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  • 99
    Thermo Fisher qrt pcr
    The targeting relationship between miR-296-5p and STAT3 in CRC. ( A ) The potential binding site between miR-296-5p and STAT3 was obtained from TargetScan database. ( B ) The targeting relationship between miR-296-5p and STAT3 was verified by dual-luciferase reporter assay. ( C ) The expression of STAT3 mRNA in CRC cell lines was detected by <t>qRT-PCR.</t> ( D and E ) The protein expression of STAT3 and p- STAT3 was detected by Western blot after transfection. ** P
    Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad qrt pcr
    NAMPT K D or inhibition decreases pluripotency and promotes differentiation in NT2/D1 cells. NT2/D1 NAMPT K D cells were subjected to (A) WB and (B) <t>qRT-PCR</t> analysis for pluripotency factors (POU5F1/ POU5F1 , NANOG/ NANOG , SOX2/ SOX2 ). (B) Cells treated with FK866 for 48 h were subjected to (C) WB and (D) qRT-PCR analysis for pluripotency factors (POU5F1/ POU5F1 , NANOG/ NANOG , SOX2/ SOX2 ). NT2/D1 NAMPT K D cells were subjected to (E) WB analysis for differentiation markers TUBB3 and CSN2 and (F) qRT-PCR analysis for differentiation markers TUBB3, CSN2, SPP1, GATA6, T and CDX2 . NT2/D1 cells treated with FK866 for 48 h were subjected to (G) WB analysis for differentiation markers TUBB3 and CSN2 and (H) qRT-PCR analysis for differentiation markers TUBB3, CSN2, SPP1, GATA6 , and CDX2 . Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control.
    Qrt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 8224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii kit
    FMDV infection induces autophagy through the EIF2S1-ATF4-AKT-MTOR cascade. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. The phosphorylation of AKT, AMPK, MTOR and ULK1 were analyzed by western blot. ACTB was used as a sample loading control. ( B ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. ATF4 and phosphorylation of EIF2S1 were analyzed by western blot. ( C ) ATF4 KD and wild-type cells cells were infected with FMDV (MOI = 1). ATF4, LC3B and phosphorylation of AKT, MTOR and ULK1 were analyzed by western blot. ( D ) ATF4 and scrambled knockdown cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) for 3 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E and F ) ATF4 KD and wil-type cells cells were infected with FMDV (MOI = 1) for 3 h. At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by <t>qRT-PCR;</t> both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. **P
    Superscript Iii Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10720 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mirvana qrt pcr mirna detection kit
    FMDV infection induces autophagy through the EIF2S1-ATF4-AKT-MTOR cascade. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. The phosphorylation of AKT, AMPK, MTOR and ULK1 were analyzed by western blot. ACTB was used as a sample loading control. ( B ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. ATF4 and phosphorylation of EIF2S1 were analyzed by western blot. ( C ) ATF4 KD and wild-type cells cells were infected with FMDV (MOI = 1). ATF4, LC3B and phosphorylation of AKT, MTOR and ULK1 were analyzed by western blot. ( D ) ATF4 and scrambled knockdown cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) for 3 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E and F ) ATF4 KD and wil-type cells cells were infected with FMDV (MOI = 1) for 3 h. At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by <t>qRT-PCR;</t> both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. **P
    Mirvana Qrt Pcr Mirna Detection Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 981 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii first strand synthesis supermix
    Histone H3 lysine 27 trimethylation is required for effector gene repression. (A) eGFP fluorescence of Zymoseptoria tritici transgenic lines with and without the histone methyltransferase gene KMT6 (wt and KO, respectively), both harboring the eGFP gene under the control of a constitutive promoter in the Avr3D1 locus. A line harboring an ectopic copy of the eGFP cassette is shown as a control. All lines were obtained in a genetic background containing an mCherry reporter cassette for visualization of fungal cells. (B) eGFP transcript levels in the transgenic lines described in the panel A legend during axenic growth in rich medium and during plant colonization at 11 days postinfection (dpi). (C) Transcript levels of AvrStb6 and Mycgr3G76589 during axenic growth in rich medium and during plant colonization in Z. tritici lines with and without KMT6 . Expression levels were normalized to the line with the wild-type KMT6 gene during axenic growth. Actin was used as reference gene for <t>qRT-PCR.</t> Error bars represent standard errors of the means. Data from at least <t>three</t> independent replicates are shown. Black asterisks indicate differences between strains with and without KMT6 , and red asterisks indicate differences between axenic and in planta growth of the same mutant line ( P
    Superscript Iii First Strand Synthesis Supermix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene qrt pcr
    Histone H3 lysine 27 trimethylation is required for effector gene repression. (A) eGFP fluorescence of Zymoseptoria tritici transgenic lines with and without the histone methyltransferase gene KMT6 (wt and KO, respectively), both harboring the eGFP gene under the control of a constitutive promoter in the Avr3D1 locus. A line harboring an ectopic copy of the eGFP cassette is shown as a control. All lines were obtained in a genetic background containing an mCherry reporter cassette for visualization of fungal cells. (B) eGFP transcript levels in the transgenic lines described in the panel A legend during axenic growth in rich medium and during plant colonization at 11 days postinfection (dpi). (C) Transcript levels of AvrStb6 and Mycgr3G76589 during axenic growth in rich medium and during plant colonization in Z. tritici lines with and without KMT6 . Expression levels were normalized to the line with the wild-type KMT6 gene during axenic growth. Actin was used as reference gene for <t>qRT-PCR.</t> Error bars represent standard errors of the means. Data from at least <t>three</t> independent replicates are shown. Black asterisks indicate differences between strains with and without KMT6 , and red asterisks indicate differences between axenic and in planta growth of the same mutant line ( P
    Qrt Pcr, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 700 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii platinum sybr green one step qrt pcr kit
    Histone H3 lysine 27 trimethylation is required for effector gene repression. (A) eGFP fluorescence of Zymoseptoria tritici transgenic lines with and without the histone methyltransferase gene KMT6 (wt and KO, respectively), both harboring the eGFP gene under the control of a constitutive promoter in the Avr3D1 locus. A line harboring an ectopic copy of the eGFP cassette is shown as a control. All lines were obtained in a genetic background containing an mCherry reporter cassette for visualization of fungal cells. (B) eGFP transcript levels in the transgenic lines described in the panel A legend during axenic growth in rich medium and during plant colonization at 11 days postinfection (dpi). (C) Transcript levels of AvrStb6 and Mycgr3G76589 during axenic growth in rich medium and during plant colonization in Z. tritici lines with and without KMT6 . Expression levels were normalized to the line with the wild-type KMT6 gene during axenic growth. Actin was used as reference gene for <t>qRT-PCR.</t> Error bars represent standard errors of the means. Data from at least <t>three</t> independent replicates are shown. Black asterisks indicate differences between strains with and without KMT6 , and red asterisks indicate differences between axenic and in planta growth of the same mutant line ( P
    Superscript Iii Platinum Sybr Green One Step Qrt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1742 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Toyobo qrt pcr
    Histone H3 lysine 27 trimethylation is required for effector gene repression. (A) eGFP fluorescence of Zymoseptoria tritici transgenic lines with and without the histone methyltransferase gene KMT6 (wt and KO, respectively), both harboring the eGFP gene under the control of a constitutive promoter in the Avr3D1 locus. A line harboring an ectopic copy of the eGFP cassette is shown as a control. All lines were obtained in a genetic background containing an mCherry reporter cassette for visualization of fungal cells. (B) eGFP transcript levels in the transgenic lines described in the panel A legend during axenic growth in rich medium and during plant colonization at 11 days postinfection (dpi). (C) Transcript levels of AvrStb6 and Mycgr3G76589 during axenic growth in rich medium and during plant colonization in Z. tritici lines with and without KMT6 . Expression levels were normalized to the line with the wild-type KMT6 gene during axenic growth. Actin was used as reference gene for <t>qRT-PCR.</t> Error bars represent standard errors of the means. Data from at least <t>three</t> independent replicates are shown. Black asterisks indicate differences between strains with and without KMT6 , and red asterisks indicate differences between axenic and in planta growth of the same mutant line ( P
    Qrt Pcr, supplied by Toyobo, used in various techniques. Bioz Stars score: 94/100, based on 494 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genecopoeia all in one mirna qrt pcr detection kit
    H19 facilitated CaOx nephrocalcinosis-induced renal tubular epithelial cell injury in vivo . CaOx deposition in the corticomedullary junction area was measured by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200). PAS staining (Magnification, × 100) illustrating renal tubular epithelial cell injury. (b) Immunohistochemical analysis of kidney HMGB1, TLR4, NF-kB, SOD2 and NOX2 expression was performed in rAAV-H19 or rAAV-vector injected CaOx nephrocalcinosis mouse models (Magnification, × 200). (c) Western blot analysis was performed to detect HMGB1, TLR4, p-NF-kB and NF-kB protein expression in H19-treated kidney tissue. GAPDH served as an internal control. (d) <t>qRT-PCR</t> analysis was performed to detect the expression levels of proinflammatory cytokines in kidney tissue. The data are shown as the mean ± standard deviation (SD) of three independent experiments. (* P
    All In One Mirna Qrt Pcr Detection Kit, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 94/100, based on 1519 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eppendorf AG qrt pcr
    H19 facilitated CaOx nephrocalcinosis-induced renal tubular epithelial cell injury in vivo . CaOx deposition in the corticomedullary junction area was measured by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200). PAS staining (Magnification, × 100) illustrating renal tubular epithelial cell injury. (b) Immunohistochemical analysis of kidney HMGB1, TLR4, NF-kB, SOD2 and NOX2 expression was performed in rAAV-H19 or rAAV-vector injected CaOx nephrocalcinosis mouse models (Magnification, × 200). (c) Western blot analysis was performed to detect HMGB1, TLR4, p-NF-kB and NF-kB protein expression in H19-treated kidney tissue. GAPDH served as an internal control. (d) <t>qRT-PCR</t> analysis was performed to detect the expression levels of proinflammatory cytokines in kidney tissue. The data are shown as the mean ± standard deviation (SD) of three independent experiments. (* P
    Qrt Pcr, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 94/100, based on 445 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies qrt pcr
    H19 facilitated CaOx nephrocalcinosis-induced renal tubular epithelial cell injury in vivo . CaOx deposition in the corticomedullary junction area was measured by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200). PAS staining (Magnification, × 100) illustrating renal tubular epithelial cell injury. (b) Immunohistochemical analysis of kidney HMGB1, TLR4, NF-kB, SOD2 and NOX2 expression was performed in rAAV-H19 or rAAV-vector injected CaOx nephrocalcinosis mouse models (Magnification, × 200). (c) Western blot analysis was performed to detect HMGB1, TLR4, p-NF-kB and NF-kB protein expression in H19-treated kidney tissue. GAPDH served as an internal control. (d) <t>qRT-PCR</t> analysis was performed to detect the expression levels of proinflammatory cytokines in kidney tissue. The data are shown as the mean ± standard deviation (SD) of three independent experiments. (* P
    Qrt Pcr, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co qrt pcr
    H19 facilitated CaOx nephrocalcinosis-induced renal tubular epithelial cell injury in vivo . CaOx deposition in the corticomedullary junction area was measured by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200). PAS staining (Magnification, × 100) illustrating renal tubular epithelial cell injury. (b) Immunohistochemical analysis of kidney HMGB1, TLR4, NF-kB, SOD2 and NOX2 expression was performed in rAAV-H19 or rAAV-vector injected CaOx nephrocalcinosis mouse models (Magnification, × 200). (c) Western blot analysis was performed to detect HMGB1, TLR4, p-NF-kB and NF-kB protein expression in H19-treated kidney tissue. GAPDH served as an internal control. (d) <t>qRT-PCR</t> analysis was performed to detect the expression levels of proinflammatory cytokines in kidney tissue. The data are shown as the mean ± standard deviation (SD) of three independent experiments. (* P
    Qrt Pcr, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc qrt pcr
    H19 facilitated CaOx nephrocalcinosis-induced renal tubular epithelial cell injury in vivo . CaOx deposition in the corticomedullary junction area was measured by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200). PAS staining (Magnification, × 100) illustrating renal tubular epithelial cell injury. (b) Immunohistochemical analysis of kidney HMGB1, TLR4, NF-kB, SOD2 and NOX2 expression was performed in rAAV-H19 or rAAV-vector injected CaOx nephrocalcinosis mouse models (Magnification, × 200). (c) Western blot analysis was performed to detect HMGB1, TLR4, p-NF-kB and NF-kB protein expression in H19-treated kidney tissue. GAPDH served as an internal control. (d) <t>qRT-PCR</t> analysis was performed to detect the expression levels of proinflammatory cytokines in kidney tissue. The data are shown as the mean ± standard deviation (SD) of three independent experiments. (* P
    Qrt Pcr, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 514 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vazyme Biotech Co qrt pcr
    H19 facilitated CaOx nephrocalcinosis-induced renal tubular epithelial cell injury in vivo . CaOx deposition in the corticomedullary junction area was measured by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200). PAS staining (Magnification, × 100) illustrating renal tubular epithelial cell injury. (b) Immunohistochemical analysis of kidney HMGB1, TLR4, NF-kB, SOD2 and NOX2 expression was performed in rAAV-H19 or rAAV-vector injected CaOx nephrocalcinosis mouse models (Magnification, × 200). (c) Western blot analysis was performed to detect HMGB1, TLR4, p-NF-kB and NF-kB protein expression in H19-treated kidney tissue. GAPDH served as an internal control. (d) <t>qRT-PCR</t> analysis was performed to detect the expression levels of proinflammatory cytokines in kidney tissue. The data are shown as the mean ± standard deviation (SD) of three independent experiments. (* P
    Qrt Pcr, supplied by Vazyme Biotech Co, used in various techniques. Bioz Stars score: 92/100, based on 301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cellsdirect one step qrt pcr kit
    H19 facilitated CaOx nephrocalcinosis-induced renal tubular epithelial cell injury in vivo . CaOx deposition in the corticomedullary junction area was measured by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200). PAS staining (Magnification, × 100) illustrating renal tubular epithelial cell injury. (b) Immunohistochemical analysis of kidney HMGB1, TLR4, NF-kB, SOD2 and NOX2 expression was performed in rAAV-H19 or rAAV-vector injected CaOx nephrocalcinosis mouse models (Magnification, × 200). (c) Western blot analysis was performed to detect HMGB1, TLR4, p-NF-kB and NF-kB protein expression in H19-treated kidney tissue. GAPDH served as an internal control. (d) <t>qRT-PCR</t> analysis was performed to detect the expression levels of proinflammatory cytokines in kidney tissue. The data are shown as the mean ± standard deviation (SD) of three independent experiments. (* P
    Cellsdirect One Step Qrt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 640 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa lenti x qrt pcr titration kit
    H19 facilitated CaOx nephrocalcinosis-induced renal tubular epithelial cell injury in vivo . CaOx deposition in the corticomedullary junction area was measured by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200). PAS staining (Magnification, × 100) illustrating renal tubular epithelial cell injury. (b) Immunohistochemical analysis of kidney HMGB1, TLR4, NF-kB, SOD2 and NOX2 expression was performed in rAAV-H19 or rAAV-vector injected CaOx nephrocalcinosis mouse models (Magnification, × 200). (c) Western blot analysis was performed to detect HMGB1, TLR4, p-NF-kB and NF-kB protein expression in H19-treated kidney tissue. GAPDH served as an internal control. (d) <t>qRT-PCR</t> analysis was performed to detect the expression levels of proinflammatory cytokines in kidney tissue. The data are shown as the mean ± standard deviation (SD) of three independent experiments. (* P
    Lenti X Qrt Pcr Titration Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Meridian Life Science qrt pcr
    H19 facilitated CaOx nephrocalcinosis-induced renal tubular epithelial cell injury in vivo . CaOx deposition in the corticomedullary junction area was measured by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200). PAS staining (Magnification, × 100) illustrating renal tubular epithelial cell injury. (b) Immunohistochemical analysis of kidney HMGB1, TLR4, NF-kB, SOD2 and NOX2 expression was performed in rAAV-H19 or rAAV-vector injected CaOx nephrocalcinosis mouse models (Magnification, × 200). (c) Western blot analysis was performed to detect HMGB1, TLR4, p-NF-kB and NF-kB protein expression in H19-treated kidney tissue. GAPDH served as an internal control. (d) <t>qRT-PCR</t> analysis was performed to detect the expression levels of proinflammatory cytokines in kidney tissue. The data are shown as the mean ± standard deviation (SD) of three independent experiments. (* P
    Qrt Pcr, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 92/100, based on 209 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The targeting relationship between miR-296-5p and STAT3 in CRC. ( A ) The potential binding site between miR-296-5p and STAT3 was obtained from TargetScan database. ( B ) The targeting relationship between miR-296-5p and STAT3 was verified by dual-luciferase reporter assay. ( C ) The expression of STAT3 mRNA in CRC cell lines was detected by qRT-PCR. ( D and E ) The protein expression of STAT3 and p- STAT3 was detected by Western blot after transfection. ** P

