively analyzed by image J software. The ELISA-based concentration of CCL2 protein in supernatant of adipocyte-culture stimulated by immune cells and lactic acid bacteria (LAB). All data shown are the mean ± SD of three independent experiments performed in triplicate. The asterisks: ‘*’ and ‘**’ indicated statistical differences either between bars of panel-a and control or between bars of panel-b and IMU, with significant levels of p

    Article Snippet: Induction of Fat Accumulation by SFAs Stimulation Adipocytes were cultured at a density of 2.5 × 104 /cm2 in 6-well plates (BD Falcon, Tokyo, Japan).

    Techniques: Co-Culture Assay, Cell Culture, Staining, Software, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Protection of 3D spheroid-derived MSCs on hepatocyte injury  in vitro . Hepatocytes were isolated from C57BL/6 mice and cultured in collagen-coated 6-well plates. After treatment with CCl 4  for 6 h, the medium of cells was replaced with normal medium, conditioned medium of 2D cultured MSCs, or conditioned medium of 3D spheroids of MSCs for 24 h before LDH assays, apoptosis analysis, and RT-PCR. (a) LDH assays. (b) Statistics of Annexin V staining. (c) Images of Annexin V staining. Annexin V staining was performed to evaluate cell apoptosis (red), with DPAI for nucleus staining (blue). (d) RT-PCR. The mRNA levels of Bax, Bcl-1, NF κ B, and TGF- β  were tested by RT-PCR, with GAPDH as the internal control. (e) Quantitative analysis of Bax/Bcl mRNA level. (f) Quantitative analysis of NF κ B mRNA level. Band intensities of NF κ B were normalized to those of the internal control. (g) Quantitative analysis of TGF- β  mRNA level. Band intensities of TGF- β  were normalized to those of the internal control.  $$ p

    Journal: Stem Cells International

    Article Title: 3D Spheroid Culture Enhances the Expression of Antifibrotic Factors in Human Adipose-Derived MSCs and Improves Their Therapeutic Effects on Hepatic Fibrosis

    doi: 10.1155/2016/4626073

    Figure Lengend Snippet: Protection of 3D spheroid-derived MSCs on hepatocyte injury in vitro . Hepatocytes were isolated from C57BL/6 mice and cultured in collagen-coated 6-well plates. After treatment with CCl 4 for 6 h, the medium of cells was replaced with normal medium, conditioned medium of 2D cultured MSCs, or conditioned medium of 3D spheroids of MSCs for 24 h before LDH assays, apoptosis analysis, and RT-PCR. (a) LDH assays. (b) Statistics of Annexin V staining. (c) Images of Annexin V staining. Annexin V staining was performed to evaluate cell apoptosis (red), with DPAI for nucleus staining (blue). (d) RT-PCR. The mRNA levels of Bax, Bcl-1, NF κ B, and TGF- β were tested by RT-PCR, with GAPDH as the internal control. (e) Quantitative analysis of Bax/Bcl mRNA level. (f) Quantitative analysis of NF κ B mRNA level. Band intensities of NF κ B were normalized to those of the internal control. (g) Quantitative analysis of TGF- β mRNA level. Band intensities of TGF- β were normalized to those of the internal control. $$ p

    Article Snippet: The isolated cells were seeded into collagen-coated 6-well plates at the density of 1 × 104 cells/cm2 and cultured using RMPI 1640 medium (Gibco) with 10% FBS.

    Techniques: Derivative Assay, In Vitro, Isolation, Mouse Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Staining

    LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in 6-well format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.

    Journal: eLife

    Article Title: Single molecule poly(A) tail-seq shows LARP4 opposes deadenylation throughout mRNA lifespan with most impact on short tails

    doi: 10.7554/eLife.59186

    Figure Lengend Snippet: LARP4 promotes accumulation of interferon-induced innate immune mRNAs. ( A ) Northern blot analysis of two ISG mRNAs, IFIT1 and ISG15 after transfection-mediated induction by IFN-stimulating nucleic acids, and + / - co transfection with LARP4, as indicated above the lanes. Bottom panels are stained gels prior to transfer. ( B ) Quantitation of biological triplicate northern blot data, normalized by GAPDH, with the poly(I:C) +, LARP4 - sample (annotated with # above the bar) set to 100%; error bars represent 95% confidence interval. P values were calculated using a 2-tailed Welch's T-test with unequal variance. ( C ) Northern blot of IFIT1 and LARP4 from total RNA 48 hr after transfection of 0, 2, 4 or 6 µg of 1 kb AT-rich DNA in 6-well format (the total DNA amount for this component of the transfection was maintained at 6 µg with carrier pUC19), plus 2.5 µg pFLAG-LARP4 (+) or empty pFLAG vector ( - ) as indicated. ( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al., 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes. Bottom: Western blot analysis of protein from the same cells.

    Article Snippet: IFNα and actinomycin D time course Two LARP4 WT and two KO MEF cell lines immortalized by the 3T3 method , were seeded at a density of 2.5 × 105 cells per well in 2 ml media with 10% FBS in 6-well plates; at least four wells per time point were seeded.

    Techniques: Northern Blot, Transfection, Cotransfection, Staining, Quantitation Assay, Plasmid Preparation, Laser Capture Microdissection, Binding Assay, Construct, Western Blot