    Journal: Cancer Management and Research

    Article Title: DICER1-AS1 Promotes the Malignant Behaviors of Colorectal Cancer Cells by Regulating miR-296-5p/STAT3 Axis

    doi: 10.2147/CMAR.S252786

    Figure Lengend Snippet: The targeting relationship between miR-296-5p and STAT3 in CRC. ( A ) The potential binding site between miR-296-5p and STAT3 was obtained from TargetScan database. ( B ) The targeting relationship between miR-296-5p and STAT3 was verified by dual-luciferase reporter assay. ( C ) The expression of STAT3 mRNA in CRC cell lines was detected by qRT-PCR. ( D and E ) The protein expression of STAT3 and p- STAT3 was detected by Western blot after transfection. ** P

    Article Snippet: The transfection efficiency was detected by quantitative real-time polymerase chain reaction (qRT-PCR).

    Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection

    The expressions of DICER1-AS in CRC tissues. ( A ) The expression of DICER1-AS1 in CRC patients from the StarBase database. ( B – D ) The expressions of DICER1-AS1, miR-296-5p and STAT3 mRNA in CRC tissues were detected by qRT-PCR. ( E – G ) Person’s correlation analysis among DICER1-AS1, miR-296-5p and STAT3 mRNA. ( H ) The representative images of the immunohistochemistry staining were shown, and the association between STAT3 protein expression and DICER1-AS1 expression in CRC tissues was analyzed. *** P

    Journal: Cancer Management and Research

    Article Title: DICER1-AS1 Promotes the Malignant Behaviors of Colorectal Cancer Cells by Regulating miR-296-5p/STAT3 Axis

    doi: 10.2147/CMAR.S252786

    Figure Lengend Snippet: The expressions of DICER1-AS in CRC tissues. ( A ) The expression of DICER1-AS1 in CRC patients from the StarBase database. ( B – D ) The expressions of DICER1-AS1, miR-296-5p and STAT3 mRNA in CRC tissues were detected by qRT-PCR. ( E – G ) Person’s correlation analysis among DICER1-AS1, miR-296-5p and STAT3 mRNA. ( H ) The representative images of the immunohistochemistry staining were shown, and the association between STAT3 protein expression and DICER1-AS1 expression in CRC tissues was analyzed. *** P

    Article Snippet: The transfection efficiency was detected by quantitative real-time polymerase chain reaction (qRT-PCR).

    Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining

    Effects of miR-296-5p on proliferation, migration and invasion of CRC cells. ( A and B ) The transfection efficiency of miR-296-5p mimics and miR-296-5p inhibitors was verified by qRT-PCR analysis. ( C – F ) Cell proliferation, migration and invasion were detected by CCK-8 assay and Transwell assay. ( G ) The protein expressions of Bax and Bcl2 were detected by Western blot after transfection. ** P

    Journal: Cancer Management and Research

    Article Title: DICER1-AS1 Promotes the Malignant Behaviors of Colorectal Cancer Cells by Regulating miR-296-5p/STAT3 Axis

    doi: 10.2147/CMAR.S252786

    Figure Lengend Snippet: Effects of miR-296-5p on proliferation, migration and invasion of CRC cells. ( A and B ) The transfection efficiency of miR-296-5p mimics and miR-296-5p inhibitors was verified by qRT-PCR analysis. ( C – F ) Cell proliferation, migration and invasion were detected by CCK-8 assay and Transwell assay. ( G ) The protein expressions of Bax and Bcl2 were detected by Western blot after transfection. ** P

    Article Snippet: The transfection efficiency was detected by quantitative real-time polymerase chain reaction (qRT-PCR).

    Techniques: Migration, Transfection, Quantitative RT-PCR, CCK-8 Assay, Transwell Assay, Western Blot

    The targeting relationship between DICER1-AS1 and miR-296-5p in CRC ( A ) The potential binding site between DICER1-AS1 and miR-296-5p was obtained from StarBase database. ( B ) The targeting relationship between DICER1-AS1 and miR-296-5p was validated by dual-luciferase reporter assay. ( C ) The expression of miR-296-5p in CRC cell lines was detected by qRT-PCR. ( D ) Effect of DICER1-AS1 on miR-296-5p expression in CRC was examined by qRT-PCR. ( E ) Effect of miR-296-5p on DICER1-AS1 expression in CRC was examined by qRT-PCR. ** P

    Journal: Cancer Management and Research

    Article Title: DICER1-AS1 Promotes the Malignant Behaviors of Colorectal Cancer Cells by Regulating miR-296-5p/STAT3 Axis

    doi: 10.2147/CMAR.S252786

    Figure Lengend Snippet: The targeting relationship between DICER1-AS1 and miR-296-5p in CRC ( A ) The potential binding site between DICER1-AS1 and miR-296-5p was obtained from StarBase database. ( B ) The targeting relationship between DICER1-AS1 and miR-296-5p was validated by dual-luciferase reporter assay. ( C ) The expression of miR-296-5p in CRC cell lines was detected by qRT-PCR. ( D ) Effect of DICER1-AS1 on miR-296-5p expression in CRC was examined by qRT-PCR. ( E ) Effect of miR-296-5p on DICER1-AS1 expression in CRC was examined by qRT-PCR. ** P

    Article Snippet: The transfection efficiency was detected by quantitative real-time polymerase chain reaction (qRT-PCR).

    Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR

    Effects of overexpression or knockdown of DICER1-AS1 on proliferation, migration and invasion of CRC cells. ( A ) The expression of DICER1-AS1 in CRC cell lines was detected by qRT-PCR. ( B and C ) The transfection efficiency of DICER1-AS1 knockdown and overexpression was verified by qRT-PCR analysis. ( D – G ) Cell proliferation, migration and invasion were detected by CCK-8 assay and Transwell assay. ( H ) The protein expressions of Bax and Bcl2 were detected by Western blot. * P

    Journal: Cancer Management and Research

    Article Title: DICER1-AS1 Promotes the Malignant Behaviors of Colorectal Cancer Cells by Regulating miR-296-5p/STAT3 Axis

    doi: 10.2147/CMAR.S252786

    Figure Lengend Snippet: Effects of overexpression or knockdown of DICER1-AS1 on proliferation, migration and invasion of CRC cells. ( A ) The expression of DICER1-AS1 in CRC cell lines was detected by qRT-PCR. ( B and C ) The transfection efficiency of DICER1-AS1 knockdown and overexpression was verified by qRT-PCR analysis. ( D – G ) Cell proliferation, migration and invasion were detected by CCK-8 assay and Transwell assay. ( H ) The protein expressions of Bax and Bcl2 were detected by Western blot. * P

    Article Snippet: The transfection efficiency was detected by quantitative real-time polymerase chain reaction (qRT-PCR).

    Techniques: Over Expression, Migration, Expressing, Quantitative RT-PCR, Transfection, CCK-8 Assay, Transwell Assay, Western Blot

    Effects of DICER1-AS1 and miR-296-5p on proliferation, migration and invasion of CRC cells. ( A – C ) The co-transfection efficiency was verified by qRT-PCR analysis. ( D – F ) Cell proliferation, migration and invasion were detected by CCK-8 assay and Transwell assay. ( G ) The protein expressions of Bax and Bcl2 were detected by Western blot after transfection. * P

    Journal: Cancer Management and Research

    Article Title: DICER1-AS1 Promotes the Malignant Behaviors of Colorectal Cancer Cells by Regulating miR-296-5p/STAT3 Axis

    doi: 10.2147/CMAR.S252786

    Figure Lengend Snippet: Effects of DICER1-AS1 and miR-296-5p on proliferation, migration and invasion of CRC cells. ( A – C ) The co-transfection efficiency was verified by qRT-PCR analysis. ( D – F ) Cell proliferation, migration and invasion were detected by CCK-8 assay and Transwell assay. ( G ) The protein expressions of Bax and Bcl2 were detected by Western blot after transfection. * P

    Article Snippet: The transfection efficiency was detected by quantitative real-time polymerase chain reaction (qRT-PCR).

    Techniques: Migration, Cotransfection, Quantitative RT-PCR, CCK-8 Assay, Transwell Assay, Western Blot, Transfection

    NAMPT K D or inhibition decreases pluripotency and promotes differentiation in NT2/D1 cells. NT2/D1 NAMPT K D cells were subjected to (A) WB and (B) qRT-PCR analysis for pluripotency factors (POU5F1/ POU5F1 , NANOG/ NANOG , SOX2/ SOX2 ). (B) Cells treated with FK866 for 48 h were subjected to (C) WB and (D) qRT-PCR analysis for pluripotency factors (POU5F1/ POU5F1 , NANOG/ NANOG , SOX2/ SOX2 ). NT2/D1 NAMPT K D cells were subjected to (E) WB analysis for differentiation markers TUBB3 and CSN2 and (F) qRT-PCR analysis for differentiation markers TUBB3, CSN2, SPP1, GATA6, T and CDX2 . NT2/D1 cells treated with FK866 for 48 h were subjected to (G) WB analysis for differentiation markers TUBB3 and CSN2 and (H) qRT-PCR analysis for differentiation markers TUBB3, CSN2, SPP1, GATA6 , and CDX2 . Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control.

    Journal: Autophagy

    Article Title: Autophagic homeostasis is required for the pluripotency of cancer stem cells

    doi: 10.1080/15548627.2016.1260808

    Figure Lengend Snippet: NAMPT K D or inhibition decreases pluripotency and promotes differentiation in NT2/D1 cells. NT2/D1 NAMPT K D cells were subjected to (A) WB and (B) qRT-PCR analysis for pluripotency factors (POU5F1/ POU5F1 , NANOG/ NANOG , SOX2/ SOX2 ). (B) Cells treated with FK866 for 48 h were subjected to (C) WB and (D) qRT-PCR analysis for pluripotency factors (POU5F1/ POU5F1 , NANOG/ NANOG , SOX2/ SOX2 ). NT2/D1 NAMPT K D cells were subjected to (E) WB analysis for differentiation markers TUBB3 and CSN2 and (F) qRT-PCR analysis for differentiation markers TUBB3, CSN2, SPP1, GATA6, T and CDX2 . NT2/D1 cells treated with FK866 for 48 h were subjected to (G) WB analysis for differentiation markers TUBB3 and CSN2 and (H) qRT-PCR analysis for differentiation markers TUBB3, CSN2, SPP1, GATA6 , and CDX2 . Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control.

    Article Snippet: The CFX96 PCR machine (Bio-Rad Laboratories, Hercules, CA) was used for the quantitative real-time PCR (qRT-PCR), using SYBR Green Supermix (Bio-Rad Laboratories, 1708880).

    Techniques: Inhibition, Western Blot, Quantitative RT-PCR

    Senescence induction via doxorubicin decreases pluripotency, increases differentiation, and inhibits autophagy in NT2/D1 cells. (A) NT2/D1 cells treated with 10 nM of doxorubicin were subjected to WB analysis for CDKN1A (B) and cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for CDKN1A, CDKN2A and CDKN1B . (C) GLB1 staining was performed to confirm the presence of senescent cells following 3 nM doxorubicin treatment. (D) Cells treated with 3 nM of doxorubicin were stained with trypan blue and counted to determine the number of viable cells after 24 and 48 h post-treatment. (E) Photographs were taken of cells treated with 3 nM and 5 nM of doxorubicin to show changes in cell morphology compare with the untreated control. (F) Cells treated with 10 nM of doxorubicin were subjected to WB analysis for pluripotency factors (POU5F1, NANOG, SOX2) and differentiation markers (TUBB3). (G) Cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for pluripotency factors ( POU5F1, NANOG, SOX2 ) and differentiation markers ( TUBB3, CSN2, GATA6, CDX2 ). (H) Cells treated with 10 nM of doxorubicin were subjected to WB analysis for autophagy markers (ATG5, ATG7, SQSTM1, LC3A-II, LC3B-II and p-MTOR). (I) Cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for autophagy markers ( ATG5, ATG7, ATG12, BECN1, ULK1 and SQSTM1 ). (J) NT2/D1 cells treated with doxorubicin (10 nM), chloroquine (CQ) (18 µM) or in combination were subjected to WB analysis for protein levels of SQSTM1, LC3A-II and LC3B-II. Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. Scale bar: 100 µm.

    Journal: Autophagy

    Article Title: Autophagic homeostasis is required for the pluripotency of cancer stem cells

    doi: 10.1080/15548627.2016.1260808

    Figure Lengend Snippet: Senescence induction via doxorubicin decreases pluripotency, increases differentiation, and inhibits autophagy in NT2/D1 cells. (A) NT2/D1 cells treated with 10 nM of doxorubicin were subjected to WB analysis for CDKN1A (B) and cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for CDKN1A, CDKN2A and CDKN1B . (C) GLB1 staining was performed to confirm the presence of senescent cells following 3 nM doxorubicin treatment. (D) Cells treated with 3 nM of doxorubicin were stained with trypan blue and counted to determine the number of viable cells after 24 and 48 h post-treatment. (E) Photographs were taken of cells treated with 3 nM and 5 nM of doxorubicin to show changes in cell morphology compare with the untreated control. (F) Cells treated with 10 nM of doxorubicin were subjected to WB analysis for pluripotency factors (POU5F1, NANOG, SOX2) and differentiation markers (TUBB3). (G) Cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for pluripotency factors ( POU5F1, NANOG, SOX2 ) and differentiation markers ( TUBB3, CSN2, GATA6, CDX2 ). (H) Cells treated with 10 nM of doxorubicin were subjected to WB analysis for autophagy markers (ATG5, ATG7, SQSTM1, LC3A-II, LC3B-II and p-MTOR). (I) Cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for autophagy markers ( ATG5, ATG7, ATG12, BECN1, ULK1 and SQSTM1 ). (J) NT2/D1 cells treated with doxorubicin (10 nM), chloroquine (CQ) (18 µM) or in combination were subjected to WB analysis for protein levels of SQSTM1, LC3A-II and LC3B-II. Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. Scale bar: 100 µm.

    Article Snippet: The CFX96 PCR machine (Bio-Rad Laboratories, Hercules, CA) was used for the quantitative real-time PCR (qRT-PCR), using SYBR Green Supermix (Bio-Rad Laboratories, 1708880).

    Techniques: Western Blot, Quantitative RT-PCR, Staining

    Decrease in pluripotency is coupled with an increase in senescence. (A) NT2/D1 cells treated with rapamycin or serum starvation were subjected to WB analysis for CDKN1A and CDKN2A and NT2/D1 cells treated with Tat-scrambled or Tat-BECN1 peptide were subjected to WB analysis for CDKN1A (B) Rapamycin-treated cells were subjected to qRT-PCR analysis for CDKN1A, CDKN2A and CDKN1B mRNA levels. (C) GLB1 staining was performed to confirm the presence of senescent cells following rapamycin treatment. (D) ATG7 and ATG12 K D NT2/D1 cells were subjected to WB analysis for CDKN1A and (E) qRT-PCR analysis for CDKN1A and CDKN1B . (F) GLB1 staining was performed to confirm the presence of senescent cells following ATG7 and ATG12 K D . (G) NAMPT K D NT2/D1 cells were subjected to WB analysis for CDKN1A and CDKN2A and (H) qRT-PCR analysis for CDKN1A, CDKN2A and CDKN1B . (I) GLB1 staining was performed to confirm the presence of senescent cells following NAMPT K D . (J) POU5F1 K D NT2/D1 cells were subjected to WB analysis for CDKN1A and CDKN2A and (K) qRT-PCR analysis for CDKN1A, CDKN2A and CDKN1B . (L) GLB1 staining was performed to confirm the presence of senescent cells following POU5F1 K D . Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. Scale bar: 100 µm.

    Journal: Autophagy

    Article Title: Autophagic homeostasis is required for the pluripotency of cancer stem cells

    doi: 10.1080/15548627.2016.1260808

    Figure Lengend Snippet: Decrease in pluripotency is coupled with an increase in senescence. (A) NT2/D1 cells treated with rapamycin or serum starvation were subjected to WB analysis for CDKN1A and CDKN2A and NT2/D1 cells treated with Tat-scrambled or Tat-BECN1 peptide were subjected to WB analysis for CDKN1A (B) Rapamycin-treated cells were subjected to qRT-PCR analysis for CDKN1A, CDKN2A and CDKN1B mRNA levels. (C) GLB1 staining was performed to confirm the presence of senescent cells following rapamycin treatment. (D) ATG7 and ATG12 K D NT2/D1 cells were subjected to WB analysis for CDKN1A and (E) qRT-PCR analysis for CDKN1A and CDKN1B . (F) GLB1 staining was performed to confirm the presence of senescent cells following ATG7 and ATG12 K D . (G) NAMPT K D NT2/D1 cells were subjected to WB analysis for CDKN1A and CDKN2A and (H) qRT-PCR analysis for CDKN1A, CDKN2A and CDKN1B . (I) GLB1 staining was performed to confirm the presence of senescent cells following NAMPT K D . (J) POU5F1 K D NT2/D1 cells were subjected to WB analysis for CDKN1A and CDKN2A and (K) qRT-PCR analysis for CDKN1A, CDKN2A and CDKN1B . (L) GLB1 staining was performed to confirm the presence of senescent cells following POU5F1 K D . Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. Scale bar: 100 µm.

    Article Snippet: The CFX96 PCR machine (Bio-Rad Laboratories, Hercules, CA) was used for the quantitative real-time PCR (qRT-PCR), using SYBR Green Supermix (Bio-Rad Laboratories, 1708880).

    Techniques: Western Blot, Quantitative RT-PCR, Staining

    Inhibition or induction of autophagy inhibits the pluripotency of mouse teratocarcinoma stem cells (P19). Cells treated with 3-MA were (A) stained with trypan blue and counted to determine the number of viable cells after 24 and 48 h post-treatment, (B) and labeled with CFSE and then analyzed by flow cytometry after 4 d of culturing. Cells treated with 3-MA were subjected to (C) WB and (D) qRT-PCR analysis for pluripotency factors (POU5F1/ Pou5f1 , NANOG/ Nanog , SOX2/ Sox2 ) and differentiation markers (TUBB3/ Tubb3 , CSN2/ Csn2 , SPP1/ Spp1 , GATA6/ Gata6 , T/ T , CDX2/ Cdx2 ). Cells treated with rapamycin or serum starvation were (E) stained with trypan blue and counted to determine the number of viable cells, (F) and labeled with CFSE and then analyzed by flow cytometry after 4 d of culturing. Cells treated with rapamycin and serum starvation were subjected to (G) WB analysis for SQSTM1 protein levels and rapamycin-treated cells were subjected to (H) WB and (I) qRT-PCR analysis for pluripotency factors (POU5F1/ Pou5f1 , NANOG/ Nanog , SOX2/ Sox2 ) and the senescence-related protein CDKN1A/ Cdkn1a . Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity.

    Journal: Autophagy

    Article Title: Autophagic homeostasis is required for the pluripotency of cancer stem cells

    doi: 10.1080/15548627.2016.1260808

    Figure Lengend Snippet: Inhibition or induction of autophagy inhibits the pluripotency of mouse teratocarcinoma stem cells (P19). Cells treated with 3-MA were (A) stained with trypan blue and counted to determine the number of viable cells after 24 and 48 h post-treatment, (B) and labeled with CFSE and then analyzed by flow cytometry after 4 d of culturing. Cells treated with 3-MA were subjected to (C) WB and (D) qRT-PCR analysis for pluripotency factors (POU5F1/ Pou5f1 , NANOG/ Nanog , SOX2/ Sox2 ) and differentiation markers (TUBB3/ Tubb3 , CSN2/ Csn2 , SPP1/ Spp1 , GATA6/ Gata6 , T/ T , CDX2/ Cdx2 ). Cells treated with rapamycin or serum starvation were (E) stained with trypan blue and counted to determine the number of viable cells, (F) and labeled with CFSE and then analyzed by flow cytometry after 4 d of culturing. Cells treated with rapamycin and serum starvation were subjected to (G) WB analysis for SQSTM1 protein levels and rapamycin-treated cells were subjected to (H) WB and (I) qRT-PCR analysis for pluripotency factors (POU5F1/ Pou5f1 , NANOG/ Nanog , SOX2/ Sox2 ) and the senescence-related protein CDKN1A/ Cdkn1a . Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity.

    Article Snippet: The CFX96 PCR machine (Bio-Rad Laboratories, Hercules, CA) was used for the quantitative real-time PCR (qRT-PCR), using SYBR Green Supermix (Bio-Rad Laboratories, 1708880).

    Techniques: Inhibition, Staining, Labeling, Flow Cytometry, Cytometry, Western Blot, Quantitative RT-PCR

    NAMPT K D or inhibition promotes autophagy in NT2/D1 cells. (A) NT2/D1 NAMPT K D cells were subjected to WB analysis for ATG5, ATG7, SQSTM1, LC3A-II and LC3B-II and (B) qRT-PCR analysis for ATG5, ATG7, ATG12, BECN1, ULK1 and SQSTM1 . (C) NT2/D1 cells treated with FK866 for 48 h were subjected to WB analysis for ATG5, ATG7, ATG12, SQSTM1, LC3A-II and LC3B-II and (D) qRT-PCR analysis for ATG5, ATG7, ATG12, BECN1, ULK1 and SQSTM1 . (E) NT2/D1 NAMPT K D cells and (F) cell treated with FK866 were labeled with green detection reagent from the autophagy detection kit (Abcam) and analyzed by flow cytometry to determine the presence of autophagic vacuoles. (G) NT2/D1 NAMPT K D or (H) FK866-treated cells were treated with 18 µM chloroquine (CQ) or 0.5 µM of BAF and the levels of SQSTM1, and LC3B-II were analyzed by WB analysis. Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity.

    Journal: Autophagy

    Article Title: Autophagic homeostasis is required for the pluripotency of cancer stem cells

    doi: 10.1080/15548627.2016.1260808

    Figure Lengend Snippet: NAMPT K D or inhibition promotes autophagy in NT2/D1 cells. (A) NT2/D1 NAMPT K D cells were subjected to WB analysis for ATG5, ATG7, SQSTM1, LC3A-II and LC3B-II and (B) qRT-PCR analysis for ATG5, ATG7, ATG12, BECN1, ULK1 and SQSTM1 . (C) NT2/D1 cells treated with FK866 for 48 h were subjected to WB analysis for ATG5, ATG7, ATG12, SQSTM1, LC3A-II and LC3B-II and (D) qRT-PCR analysis for ATG5, ATG7, ATG12, BECN1, ULK1 and SQSTM1 . (E) NT2/D1 NAMPT K D cells and (F) cell treated with FK866 were labeled with green detection reagent from the autophagy detection kit (Abcam) and analyzed by flow cytometry to determine the presence of autophagic vacuoles. (G) NT2/D1 NAMPT K D or (H) FK866-treated cells were treated with 18 µM chloroquine (CQ) or 0.5 µM of BAF and the levels of SQSTM1, and LC3B-II were analyzed by WB analysis. Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity.

    Article Snippet: The CFX96 PCR machine (Bio-Rad Laboratories, Hercules, CA) was used for the quantitative real-time PCR (qRT-PCR), using SYBR Green Supermix (Bio-Rad Laboratories, 1708880).

    Techniques: Inhibition, Western Blot, Quantitative RT-PCR, Labeling, Flow Cytometry, Cytometry

    Inhibition of autophagy via ATG7 or ATG12 K D inhibits pluripotency and promotes differentiation. ATG7 or ATG12 K D cells were (A) labeled with green detection reagent from the autophagy detection kit (Abcam) and then analyzed by flow cytometry to determine the presence of autophagic vacuoles and (B) subjected to WB analysis of SQSTM1 protein levels. (C) ATG7 or ATG12 K D cells were stained with trypan blue and counted to determine the number of viable cells after 72 and 120 h transfection, (D) and labeled with CFSE and then analyzed by flow cytometry for CFSE fluorescence after 4 d of culturing. (E) Photographs were taken to show changes in cell morphology in ATG7 and ATG12 K D cells compare with the scrambled control. (F) WB and (G) qRT-PCR analysis were performed for pluripotency factors (POU5F1/ POU5F1 , NANOG/ NANOG , SOX2/ SOX2 ). (H) WB and (I) qRT-PCR analysis were performed for differentiation markers (TUBB3/ TUBB3 , CSN2/ CSN2 , GATA6/ GATA6 ). Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity. Scale bar: 100 µm.

    Journal: Autophagy

    Article Title: Autophagic homeostasis is required for the pluripotency of cancer stem cells

    doi: 10.1080/15548627.2016.1260808

    Figure Lengend Snippet: Inhibition of autophagy via ATG7 or ATG12 K D inhibits pluripotency and promotes differentiation. ATG7 or ATG12 K D cells were (A) labeled with green detection reagent from the autophagy detection kit (Abcam) and then analyzed by flow cytometry to determine the presence of autophagic vacuoles and (B) subjected to WB analysis of SQSTM1 protein levels. (C) ATG7 or ATG12 K D cells were stained with trypan blue and counted to determine the number of viable cells after 72 and 120 h transfection, (D) and labeled with CFSE and then analyzed by flow cytometry for CFSE fluorescence after 4 d of culturing. (E) Photographs were taken to show changes in cell morphology in ATG7 and ATG12 K D cells compare with the scrambled control. (F) WB and (G) qRT-PCR analysis were performed for pluripotency factors (POU5F1/ POU5F1 , NANOG/ NANOG , SOX2/ SOX2 ). (H) WB and (I) qRT-PCR analysis were performed for differentiation markers (TUBB3/ TUBB3 , CSN2/ CSN2 , GATA6/ GATA6 ). Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity. Scale bar: 100 µm.

    Article Snippet: The CFX96 PCR machine (Bio-Rad Laboratories, Hercules, CA) was used for the quantitative real-time PCR (qRT-PCR), using SYBR Green Supermix (Bio-Rad Laboratories, 1708880).

    Techniques: Inhibition, Labeling, Flow Cytometry, Cytometry, Western Blot, Staining, Transfection, Fluorescence, Quantitative RT-PCR

    POU5F1 and NAMPT regulate the expression of each other. (A) NT2/D1 POU5F1 K D cells were subjected to WB and qRT-PCR analysis for NAMPT/ NAMPT protein and mRNA levels. (B) NT2/D1 POU5F1 K D cells were stained with trypan blue and counted to determine the number of viable cells after 24 and 48 h transfection (C) or labeled with CFSE and then analyzed by flow cytometry for CFSE fluorescence after 4 d of culturing. (D) Photographs were taken to show a change in cell morphology of POU5F1 KDs using 3 different shRNA clones compare with the scrambled control. (E) WB and qRT-PCR analysis were performed for pluripotency factors (NANOG/ NANOG , SOX2/ SOX2 ) and (F) differentiation markers (TUBB3/ TUBB3 , CSN2/ CSN2 , SPP1/ SPP1 , GATA6/ GATA6 ). Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity. Scale bar: 100 µm.

    Journal: Autophagy

    Article Title: Autophagic homeostasis is required for the pluripotency of cancer stem cells

    doi: 10.1080/15548627.2016.1260808

    Figure Lengend Snippet: POU5F1 and NAMPT regulate the expression of each other. (A) NT2/D1 POU5F1 K D cells were subjected to WB and qRT-PCR analysis for NAMPT/ NAMPT protein and mRNA levels. (B) NT2/D1 POU5F1 K D cells were stained with trypan blue and counted to determine the number of viable cells after 24 and 48 h transfection (C) or labeled with CFSE and then analyzed by flow cytometry for CFSE fluorescence after 4 d of culturing. (D) Photographs were taken to show a change in cell morphology of POU5F1 KDs using 3 different shRNA clones compare with the scrambled control. (E) WB and qRT-PCR analysis were performed for pluripotency factors (NANOG/ NANOG , SOX2/ SOX2 ) and (F) differentiation markers (TUBB3/ TUBB3 , CSN2/ CSN2 , SPP1/ SPP1 , GATA6/ GATA6 ). Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity. Scale bar: 100 µm.

    Article Snippet: The CFX96 PCR machine (Bio-Rad Laboratories, Hercules, CA) was used for the quantitative real-time PCR (qRT-PCR), using SYBR Green Supermix (Bio-Rad Laboratories, 1708880).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Staining, Transfection, Labeling, Flow Cytometry, Cytometry, Fluorescence, shRNA, Clone Assay

    Induction of autophagy via rapamycin or serum starvation inhibits pluripotency. (A) Rapamycin-treated NT2/D1 cells were labeled with green detection reagent from the autophagy detection kit (Abcam) and then analyzed by flow cytometry to determine the presence of autophagic vacuoles. (B) NT2/D1 cells treated with rapamycin or serum starvation were subjected to WB analysis of SQSTM1, LC3A-II, and LC3B-II protein levels and (C) rapamycin-treated cells were subjected to qRT-PCR analysis of SQSTM1 mRNA levels. (D) NT2/D1 cells were treated with rapamycin or serum starvation in the presence or absence of chloroquine (CQ) (18 µM) and the levels LC3A-II and LC3B-II were analyzed by WB analysis. (E) Rapamycin and serum starvation-treated cells were stained with trypan blue and counted to determine the number of viable cells after 24 and 48 h post-treatment. (F) Rapamycin-treated cells were labeled with CFSE and then analyzed by flow cytometry for CFSE fluorescence after 4 d of culturing. (G) Photographs were taken to show cell morphology of rapamycin and serum starvation-treated NT2/D1 cells compare with the untreated control. Rapamycin and serum starvation-treated cells were subjected to (H) WB analysis and rapamycin-treated cells were subjected to (I) qRT-PCR analysis for pluripotency factors ( POU5F1, NANOG, SOX2 ). Rapamycin and serum starvation-treated cells were subjected to (J) WB analysis and rapamycin-treated cells were subjected to (K) qRT-PCR analysis for differentiation markers ( TUBB3, CSN2, SPP1, GATA6, T, CDX2 ). Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity. Scale bar: 100 µm.

    Journal: Autophagy

    Article Title: Autophagic homeostasis is required for the pluripotency of cancer stem cells

    doi: 10.1080/15548627.2016.1260808

    Figure Lengend Snippet: Induction of autophagy via rapamycin or serum starvation inhibits pluripotency. (A) Rapamycin-treated NT2/D1 cells were labeled with green detection reagent from the autophagy detection kit (Abcam) and then analyzed by flow cytometry to determine the presence of autophagic vacuoles. (B) NT2/D1 cells treated with rapamycin or serum starvation were subjected to WB analysis of SQSTM1, LC3A-II, and LC3B-II protein levels and (C) rapamycin-treated cells were subjected to qRT-PCR analysis of SQSTM1 mRNA levels. (D) NT2/D1 cells were treated with rapamycin or serum starvation in the presence or absence of chloroquine (CQ) (18 µM) and the levels LC3A-II and LC3B-II were analyzed by WB analysis. (E) Rapamycin and serum starvation-treated cells were stained with trypan blue and counted to determine the number of viable cells after 24 and 48 h post-treatment. (F) Rapamycin-treated cells were labeled with CFSE and then analyzed by flow cytometry for CFSE fluorescence after 4 d of culturing. (G) Photographs were taken to show cell morphology of rapamycin and serum starvation-treated NT2/D1 cells compare with the untreated control. Rapamycin and serum starvation-treated cells were subjected to (H) WB analysis and rapamycin-treated cells were subjected to (I) qRT-PCR analysis for pluripotency factors ( POU5F1, NANOG, SOX2 ). Rapamycin and serum starvation-treated cells were subjected to (J) WB analysis and rapamycin-treated cells were subjected to (K) qRT-PCR analysis for differentiation markers ( TUBB3, CSN2, SPP1, GATA6, T, CDX2 ). Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity. Scale bar: 100 µm.

    Article Snippet: The CFX96 PCR machine (Bio-Rad Laboratories, Hercules, CA) was used for the quantitative real-time PCR (qRT-PCR), using SYBR Green Supermix (Bio-Rad Laboratories, 1708880).

    Techniques: Labeling, Flow Cytometry, Cytometry, Western Blot, Quantitative RT-PCR, Staining, Fluorescence

    POU5F1 K D inhibits autophagy in NT2/D1 cells. (A) NT2/D1 POU5F1 K D cells were subjected to WB analysis for ATG5, ATG7, ATG12, SQSTM1, LC3A-II and LC3B-II and (B) qRT-PCR analysis for ATG5, ATG7, ATG12, BECN1, ULK1 and SQSTM1 . (C) POU5F1 K D cells were labeled with green detection reagent from the autophagy detection kit (Abcam) and analyzed by flow cytometry to determine the presence of autophagic vacuoles. Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity.

    Journal: Autophagy

    Article Title: Autophagic homeostasis is required for the pluripotency of cancer stem cells

    doi: 10.1080/15548627.2016.1260808

    Figure Lengend Snippet: POU5F1 K D inhibits autophagy in NT2/D1 cells. (A) NT2/D1 POU5F1 K D cells were subjected to WB analysis for ATG5, ATG7, ATG12, SQSTM1, LC3A-II and LC3B-II and (B) qRT-PCR analysis for ATG5, ATG7, ATG12, BECN1, ULK1 and SQSTM1 . (C) POU5F1 K D cells were labeled with green detection reagent from the autophagy detection kit (Abcam) and analyzed by flow cytometry to determine the presence of autophagic vacuoles. Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. MFI, mean fluorescent intensity.

    Article Snippet: The CFX96 PCR machine (Bio-Rad Laboratories, Hercules, CA) was used for the quantitative real-time PCR (qRT-PCR), using SYBR Green Supermix (Bio-Rad Laboratories, 1708880).

    Techniques: Western Blot, Quantitative RT-PCR, Labeling, Flow Cytometry, Cytometry

    ROS content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots inoculated with Rhizobium tropici . Measurements were done at initial time (0 h) and 3, 6, 12, 24 and 48 h after inoculation with R. tropici. (A) Histological (fluorescence) detection of ROS accumulation in inoculated root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in inoculated roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: ROS content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots inoculated with Rhizobium tropici . Measurements were done at initial time (0 h) and 3, 6, 12, 24 and 48 h after inoculation with R. tropici. (A) Histological (fluorescence) detection of ROS accumulation in inoculated root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in inoculated roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Expressing, Fluorescence, Quantitative RT-PCR

    Expression pattern of miR398 and target genes CSD1 and Nod19 in tissues from common bean plants under copper deficiency (CuD) or copper toxicity (CuT). (A) miR398 levels in roots, nodules and leaves of plants grown under control (C) or stress (CuD or CuT) conditions were detected by Northern blot analysis using U6snRNA as loading control. Signal intensity of the hybridization bands was calculated and the expression ratio (stress:control) was obtained. Relative expression of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod19 (red) in roots, nodules and leaves of plants grown under CuD (light colors) or CuT (dark colors) as determined by qRT-PCR. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: Expression pattern of miR398 and target genes CSD1 and Nod19 in tissues from common bean plants under copper deficiency (CuD) or copper toxicity (CuT). (A) miR398 levels in roots, nodules and leaves of plants grown under control (C) or stress (CuD or CuT) conditions were detected by Northern blot analysis using U6snRNA as loading control. Signal intensity of the hybridization bands was calculated and the expression ratio (stress:control) was obtained. Relative expression of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod19 (red) in roots, nodules and leaves of plants grown under CuD (light colors) or CuT (dark colors) as determined by qRT-PCR. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Expressing, Northern Blot, Hybridization, Quantitative RT-PCR

    Effect of miR398b transient over-expression in Nicotiana benthamiana leaves infected with Sclerotinia sclerotiorum . N. benthamiana leaves were infiltrated with water (Control) or with A. tumefaciens bearing EV or OE398 plasmids and miR398b expression level was determined 3d after infiltration (A) . Subsequently, infiltrated leaves (EV or OE398) were inoculated with S. sclerotiorum . Characteristic fungal lesions (B) quantified by measuring the infection halo; asterisk: Student's t test, P ≤0.01 (C) and miR398b expression levels determined by qRT-PCR (D) at 48 h after fungal infection.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: Effect of miR398b transient over-expression in Nicotiana benthamiana leaves infected with Sclerotinia sclerotiorum . N. benthamiana leaves were infiltrated with water (Control) or with A. tumefaciens bearing EV or OE398 plasmids and miR398b expression level was determined 3d after infiltration (A) . Subsequently, infiltrated leaves (EV or OE398) were inoculated with S. sclerotiorum . Characteristic fungal lesions (B) quantified by measuring the infection halo; asterisk: Student's t test, P ≤0.01 (C) and miR398b expression levels determined by qRT-PCR (D) at 48 h after fungal infection.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Over Expression, Infection, Expressing, Quantitative RT-PCR

    Effect of miR398b over-expression in transgenic roots from composite plants grown under CuD. Composite plants were obtained through A. rhizogenes transformation with EV or with OE398 plasmid, these were grown in control (sufficient nutrient) condition (C) or in CuD stress condition. ( A ) Relative expression of miR398b (blue) and of ( B ) target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from the EV roots grown in the C condition that was set to 1. ( C ) Anthocyanin contents in root crown of composite plants. ( D ) Expression ratio (CuD:C) of copper-stress responsive genes: Fe superoxide dismutase (FSD, yellow), high affinity Cu transporter (COPT, purple) and ferric chelate reductase (FRO, brown). Values represent the average ± SD from three biological replicates.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: Effect of miR398b over-expression in transgenic roots from composite plants grown under CuD. Composite plants were obtained through A. rhizogenes transformation with EV or with OE398 plasmid, these were grown in control (sufficient nutrient) condition (C) or in CuD stress condition. ( A ) Relative expression of miR398b (blue) and of ( B ) target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from the EV roots grown in the C condition that was set to 1. ( C ) Anthocyanin contents in root crown of composite plants. ( D ) Expression ratio (CuD:C) of copper-stress responsive genes: Fe superoxide dismutase (FSD, yellow), high affinity Cu transporter (COPT, purple) and ferric chelate reductase (FRO, brown). Values represent the average ± SD from three biological replicates.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Over Expression, Transgenic Assay, Transformation Assay, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Reactive oxygen species (ROS) content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots exposed to high Cu (CuT). Measurements were done at initial time (0 h) and after 12, 24 and 48 h of high Cu (70 µM CuSO 4 ) application. (A) Histological (fluorescence) detection of ROS accumulation in CuT stressed root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in CuT-stressed roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: Reactive oxygen species (ROS) content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots exposed to high Cu (CuT). Measurements were done at initial time (0 h) and after 12, 24 and 48 h of high Cu (70 µM CuSO 4 ) application. (A) Histological (fluorescence) detection of ROS accumulation in CuT stressed root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in CuT-stressed roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Expressing, Fluorescence, Quantitative RT-PCR

    Expression pattern of miR398b and target genes CSD1 and Nod19 in common bean leaves infected with Sclerotinia sclerotiorum . (A) Mock (left) or S. sclerotiorum infected (right) common bean leaves after 24 h. (B) Relative expression of miR398b (blue) and of target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from mock that was set to 1 as indicated with a dashed line.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: Expression pattern of miR398b and target genes CSD1 and Nod19 in common bean leaves infected with Sclerotinia sclerotiorum . (A) Mock (left) or S. sclerotiorum infected (right) common bean leaves after 24 h. (B) Relative expression of miR398b (blue) and of target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from mock that was set to 1 as indicated with a dashed line.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Expressing, Infection, Quantitative RT-PCR

    Over-expression of PINCR in PINCR-S1 cells does not alter PINCR-S1 expression or the induction of PINCR targets. ( A, B ) PINCR-S1 HCT116 cells were transfected with pCB6 or pCB6- PINCR for 48 hr and then left untreated or treated with 5-FU for 16 hr. The expression of PINCR , PINCR-S1 , the PINCR targets BTG2 , GPX1 and RRM2B and the negative control p21 , was measured by qRT-PCR normalized to GAPDH . Error bars represent SD from three independent experiments. *p

    Journal: eLife

    Article Title: Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    doi: 10.7554/eLife.23244

    Figure Lengend Snippet: Over-expression of PINCR in PINCR-S1 cells does not alter PINCR-S1 expression or the induction of PINCR targets. ( A, B ) PINCR-S1 HCT116 cells were transfected with pCB6 or pCB6- PINCR for 48 hr and then left untreated or treated with 5-FU for 16 hr. The expression of PINCR , PINCR-S1 , the PINCR targets BTG2 , GPX1 and RRM2B and the negative control p21 , was measured by qRT-PCR normalized to GAPDH . Error bars represent SD from three independent experiments. *p

    Article Snippet: For qRT-PCR analysis, 500 ng total RNA was reverse-transcribed using iScript Reverse Transcription kit (Bio-Rad), and qPCR was performed using Fast SYBR Green Master Mix (Life technologies) per the manufacturer’s instructions.

    Techniques: Over Expression, Expressing, Transfection, Negative Control, Quantitative RT-PCR

    PINCR molecules per HCT116 cell. The number of molecules of PINCR RNA per HCT116 cell was determined by two approaches. First ( A–C ), using RNA-seq from HCT116 cells (Li et al., unpublished): The FPKM of PINCR from CTL and DOXO-treated HCT116 cells was compared to NORAD, a lncRNA known to be expressed at 500–1000 molecules/HCT116 cell ( Lee et al., 2016 ). Second ( D ), by qRT-PCR from DOXO-treated HCT116 cells using in vitro transcribed PINCR RNA as standard. DOI: http://dx.doi.org/10.7554/eLife.23244.009

    Journal: eLife

    Article Title: Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    doi: 10.7554/eLife.23244

    Figure Lengend Snippet: PINCR molecules per HCT116 cell. The number of molecules of PINCR RNA per HCT116 cell was determined by two approaches. First ( A–C ), using RNA-seq from HCT116 cells (Li et al., unpublished): The FPKM of PINCR from CTL and DOXO-treated HCT116 cells was compared to NORAD, a lncRNA known to be expressed at 500–1000 molecules/HCT116 cell ( Lee et al., 2016 ). Second ( D ), by qRT-PCR from DOXO-treated HCT116 cells using in vitro transcribed PINCR RNA as standard. DOI: http://dx.doi.org/10.7554/eLife.23244.009

    Article Snippet: For qRT-PCR analysis, 500 ng total RNA was reverse-transcribed using iScript Reverse Transcription kit (Bio-Rad), and qPCR was performed using Fast SYBR Green Master Mix (Life technologies) per the manufacturer’s instructions.

    Techniques: RNA Sequencing Assay, CTL Assay, Quantitative RT-PCR, In Vitro

    CRISPR knockout of PINCR in SW48 cells. ( A ) Partial genomic sequence of PINCR from Sanger sequencing and snap-shot of PINCR locus (using BLAT) for the PINCR -KO SW48 clone is shown. The p53RE is underlined and in green font. ( B ) qRT-PCR analysis from PINCR -WT and PINCR -KO SW48 cells untreated or treated with 5-FU for 24 hr. Error bars represent SD from three experiments. ## p

    Journal: eLife

    Article Title: Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    doi: 10.7554/eLife.23244

    Figure Lengend Snippet: CRISPR knockout of PINCR in SW48 cells. ( A ) Partial genomic sequence of PINCR from Sanger sequencing and snap-shot of PINCR locus (using BLAT) for the PINCR -KO SW48 clone is shown. The p53RE is underlined and in green font. ( B ) qRT-PCR analysis from PINCR -WT and PINCR -KO SW48 cells untreated or treated with 5-FU for 24 hr. Error bars represent SD from three experiments. ## p

    Article Snippet: For qRT-PCR analysis, 500 ng total RNA was reverse-transcribed using iScript Reverse Transcription kit (Bio-Rad), and qPCR was performed using Fast SYBR Green Master Mix (Life technologies) per the manufacturer’s instructions.

    Techniques: CRISPR, Knock-Out, Sequencing, Quantitative RT-PCR

    PINCR is highly induced after DNA damage in SW48 cells. ( A ) qRT-PCR analysis of PINCR and the known p53 target PUMA from isogenic p53-WT and p53-KO SW48 cells untreated or treated with DOXO for the indicated times. ( B ) Pictorial representation of PINCR locus and the p53 response element (p53RE) located 118 bp upstream of the first exon. Error bars represent SD from three biological replicates. *p

    Journal: eLife

    Article Title: Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    doi: 10.7554/eLife.23244

    Figure Lengend Snippet: PINCR is highly induced after DNA damage in SW48 cells. ( A ) qRT-PCR analysis of PINCR and the known p53 target PUMA from isogenic p53-WT and p53-KO SW48 cells untreated or treated with DOXO for the indicated times. ( B ) Pictorial representation of PINCR locus and the p53 response element (p53RE) located 118 bp upstream of the first exon. Error bars represent SD from three biological replicates. *p

    Article Snippet: For qRT-PCR analysis, 500 ng total RNA was reverse-transcribed using iScript Reverse Transcription kit (Bio-Rad), and qPCR was performed using Fast SYBR Green Master Mix (Life technologies) per the manufacturer’s instructions.

    Techniques: Quantitative RT-PCR

    Matrin 3 regulates the induction of PINCR targets upon p53 activation by Nutlin-3. PINCR -WT HCT116 cells were reverse transfected with CTL siRNA or Matrin-3 siRNAs for 48 hr and then treated with Nutlin-3 for 24 hr. The expression of PINCR targets and the negative control p21 , was assessed by qRT-PCR. Error bars represent SD from two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.23244.043

    Journal: eLife

    Article Title: Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    doi: 10.7554/eLife.23244

    Figure Lengend Snippet: Matrin 3 regulates the induction of PINCR targets upon p53 activation by Nutlin-3. PINCR -WT HCT116 cells were reverse transfected with CTL siRNA or Matrin-3 siRNAs for 48 hr and then treated with Nutlin-3 for 24 hr. The expression of PINCR targets and the negative control p21 , was assessed by qRT-PCR. Error bars represent SD from two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.23244.043

    Article Snippet: For qRT-PCR analysis, 500 ng total RNA was reverse-transcribed using iScript Reverse Transcription kit (Bio-Rad), and qPCR was performed using Fast SYBR Green Master Mix (Life technologies) per the manufacturer’s instructions.

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

    PINCR-S1 is strongly induced after DNA damage and is a predominantly nuclear lncRNA. ( A ) PINCR-S1 cells were left untreated or treated with DOXO for 16 hr and the extent of induction of PINCR-S1 RNA was assessed by qRT-PCR. ( B ) qRT-PCR for PINCR , the cytoplasmic GAPDH and nuclear MALAT1 was performed from nuclear and cytoplasmic fractions of PINCR-S1 cells treated with DOXO for 16 hr. ( C ) The expression of endogenous PINCR and endogenous PINCR-S1 relative to GAPDH was measured by qRT-PCR from HCT116 cells or PINCR-S1 HCT116 cells untreated or treated with 5-FU for 16 hr. Error bars represent SD from three independent experiments. *p

    Journal: eLife

    Article Title: Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    doi: 10.7554/eLife.23244

    Figure Lengend Snippet: PINCR-S1 is strongly induced after DNA damage and is a predominantly nuclear lncRNA. ( A ) PINCR-S1 cells were left untreated or treated with DOXO for 16 hr and the extent of induction of PINCR-S1 RNA was assessed by qRT-PCR. ( B ) qRT-PCR for PINCR , the cytoplasmic GAPDH and nuclear MALAT1 was performed from nuclear and cytoplasmic fractions of PINCR-S1 cells treated with DOXO for 16 hr. ( C ) The expression of endogenous PINCR and endogenous PINCR-S1 relative to GAPDH was measured by qRT-PCR from HCT116 cells or PINCR-S1 HCT116 cells untreated or treated with 5-FU for 16 hr. Error bars represent SD from three independent experiments. *p

    Article Snippet: For qRT-PCR analysis, 500 ng total RNA was reverse-transcribed using iScript Reverse Transcription kit (Bio-Rad), and qPCR was performed using Fast SYBR Green Master Mix (Life technologies) per the manufacturer’s instructions.

    Techniques: Quantitative RT-PCR, Expressing

    Knockdown of the PINCR targets BTG2 , GPX1 or RRM2B phenocopies the effect of PINCR loss. ( A ) qRT-PCR analysis from PINCR -WT HCT116 cells transfected for 48 hr with control siRNA (siCTL) or two independent siRNAs ( I and II ) against BTG2 , GPX1 or RRM2B . PI-staining and FACS analysis was performed from PINCR -WT HC116 cells (treated with 5-FU for 48 hr) after knockdown of BTG2 , GPX1 or RRM2B . Error bars represent SD from three biological replicates. *p

    Journal: eLife

    Article Title: Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    doi: 10.7554/eLife.23244

    Figure Lengend Snippet: Knockdown of the PINCR targets BTG2 , GPX1 or RRM2B phenocopies the effect of PINCR loss. ( A ) qRT-PCR analysis from PINCR -WT HCT116 cells transfected for 48 hr with control siRNA (siCTL) or two independent siRNAs ( I and II ) against BTG2 , GPX1 or RRM2B . PI-staining and FACS analysis was performed from PINCR -WT HC116 cells (treated with 5-FU for 48 hr) after knockdown of BTG2 , GPX1 or RRM2B . Error bars represent SD from three biological replicates. *p

    Article Snippet: For qRT-PCR analysis, 500 ng total RNA was reverse-transcribed using iScript Reverse Transcription kit (Bio-Rad), and qPCR was performed using Fast SYBR Green Master Mix (Life technologies) per the manufacturer’s instructions.

    Techniques: Quantitative RT-PCR, Transfection, Staining, FACS

    Effect of different doses of 5-FU on PINCR levels and cell survival. ( A ) qRT-PCR analysis from HCT116 cells untreated or treated for 24 hr with different doses of 5-FU. ( B ) HCT116 PINCR -WT and PINCR -KO cells were untreated or treated for 48 hr with different doses of 5-FU and cell cycle profiles were examined by PI-staining and FACS analysis. Error bars represent SD from three experiments. *p

    Journal: eLife

    Article Title: Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    doi: 10.7554/eLife.23244

    Figure Lengend Snippet: Effect of different doses of 5-FU on PINCR levels and cell survival. ( A ) qRT-PCR analysis from HCT116 cells untreated or treated for 24 hr with different doses of 5-FU. ( B ) HCT116 PINCR -WT and PINCR -KO cells were untreated or treated for 48 hr with different doses of 5-FU and cell cycle profiles were examined by PI-staining and FACS analysis. Error bars represent SD from three experiments. *p

    Article Snippet: For qRT-PCR analysis, 500 ng total RNA was reverse-transcribed using iScript Reverse Transcription kit (Bio-Rad), and qPCR was performed using Fast SYBR Green Master Mix (Life technologies) per the manufacturer’s instructions.

    Techniques: Quantitative RT-PCR, Staining, FACS

    Over-expression of PINCR has no significant effect on cell cycle. HCT116 cells were transfected with pCB6 or pCB6- PINCR for 24 hr and then left untreated cells or treated with 5-FU for 48 hr. PINCR levels were measured by qRT-PCR ( A ) and the effect on cell cycle was determined by PI-staining and FACS analysis ( B, C ). Raw cell cycle profiles from a representative experiment are shown in ( B ). Cell cycle profiles from three independent experiments are shown in ( C ). Error bars represent SD from three experiments. *p

    Journal: eLife

    Article Title: Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    doi: 10.7554/eLife.23244

    Figure Lengend Snippet: Over-expression of PINCR has no significant effect on cell cycle. HCT116 cells were transfected with pCB6 or pCB6- PINCR for 24 hr and then left untreated cells or treated with 5-FU for 48 hr. PINCR levels were measured by qRT-PCR ( A ) and the effect on cell cycle was determined by PI-staining and FACS analysis ( B, C ). Raw cell cycle profiles from a representative experiment are shown in ( B ). Cell cycle profiles from three independent experiments are shown in ( C ). Error bars represent SD from three experiments. *p

    Article Snippet: For qRT-PCR analysis, 500 ng total RNA was reverse-transcribed using iScript Reverse Transcription kit (Bio-Rad), and qPCR was performed using Fast SYBR Green Master Mix (Life technologies) per the manufacturer’s instructions.

    Techniques: Over Expression, Transfection, Quantitative RT-PCR, Staining, FACS

    PINCR is induced by 5-FU or NCS. ( A ) qRT-PCR analysis from PINCR -WT and PINCR -KO cells untreated or treated with 5-FU for 24 hr. ( B ) qRT-PCR for PINCR from HCT116 cells untreated or treated with NCS for 24 hr. Error bars represent SD from three biological replicates. **p

    Journal: eLife

    Article Title: Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    doi: 10.7554/eLife.23244

    Figure Lengend Snippet: PINCR is induced by 5-FU or NCS. ( A ) qRT-PCR analysis from PINCR -WT and PINCR -KO cells untreated or treated with 5-FU for 24 hr. ( B ) qRT-PCR for PINCR from HCT116 cells untreated or treated with NCS for 24 hr. Error bars represent SD from three biological replicates. **p

    Article Snippet: For qRT-PCR analysis, 500 ng total RNA was reverse-transcribed using iScript Reverse Transcription kit (Bio-Rad), and qPCR was performed using Fast SYBR Green Master Mix (Life technologies) per the manufacturer’s instructions.

    Techniques: Quantitative RT-PCR

    Matrin 3 motifs in PINCR RNA. ( A, B ) Putative Matrin 3 binding motif in PINCR RNA. ( A ) ‘N’ represents the number of times the motif appears in the PINCR RNA. ‘ES’ represents the enrichment score calculated as shown in ‘ B ’. ( C ) HCT116 cells were reverse transfected with a control siRNA (siCTL) or siRNAs targeting Matrin 3 (siMatrin 3-I and siMatrin 3-II) for 48 hr and the extent of Matrin 3 knockdown was measured by qRT-PCR for Matrin 3 normalized to GADPH. Error bars represent SD from three independent experiments. ##p

    Journal: eLife

    Article Title: Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    doi: 10.7554/eLife.23244

    Figure Lengend Snippet: Matrin 3 motifs in PINCR RNA. ( A, B ) Putative Matrin 3 binding motif in PINCR RNA. ( A ) ‘N’ represents the number of times the motif appears in the PINCR RNA. ‘ES’ represents the enrichment score calculated as shown in ‘ B ’. ( C ) HCT116 cells were reverse transfected with a control siRNA (siCTL) or siRNAs targeting Matrin 3 (siMatrin 3-I and siMatrin 3-II) for 48 hr and the extent of Matrin 3 knockdown was measured by qRT-PCR for Matrin 3 normalized to GADPH. Error bars represent SD from three independent experiments. ##p

    Article Snippet: For qRT-PCR analysis, 500 ng total RNA was reverse-transcribed using iScript Reverse Transcription kit (Bio-Rad), and qPCR was performed using Fast SYBR Green Master Mix (Life technologies) per the manufacturer’s instructions.

    Techniques: Binding Assay, Transfection, Quantitative RT-PCR

    ISG induction, phosphorylation of IRFs, and IκBα degradation in primary myeloid cells derived from TLR3 −/− and TLR4 −/− mice after JEV infection. A and B . Clustered heatmap showing the expression of IRF, ISG, and RLR genes in infected BMDC and BMDM. Primary bone marrow-derived DCs (BMDC) and macrophages (BMDM) recovered from TLR3 −/− and TLR4 −/− mice were infected with JEV at a MOI of 10 or mock-infected (M), and employed to analyze the induction of IRF, ISG, and RLR genes at 24 and 48 h pi. The expression of each IRF, ISG, and RLR gene was normalized to β-actin after determining mRNA levels by real-time qRT-PCR, and displayed as the average of at least four independent samples, according to the indicated color on a log 2 scale. C and D . Expression and phosphorylation of IRF3, IRF7, and STAT1 and IκBα degradation. BMDC and BMDM derived from TLR3 −/− and TLR4 −/− mice were infected with JEV at 10 MOI or mock-infected (M). At 6, 12, 24, and 48 h after infection, cells were lysed, separated by SDS-PAGE and analyzed by western blot to detect unphosphorylated and phosphorylated form of target proteins using specific Abs. One representative picture of at least three experiments is shown.

    Journal: PLoS Pathogens

    Article Title: Distinct Dictation of Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality via Triggering TLR3 and TLR4 Signal Pathways

    doi: 10.1371/journal.ppat.1004319

    Figure Lengend Snippet: ISG induction, phosphorylation of IRFs, and IκBα degradation in primary myeloid cells derived from TLR3 −/− and TLR4 −/− mice after JEV infection. A and B . Clustered heatmap showing the expression of IRF, ISG, and RLR genes in infected BMDC and BMDM. Primary bone marrow-derived DCs (BMDC) and macrophages (BMDM) recovered from TLR3 −/− and TLR4 −/− mice were infected with JEV at a MOI of 10 or mock-infected (M), and employed to analyze the induction of IRF, ISG, and RLR genes at 24 and 48 h pi. The expression of each IRF, ISG, and RLR gene was normalized to β-actin after determining mRNA levels by real-time qRT-PCR, and displayed as the average of at least four independent samples, according to the indicated color on a log 2 scale. C and D . Expression and phosphorylation of IRF3, IRF7, and STAT1 and IκBα degradation. BMDC and BMDM derived from TLR3 −/− and TLR4 −/− mice were infected with JEV at 10 MOI or mock-infected (M). At 6, 12, 24, and 48 h after infection, cells were lysed, separated by SDS-PAGE and analyzed by western blot to detect unphosphorylated and phosphorylated form of target proteins using specific Abs. One representative picture of at least three experiments is shown.

    Article Snippet: Total RNAs extracted from tissues using easyBLUE (iNtRON, INC., Daejeon, Korea) were employed for real-time qRT-PCR using a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Derivative Assay, Mouse Assay, Infection, Expressing, Quantitative RT-PCR, SDS Page, Western Blot

    Induction of type I IFNs and ISGs in primary cortical neurons derived from TLR3 −/− and TLR4 −/− mice after JEV infection. Primary cortical neurons generated from TLR3 −/− and TLR4 −/− mice were infected at an MOI of 0.1, and viral replication and type I IFNs responses at 24 and 48 h pi were analyzed. A . JEV replication. JEV replication was determined by both real-time qRT-PCR and focus-forming assay. B . The expression of type I IFNs (IFN-α and IFN-β) mRNA and IFN-β secretion in primary cortical neurons infected by JEV. C . Induction of IRFs, ISGs, and RLRs gene in infected primary cortical neurons. The mRNA levels of the indicated gene were determined by real-time qRT-PCR, and IFN-β levels in culture media were determined by ELISA. Data represent the average ± SD derived from primary cortical neurons quadruplicate. *, p

    Journal: PLoS Pathogens

    Article Title: Distinct Dictation of Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality via Triggering TLR3 and TLR4 Signal Pathways

    doi: 10.1371/journal.ppat.1004319

    Figure Lengend Snippet: Induction of type I IFNs and ISGs in primary cortical neurons derived from TLR3 −/− and TLR4 −/− mice after JEV infection. Primary cortical neurons generated from TLR3 −/− and TLR4 −/− mice were infected at an MOI of 0.1, and viral replication and type I IFNs responses at 24 and 48 h pi were analyzed. A . JEV replication. JEV replication was determined by both real-time qRT-PCR and focus-forming assay. B . The expression of type I IFNs (IFN-α and IFN-β) mRNA and IFN-β secretion in primary cortical neurons infected by JEV. C . Induction of IRFs, ISGs, and RLRs gene in infected primary cortical neurons. The mRNA levels of the indicated gene were determined by real-time qRT-PCR, and IFN-β levels in culture media were determined by ELISA. Data represent the average ± SD derived from primary cortical neurons quadruplicate. *, p

    Article Snippet: Total RNAs extracted from tissues using easyBLUE (iNtRON, INC., Daejeon, Korea) were employed for real-time qRT-PCR using a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Derivative Assay, Mouse Assay, Infection, Generated, Quantitative RT-PCR, Focus Forming Assay, Expressing, Enzyme-linked Immunosorbent Assay

    Localized and systemic type I IFN responses of TLR3 −/− and TLR4 −/− mice following JEV infection. A and B . The expression of type I IFNs (IFN-α and β) in lymphoid and inflammatory tissues. The levels of type I IFN (IFN-α and β) mRNA were determined by real-time qRT-PCR at the indicated day pi. Each symbol represents the level of an individual mouse; horizontal line indicates the median of each group. p -values were calculated using Student's t-test. C . Systemic IFN-β levels. The amount of serum IFN-β was determined by ELISA. Data represent the average ± SD derived from at least five mice per group. **, p

    Journal: PLoS Pathogens

    Article Title: Distinct Dictation of Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality via Triggering TLR3 and TLR4 Signal Pathways

    doi: 10.1371/journal.ppat.1004319

    Figure Lengend Snippet: Localized and systemic type I IFN responses of TLR3 −/− and TLR4 −/− mice following JEV infection. A and B . The expression of type I IFNs (IFN-α and β) in lymphoid and inflammatory tissues. The levels of type I IFN (IFN-α and β) mRNA were determined by real-time qRT-PCR at the indicated day pi. Each symbol represents the level of an individual mouse; horizontal line indicates the median of each group. p -values were calculated using Student's t-test. C . Systemic IFN-β levels. The amount of serum IFN-β was determined by ELISA. Data represent the average ± SD derived from at least five mice per group. **, p

    Article Snippet: Total RNAs extracted from tissues using easyBLUE (iNtRON, INC., Daejeon, Korea) were employed for real-time qRT-PCR using a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Mouse Assay, Infection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay

    TLR3 ablation induces huge production of pro-inflammatory cytokines in inflammatory tissues. A–D . The expression of pro-inflammatory cytokine and chemokine in inflammatory tissues. The expression of pro-inflammatory cytokines and chemokines in brain (A and C) and spinal cord (B and D) was determined by real-time qRT-PCR 4 days pi. Each symbol represents the level of an individual mouse; horizontal line indicates median of each group. E . Systemic production of pro-inflammatory IL-6 cytokine. The levels of IL-6 in sera were determined by cytokine ELISA at the indicated day pi. Data represent the average ± SD derived from at least five mice per group. ***, p

    Journal: PLoS Pathogens

    Article Title: Distinct Dictation of Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality via Triggering TLR3 and TLR4 Signal Pathways

    doi: 10.1371/journal.ppat.1004319

    Figure Lengend Snippet: TLR3 ablation induces huge production of pro-inflammatory cytokines in inflammatory tissues. A–D . The expression of pro-inflammatory cytokine and chemokine in inflammatory tissues. The expression of pro-inflammatory cytokines and chemokines in brain (A and C) and spinal cord (B and D) was determined by real-time qRT-PCR 4 days pi. Each symbol represents the level of an individual mouse; horizontal line indicates median of each group. E . Systemic production of pro-inflammatory IL-6 cytokine. The levels of IL-6 in sera were determined by cytokine ELISA at the indicated day pi. Data represent the average ± SD derived from at least five mice per group. ***, p

    Article Snippet: Total RNAs extracted from tissues using easyBLUE (iNtRON, INC., Daejeon, Korea) were employed for real-time qRT-PCR using a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay, Mouse Assay

    The spread of JEV in the brain of TLR3 −/− and TLR4 −/− mice after intracranial inoculation. TLR3 −/− and TLR4 −/− mice were inoculated with JEV (10 3 pfu) by intracranial injection. Brains were harvested on days 2 and 4 pi, and then used for the determination of viral spread and cytokine expression. A–F . Viral burden in each sub-tissue of brain. The CNS tissues were separated into cortex (A), olfactory bulb (B), hippocampus (C), brain stem (D), cerebellum (E), and spinal cord (F). Viral burden was determined by real-time qRT-PCR. The viral RNA load was expressed by viral RNA copy number per microgram of total RNA. Each symbol represents the level of an individual mouse; the horizontal line indicates the median of each group. G . The expression levels of pro-inflammatory cytokine and type I IFN genes in each sub-tissue of brain. The expression levels were expressed by the indicated target gene levels relative to those in the mock-infected group. The bar represents the average ± SD of the indicated target gene levels obtained from each group ( n = 5). Cor, cortex; Olf, olfactory bulb; Hip, hippocampus; BS, brain stem; Cer, cerebellum; SC, spinal cord. H . Leukocyte accumulation in the CNS of TLR3 −/− and TLR4 −/− mice after intracranial infection of JEV. TLR3 −/− and TLR4 −/− mice were inoculated with JEV (10 3 pfu) by intracranial injection, and brains were harvested on day 2. Leukocyte populations were isolated by vigorous heart perfusion and then determined by flow cytometric analysis. The values in the representative dot-plots denote the average of the indicated cell population obtained from three individual experiments.

    Journal: PLoS Pathogens

    Article Title: Distinct Dictation of Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality via Triggering TLR3 and TLR4 Signal Pathways

    doi: 10.1371/journal.ppat.1004319

    Figure Lengend Snippet: The spread of JEV in the brain of TLR3 −/− and TLR4 −/− mice after intracranial inoculation. TLR3 −/− and TLR4 −/− mice were inoculated with JEV (10 3 pfu) by intracranial injection. Brains were harvested on days 2 and 4 pi, and then used for the determination of viral spread and cytokine expression. A–F . Viral burden in each sub-tissue of brain. The CNS tissues were separated into cortex (A), olfactory bulb (B), hippocampus (C), brain stem (D), cerebellum (E), and spinal cord (F). Viral burden was determined by real-time qRT-PCR. The viral RNA load was expressed by viral RNA copy number per microgram of total RNA. Each symbol represents the level of an individual mouse; the horizontal line indicates the median of each group. G . The expression levels of pro-inflammatory cytokine and type I IFN genes in each sub-tissue of brain. The expression levels were expressed by the indicated target gene levels relative to those in the mock-infected group. The bar represents the average ± SD of the indicated target gene levels obtained from each group ( n = 5). Cor, cortex; Olf, olfactory bulb; Hip, hippocampus; BS, brain stem; Cer, cerebellum; SC, spinal cord. H . Leukocyte accumulation in the CNS of TLR3 −/− and TLR4 −/− mice after intracranial infection of JEV. TLR3 −/− and TLR4 −/− mice were inoculated with JEV (10 3 pfu) by intracranial injection, and brains were harvested on day 2. Leukocyte populations were isolated by vigorous heart perfusion and then determined by flow cytometric analysis. The values in the representative dot-plots denote the average of the indicated cell population obtained from three individual experiments.

    Article Snippet: Total RNAs extracted from tissues using easyBLUE (iNtRON, INC., Daejeon, Korea) were employed for real-time qRT-PCR using a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Mouse Assay, Injection, Expressing, Quantitative RT-PCR, Infection, Isolation, Flow Cytometry

    Contrasting regulation of JE by triggering TLR3 and TLR4 signal pathway. A. Susceptibility of TLR3 −/− , TLR4 −/− , and TLR3/4 −/− mice to JE. Four- to five-week-old mice ( n = 10–18) were inoculated with JEV (2.8×10 7 pfu), and the survival rate was examined over 15 days. B. Ratio of mice showing neurologic disorder during JE progression. Mice infected with JEV were examined every 6 h from 4 to 7 days pi. C. Viral burden in lymphoid and inflammatory tissues during JE progression. Viral burden in spleen, brain, and spinal cord of mice infected with JEV was assessed by real-time qRT-PCR at the indicated days pi. The viral RNA load was expressed by viral RNA copy number per microgram of total RNA ( n = 5). Each symbol represents the level of an individual mouse; horizontal line indicates the median of each group.

    Journal: PLoS Pathogens

    Article Title: Distinct Dictation of Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality via Triggering TLR3 and TLR4 Signal Pathways

    doi: 10.1371/journal.ppat.1004319

    Figure Lengend Snippet: Contrasting regulation of JE by triggering TLR3 and TLR4 signal pathway. A. Susceptibility of TLR3 −/− , TLR4 −/− , and TLR3/4 −/− mice to JE. Four- to five-week-old mice ( n = 10–18) were inoculated with JEV (2.8×10 7 pfu), and the survival rate was examined over 15 days. B. Ratio of mice showing neurologic disorder during JE progression. Mice infected with JEV were examined every 6 h from 4 to 7 days pi. C. Viral burden in lymphoid and inflammatory tissues during JE progression. Viral burden in spleen, brain, and spinal cord of mice infected with JEV was assessed by real-time qRT-PCR at the indicated days pi. The viral RNA load was expressed by viral RNA copy number per microgram of total RNA ( n = 5). Each symbol represents the level of an individual mouse; horizontal line indicates the median of each group.

    Article Snippet: Total RNAs extracted from tissues using easyBLUE (iNtRON, INC., Daejeon, Korea) were employed for real-time qRT-PCR using a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Mouse Assay, Infection, Quantitative RT-PCR

    Virus control and type I IFN responses of myeloid cells derived from TLR3 −/− and TLR4 −/− mice to JEV infection. Primary bone marrow-derived DCs (BMDC) and macrophages (BMDM) recovered from TLR3 −/− and TLR4 −/− mice were infected with JEV at a MOI of 1.0 for viral replication and 10 for cytokine expression. A . JEV replication in BMDC and BMDM. Viral RNA replication was expressed by viral RNA copy number per microgram of total RNA. B and C . The expression of pro-inflammatory cytokines (IL-6 and TNF-α) in infected BMDC and BMDM. D and E . The expression of type I IFNs (IFN-α and β) in infected BMDC and BMDM. F . The secretion of IFN-β protein by infected BMDC and BMDM. The mRNA levels of the indicated cytokines were determined by real-time qRT-PCR and the cytokine levels in culture media were determined by ELISA. Data represent the average ± SD derived from BMDC and BMDM evaluated in quadruplicate. *, p

    Journal: PLoS Pathogens

    Article Title: Distinct Dictation of Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality via Triggering TLR3 and TLR4 Signal Pathways

    doi: 10.1371/journal.ppat.1004319

    Figure Lengend Snippet: Virus control and type I IFN responses of myeloid cells derived from TLR3 −/− and TLR4 −/− mice to JEV infection. Primary bone marrow-derived DCs (BMDC) and macrophages (BMDM) recovered from TLR3 −/− and TLR4 −/− mice were infected with JEV at a MOI of 1.0 for viral replication and 10 for cytokine expression. A . JEV replication in BMDC and BMDM. Viral RNA replication was expressed by viral RNA copy number per microgram of total RNA. B and C . The expression of pro-inflammatory cytokines (IL-6 and TNF-α) in infected BMDC and BMDM. D and E . The expression of type I IFNs (IFN-α and β) in infected BMDC and BMDM. F . The secretion of IFN-β protein by infected BMDC and BMDM. The mRNA levels of the indicated cytokines were determined by real-time qRT-PCR and the cytokine levels in culture media were determined by ELISA. Data represent the average ± SD derived from BMDC and BMDM evaluated in quadruplicate. *, p

    Article Snippet: Total RNAs extracted from tissues using easyBLUE (iNtRON, INC., Daejeon, Korea) were employed for real-time qRT-PCR using a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Derivative Assay, Mouse Assay, Infection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    FMDV infection induces autophagy through the EIF2S1-ATF4-AKT-MTOR cascade. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. The phosphorylation of AKT, AMPK, MTOR and ULK1 were analyzed by western blot. ACTB was used as a sample loading control. ( B ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. ATF4 and phosphorylation of EIF2S1 were analyzed by western blot. ( C ) ATF4 KD and wild-type cells cells were infected with FMDV (MOI = 1). ATF4, LC3B and phosphorylation of AKT, MTOR and ULK1 were analyzed by western blot. ( D ) ATF4 and scrambled knockdown cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) for 3 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E and F ) ATF4 KD and wil-type cells cells were infected with FMDV (MOI = 1) for 3 h. At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. **P

    Journal: Autophagy

    Article Title: Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

    doi: 10.1080/15548627.2017.1405187

    Figure Lengend Snippet: FMDV infection induces autophagy through the EIF2S1-ATF4-AKT-MTOR cascade. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. The phosphorylation of AKT, AMPK, MTOR and ULK1 were analyzed by western blot. ACTB was used as a sample loading control. ( B ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. ATF4 and phosphorylation of EIF2S1 were analyzed by western blot. ( C ) ATF4 KD and wild-type cells cells were infected with FMDV (MOI = 1). ATF4, LC3B and phosphorylation of AKT, MTOR and ULK1 were analyzed by western blot. ( D ) ATF4 and scrambled knockdown cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) for 3 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E and F ) ATF4 KD and wil-type cells cells were infected with FMDV (MOI = 1) for 3 h. At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. **P

    Article Snippet: The real-time RT-PCR was performed using the SuperScript® III Platinum®One-Step qRT-PCR Kit (Thermo Fisher Scientific, 11732-020) and DyNAmo Flash SYBR Green qPCR Kit (Thermo Fisher Scientific, F-415S).

    Techniques: Infection, Western Blot, Transfection, Fluorescence, Immunofluorescence, Microscopy, Quantitative RT-PCR

    FMDV infection-induced autophagy plays an important role in viral replication. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 0, 0.5, 1, 1.5, 2 and 3 h. The expression of LC3B and SQSTM1 was analyzed by western blot. The level of 3D was analyzed by RT-PCR. ACTB and GAPDH were used as a sample loading control. ( B ) The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the 3D protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. ( C ) ATG5 knockdown (KD) and wild-type cells were infected with FMDV (MOI = 1) for 2 and 3 h. The expression of ATG5 and LC3B was analyzed by western blot. ( D and E ) ATG5 KD and wild-type cells were infected with FMDV (MOI = 1). At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. ***P

    Journal: Autophagy

    Article Title: Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

    doi: 10.1080/15548627.2017.1405187

    Figure Lengend Snippet: FMDV infection-induced autophagy plays an important role in viral replication. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 0, 0.5, 1, 1.5, 2 and 3 h. The expression of LC3B and SQSTM1 was analyzed by western blot. The level of 3D was analyzed by RT-PCR. ACTB and GAPDH were used as a sample loading control. ( B ) The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the 3D protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. ( C ) ATG5 knockdown (KD) and wild-type cells were infected with FMDV (MOI = 1) for 2 and 3 h. The expression of ATG5 and LC3B was analyzed by western blot. ( D and E ) ATG5 KD and wild-type cells were infected with FMDV (MOI = 1). At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. ***P

    Article Snippet: The real-time RT-PCR was performed using the SuperScript® III Platinum®One-Step qRT-PCR Kit (Thermo Fisher Scientific, 11732-020) and DyNAmo Flash SYBR Green qPCR Kit (Thermo Fisher Scientific, F-415S).

    Techniques: Infection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Fluorescence, Microscopy, Quantitative RT-PCR

    Cdc42/Rac1 GTPase controls LL-37-induced chemokine secretion. Macrophage-like THP-1 cells were pre-incubated with Cdc42/Rac1 inhibitor ML141 (10 µM) for 1 h, prior to stimulation with either LL-37 or sLL-37 (5 µM each), lipopolysaccharide (LPS) (10 ng/ml), or 0.01% DMSO as vehicle control (VC). (A) Tissue culture supernatants were monitored by ELISA for the production of chemokines GRO-α and IL-8 after 24 h. Results shown as mean ± SE of eight independent experiments ( n = 8). (B) mRNA expressions were evaluated by quantitative real-time PCR for chemokines ( GRO- α and IL-8 ) and anti-inflammatory cytokine IL-1RA , after 4 h. Relative fold changes were calculated compared to the expression in unstimulated cells, using the ΔΔCt method, after normalization with 18sRNA expression. Results shown as mean ± SE of three independent experiments. (C) Macrophage-like THP-1 cells were pre-incubated with the Ras inhibitor FTS (10 µM) for 1 h, prior to stimulation with either LL-37 or sLL-37 (5 µM each), or LPS (10 ng/ml). TC supernatants were monitored by ELISA for the production of chemokines GRO-α and IL-8 after 24 h. Results shown as mean ± SE of eight independent experiments ( n = 8). Analysis of variance with Bonferroni’s post hoc test was used for statistical analyses (*** p

    Journal: Frontiers in Immunology

    Article Title: Host Defense Peptide LL-37-Mediated Chemoattractant Properties, but Not Anti-Inflammatory Cytokine IL-1RA Production, Is Selectively Controlled by Cdc42 Rho GTPase via G Protein-Coupled Receptors and JNK Mitogen-Activated Protein Kinase

    doi: 10.3389/fimmu.2018.01871

    Figure Lengend Snippet: Cdc42/Rac1 GTPase controls LL-37-induced chemokine secretion. Macrophage-like THP-1 cells were pre-incubated with Cdc42/Rac1 inhibitor ML141 (10 µM) for 1 h, prior to stimulation with either LL-37 or sLL-37 (5 µM each), lipopolysaccharide (LPS) (10 ng/ml), or 0.01% DMSO as vehicle control (VC). (A) Tissue culture supernatants were monitored by ELISA for the production of chemokines GRO-α and IL-8 after 24 h. Results shown as mean ± SE of eight independent experiments ( n = 8). (B) mRNA expressions were evaluated by quantitative real-time PCR for chemokines ( GRO- α and IL-8 ) and anti-inflammatory cytokine IL-1RA , after 4 h. Relative fold changes were calculated compared to the expression in unstimulated cells, using the ΔΔCt method, after normalization with 18sRNA expression. Results shown as mean ± SE of three independent experiments. (C) Macrophage-like THP-1 cells were pre-incubated with the Ras inhibitor FTS (10 µM) for 1 h, prior to stimulation with either LL-37 or sLL-37 (5 µM each), or LPS (10 ng/ml). TC supernatants were monitored by ELISA for the production of chemokines GRO-α and IL-8 after 24 h. Results shown as mean ± SE of eight independent experiments ( n = 8). Analysis of variance with Bonferroni’s post hoc test was used for statistical analyses (*** p

    Article Snippet: RNA concentration and purity were assessed using a NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific). mRNA expression was analyzed using a SuperScript III Platinum Two-Step qRT-PCR kit with SYBR Green (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s instructions, using the ABI PRISM 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing

    Histone H3 lysine 27 trimethylation is required for effector gene repression. (A) eGFP fluorescence of Zymoseptoria tritici transgenic lines with and without the histone methyltransferase gene KMT6 (wt and KO, respectively), both harboring the eGFP gene under the control of a constitutive promoter in the Avr3D1 locus. A line harboring an ectopic copy of the eGFP cassette is shown as a control. All lines were obtained in a genetic background containing an mCherry reporter cassette for visualization of fungal cells. (B) eGFP transcript levels in the transgenic lines described in the panel A legend during axenic growth in rich medium and during plant colonization at 11 days postinfection (dpi). (C) Transcript levels of AvrStb6 and Mycgr3G76589 during axenic growth in rich medium and during plant colonization in Z. tritici lines with and without KMT6 . Expression levels were normalized to the line with the wild-type KMT6 gene during axenic growth. Actin was used as reference gene for qRT-PCR. Error bars represent standard errors of the means. Data from at least three independent replicates are shown. Black asterisks indicate differences between strains with and without KMT6 , and red asterisks indicate differences between axenic and in planta growth of the same mutant line ( P

    Journal: mBio

    Article Title: Chromatin Dynamics Contribute to the Spatiotemporal Expression Pattern of Virulence Genes in a Fungal Plant Pathogen

    doi: 10.1128/mBio.02343-20

    Figure Lengend Snippet: Histone H3 lysine 27 trimethylation is required for effector gene repression. (A) eGFP fluorescence of Zymoseptoria tritici transgenic lines with and without the histone methyltransferase gene KMT6 (wt and KO, respectively), both harboring the eGFP gene under the control of a constitutive promoter in the Avr3D1 locus. A line harboring an ectopic copy of the eGFP cassette is shown as a control. All lines were obtained in a genetic background containing an mCherry reporter cassette for visualization of fungal cells. (B) eGFP transcript levels in the transgenic lines described in the panel A legend during axenic growth in rich medium and during plant colonization at 11 days postinfection (dpi). (C) Transcript levels of AvrStb6 and Mycgr3G76589 during axenic growth in rich medium and during plant colonization in Z. tritici lines with and without KMT6 . Expression levels were normalized to the line with the wild-type KMT6 gene during axenic growth. Actin was used as reference gene for qRT-PCR. Error bars represent standard errors of the means. Data from at least three independent replicates are shown. Black asterisks indicate differences between strains with and without KMT6 , and red asterisks indicate differences between axenic and in planta growth of the same mutant line ( P

    Article Snippet: RNA isolation and quantitative reverse transcription-PCR.

    Techniques: Fluorescence, Transgenic Assay, Expressing, Quantitative RT-PCR, Mutagenesis

    H19 facilitated CaOx nephrocalcinosis-induced renal tubular epithelial cell injury in vivo . CaOx deposition in the corticomedullary junction area was measured by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200). PAS staining (Magnification, × 100) illustrating renal tubular epithelial cell injury. (b) Immunohistochemical analysis of kidney HMGB1, TLR4, NF-kB, SOD2 and NOX2 expression was performed in rAAV-H19 or rAAV-vector injected CaOx nephrocalcinosis mouse models (Magnification, × 200). (c) Western blot analysis was performed to detect HMGB1, TLR4, p-NF-kB and NF-kB protein expression in H19-treated kidney tissue. GAPDH served as an internal control. (d) qRT-PCR analysis was performed to detect the expression levels of proinflammatory cytokines in kidney tissue. The data are shown as the mean ± standard deviation (SD) of three independent experiments. (* P

    Journal: EBioMedicine

    Article Title: H19 promote calcium oxalate nephrocalcinosis-induced renal tubular epithelial cell injury via a ceRNA pathway

    doi: 10.1016/j.ebiom.2019.10.059

    Figure Lengend Snippet: H19 facilitated CaOx nephrocalcinosis-induced renal tubular epithelial cell injury in vivo . CaOx deposition in the corticomedullary junction area was measured by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200). PAS staining (Magnification, × 100) illustrating renal tubular epithelial cell injury. (b) Immunohistochemical analysis of kidney HMGB1, TLR4, NF-kB, SOD2 and NOX2 expression was performed in rAAV-H19 or rAAV-vector injected CaOx nephrocalcinosis mouse models (Magnification, × 200). (c) Western blot analysis was performed to detect HMGB1, TLR4, p-NF-kB and NF-kB protein expression in H19-treated kidney tissue. GAPDH served as an internal control. (d) qRT-PCR analysis was performed to detect the expression levels of proinflammatory cytokines in kidney tissue. The data are shown as the mean ± standard deviation (SD) of three independent experiments. (* P

    Article Snippet: The quantitative detection of mature miRNA was conducted with an All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia). cDNA was obtained by reverse transcription of the total RNA.

    Techniques: In Vivo, Light Microscopy, Staining, Immunohistochemistry, Expressing, Plasmid Preparation, Injection, Western Blot, Quantitative RT-PCR, Standard Deviation

    H19 and HMGB1 expression was significantly increased in the CaOx nephrocalcinosis mouse model. (a) Hierarchical clustering and heatmap analysis of GSE73680 from a recent genome-wide gene expression profile analysis of Randall's plaques from 29 CaOx stone patients and 6 healthy controls. (b) A glyoxylate-induced kidney CaOx nephrocalcinosis mouse model was established and verified by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200) of CaOx crystal deposition in the corticomedullary junction area. PAS staining (Magnification, × 100) illustrating tubular injury. Immunohistochemical analysis of kidney HMGB1, TLR4 and NF-kB expression in a CaOx nephrocalcinosis-induced mouse model (Magnification, × 200). (c) qRT-PCR analysis was performed to detect the expression levels of HMGB1, TLR4 and NF-kB in CaOx mouse kidney samples, and these levels were compared with those in mock controls. Pearson correlation coefficient analysis of the expression levels of H19 and HMGB1 (d), TLR4 (e), and NF-kB (f). (g) qRT-PCR analysis was performed to detect the expression levels of HMGB1, TLR4 and NF-kB in HK-2 cells treated with different concentrations of COM crystals. The data are shown as the mean ± standard error (SE) of three independent experiments. (* P

    Journal: EBioMedicine

    Article Title: H19 promote calcium oxalate nephrocalcinosis-induced renal tubular epithelial cell injury via a ceRNA pathway

    doi: 10.1016/j.ebiom.2019.10.059

    Figure Lengend Snippet: H19 and HMGB1 expression was significantly increased in the CaOx nephrocalcinosis mouse model. (a) Hierarchical clustering and heatmap analysis of GSE73680 from a recent genome-wide gene expression profile analysis of Randall's plaques from 29 CaOx stone patients and 6 healthy controls. (b) A glyoxylate-induced kidney CaOx nephrocalcinosis mouse model was established and verified by polarized light microscopy (Magnification, × 100) and Pizzolato staining (Magnification, × 200) of CaOx crystal deposition in the corticomedullary junction area. PAS staining (Magnification, × 100) illustrating tubular injury. Immunohistochemical analysis of kidney HMGB1, TLR4 and NF-kB expression in a CaOx nephrocalcinosis-induced mouse model (Magnification, × 200). (c) qRT-PCR analysis was performed to detect the expression levels of HMGB1, TLR4 and NF-kB in CaOx mouse kidney samples, and these levels were compared with those in mock controls. Pearson correlation coefficient analysis of the expression levels of H19 and HMGB1 (d), TLR4 (e), and NF-kB (f). (g) qRT-PCR analysis was performed to detect the expression levels of HMGB1, TLR4 and NF-kB in HK-2 cells treated with different concentrations of COM crystals. The data are shown as the mean ± standard error (SE) of three independent experiments. (* P

    Article Snippet: The quantitative detection of mature miRNA was conducted with an All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia). cDNA was obtained by reverse transcription of the total RNA.

    Techniques: Expressing, Genome Wide, Light Microscopy, Staining, Immunohistochemistry, Quantitative RT-PCR

    miR-216b reversed the effect of H19 on COM crystal-induced renal tubular epithelial cell oxidative stress injury in vitro . Western blot (a) and qRT-PCR (b) analyses of HMGB1, TLR4 and NF-kB expression in HK-2 cells following transfection with lenti-H19, miR-216b mimics, or their combination. β-Actin was used for normalization. ROS generation (c), LDH release (d), cellular malondialdehyde (MDA) levels (e), and H 2 O 2 concentrations (f) were determined in HK-2 cells incubated with COM crystals following treatment with lenti-H19, miR-216b mimics or their combination. (G-H) Flow cytometric analysis of cellular ROS generation was carried out in HK-2 cells. Histograms showing the mean fluorescence intensity of DCFH. (g) The apoptosis effect was investigated by flow cytometric analysis of HK-2 cells stained with Annexin V-FITC and propidium iodide. The data are shown as the mean ± SD of three independent experiments. (* P

    Journal: EBioMedicine

    Article Title: H19 promote calcium oxalate nephrocalcinosis-induced renal tubular epithelial cell injury via a ceRNA pathway

    doi: 10.1016/j.ebiom.2019.10.059

    Figure Lengend Snippet: miR-216b reversed the effect of H19 on COM crystal-induced renal tubular epithelial cell oxidative stress injury in vitro . Western blot (a) and qRT-PCR (b) analyses of HMGB1, TLR4 and NF-kB expression in HK-2 cells following transfection with lenti-H19, miR-216b mimics, or their combination. β-Actin was used for normalization. ROS generation (c), LDH release (d), cellular malondialdehyde (MDA) levels (e), and H 2 O 2 concentrations (f) were determined in HK-2 cells incubated with COM crystals following treatment with lenti-H19, miR-216b mimics or their combination. (G-H) Flow cytometric analysis of cellular ROS generation was carried out in HK-2 cells. Histograms showing the mean fluorescence intensity of DCFH. (g) The apoptosis effect was investigated by flow cytometric analysis of HK-2 cells stained with Annexin V-FITC and propidium iodide. The data are shown as the mean ± SD of three independent experiments. (* P

    Article Snippet: The quantitative detection of mature miRNA was conducted with an All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia). cDNA was obtained by reverse transcription of the total RNA.

    Techniques: In Vitro, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Multiple Displacement Amplification, Incubation, Flow Cytometry, Fluorescence, Staining

    H19 promoted COM crystal-induced renal tubular epithelial cell oxidative stress injury in vitro . Western blot (a) and qRT-PCR (b) analyses of HMGB1, TLR4, NF-kB and p-NF-kB expression in HK-2 cells. β-Actin was used for normalization. SOD level (c), LDH release (d), MDA level (e), and H 2 O 2 concentration (f) were determined in HK-2 cells incubated with COM crystals following lenti-H19 and si-H19 treatment. (g) Cellular ROS production in HK-2 cells was measured by flow cytometry. (h) Histograms showing the mean fluorescence intensity of DCFH. (i) The apoptosis effect was investigated by flow cytometric analysis of HK-2 cells stained with Annexin V-FITC and propidium iodide. (* P

    Journal: EBioMedicine

    Article Title: H19 promote calcium oxalate nephrocalcinosis-induced renal tubular epithelial cell injury via a ceRNA pathway

    doi: 10.1016/j.ebiom.2019.10.059

    Figure Lengend Snippet: H19 promoted COM crystal-induced renal tubular epithelial cell oxidative stress injury in vitro . Western blot (a) and qRT-PCR (b) analyses of HMGB1, TLR4, NF-kB and p-NF-kB expression in HK-2 cells. β-Actin was used for normalization. SOD level (c), LDH release (d), MDA level (e), and H 2 O 2 concentration (f) were determined in HK-2 cells incubated with COM crystals following lenti-H19 and si-H19 treatment. (g) Cellular ROS production in HK-2 cells was measured by flow cytometry. (h) Histograms showing the mean fluorescence intensity of DCFH. (i) The apoptosis effect was investigated by flow cytometric analysis of HK-2 cells stained with Annexin V-FITC and propidium iodide. (* P

    Article Snippet: The quantitative detection of mature miRNA was conducted with an All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia). cDNA was obtained by reverse transcription of the total RNA.

    Techniques: In Vitro, Western Blot, Quantitative RT-PCR, Expressing, Multiple Displacement Amplification, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Staining

    miR-216b inhibited HMGB1 expression by directly binding to its 3′-UTR. Schematic diagram of our constructed luciferase reporter plasmid psiCHECK-2 containing the 3′-UTR of HMGB1. (b) Mutant and WT seed sequences of miR-216b targeting the 3′-UTR of HMGB1. (c) Luciferase reporters harbouring putative target sites in the WT and mutant 3′-UTRs of HMGB1 were cotransfected with 100 nM of the indicated small RNA molecules in HK-2 cells. (d) Pearson correlation coefficient analysis of the expression levels of H19 and miR-216b. (e) Western blot analysis was performed to detect the expression of HMGB1, TLR4, NF-kB and p-NF-kB in HK-2 cells transfected with the miR-216b mimics or inhibitor. Actin-β served as an internal control. (f) miR-216b mimics and inhibitor were used to establish miR-216b overexpression and inhibition, respectively, in HK-2 cells. The expression of HMGB1 (g), TLR4 (h), and NF-kB (i) was detected using qRT-PCR in HK-2 cells transfected with miR-216b mimics or inhibitor. The data are shown as the mean ± SD of three independent experiments. (* P

    Journal: EBioMedicine

    Article Title: H19 promote calcium oxalate nephrocalcinosis-induced renal tubular epithelial cell injury via a ceRNA pathway

    doi: 10.1016/j.ebiom.2019.10.059

    Figure Lengend Snippet: miR-216b inhibited HMGB1 expression by directly binding to its 3′-UTR. Schematic diagram of our constructed luciferase reporter plasmid psiCHECK-2 containing the 3′-UTR of HMGB1. (b) Mutant and WT seed sequences of miR-216b targeting the 3′-UTR of HMGB1. (c) Luciferase reporters harbouring putative target sites in the WT and mutant 3′-UTRs of HMGB1 were cotransfected with 100 nM of the indicated small RNA molecules in HK-2 cells. (d) Pearson correlation coefficient analysis of the expression levels of H19 and miR-216b. (e) Western blot analysis was performed to detect the expression of HMGB1, TLR4, NF-kB and p-NF-kB in HK-2 cells transfected with the miR-216b mimics or inhibitor. Actin-β served as an internal control. (f) miR-216b mimics and inhibitor were used to establish miR-216b overexpression and inhibition, respectively, in HK-2 cells. The expression of HMGB1 (g), TLR4 (h), and NF-kB (i) was detected using qRT-PCR in HK-2 cells transfected with miR-216b mimics or inhibitor. The data are shown as the mean ± SD of three independent experiments. (* P

    Article Snippet: The quantitative detection of mature miRNA was conducted with an All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia). cDNA was obtained by reverse transcription of the total RNA.

    Techniques: Expressing, Binding Assay, Construct, Luciferase, Plasmid Preparation, Mutagenesis, Western Blot, Transfection, Over Expression, Inhibition, Quantitative RT-PCR

    H19 interacted with miR-216b by directly binding its 3′-UTR. Schematic diagram of the mutant and WT seed sequences of miR-216b targeting the 3′-UTR of H19. (b) qRT-PCR analysis was performed to detect the expression level of miR-216b in CaOx-induced mouse kidney samples, and this expression was compared with that in the mock control. U6 was used as a miRNA control. (c) Pearson correlation coefficient analysis of the expression levels of H19 and miR-216b. (d) miR-216b expression was inhibited by lenti-H19 transfection. (e) H19 expression was detected using qRT-PCR in HK-2 cells transfected with the miR-216b mimics and inhibitor. Luciferase reporters harbouring putative target sites in the WT and mutant 3′-UTR of H19 were cotransfected with 100 nM miR-216b mimics (f) or miR-216b inhibitor (g) in HK-2 cells. The data are shown as the mean ± SD of three independent experiments. (* P

    Journal: EBioMedicine

    Article Title: H19 promote calcium oxalate nephrocalcinosis-induced renal tubular epithelial cell injury via a ceRNA pathway

    doi: 10.1016/j.ebiom.2019.10.059

    Figure Lengend Snippet: H19 interacted with miR-216b by directly binding its 3′-UTR. Schematic diagram of the mutant and WT seed sequences of miR-216b targeting the 3′-UTR of H19. (b) qRT-PCR analysis was performed to detect the expression level of miR-216b in CaOx-induced mouse kidney samples, and this expression was compared with that in the mock control. U6 was used as a miRNA control. (c) Pearson correlation coefficient analysis of the expression levels of H19 and miR-216b. (d) miR-216b expression was inhibited by lenti-H19 transfection. (e) H19 expression was detected using qRT-PCR in HK-2 cells transfected with the miR-216b mimics and inhibitor. Luciferase reporters harbouring putative target sites in the WT and mutant 3′-UTR of H19 were cotransfected with 100 nM miR-216b mimics (f) or miR-216b inhibitor (g) in HK-2 cells. The data are shown as the mean ± SD of three independent experiments. (* P

    Article Snippet: The quantitative detection of mature miRNA was conducted with an All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia). cDNA was obtained by reverse transcription of the total RNA.

    Techniques: Binding Assay, Mutagenesis, Quantitative RT-PCR, Expressing, Transfection, Luciferase