glyceraldehyde 3 phosphate dehydrogenase gapdh Thermo Fisher Search Results


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    Thermo Fisher gene exp gapdh hs99999905 m1
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    Oleacein affects the acetylome but not the methylome of MM cells. ( A ) Global DNA methylation was measured in MM cells treated for 24 h with oleacein, as reported in materials and methods. Quantitative Real Time PCR (QRT-PCR) ( B ) and WB analysis ( C ) of DNMT1, DNMT3A and DNMT3B in JJN3 cells treated for 24 h with oleacein; <t>GAPDH</t> was used as loading control. WB analysis of acetylated histone H3, histone H3, acetylated histone H4, histone H4 ( D ) and acetylated α-tubulin ( E ) in NCI-H929 and JJN3 cells treated with oleacein for 24 h; GAPDH was used as loading control.
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    Oleacein affects the acetylome but not the methylome of MM cells. ( A ) Global DNA methylation was measured in MM cells treated for 24 h with oleacein, as reported in materials and methods. Quantitative Real Time PCR (QRT-PCR) ( B ) and WB analysis ( C ) of DNMT1, DNMT3A and DNMT3B in JJN3 cells treated for 24 h with oleacein; <t>GAPDH</t> was used as loading control. WB analysis of acetylated histone H3, histone H3, acetylated histone H4, histone H4 ( D ) and acetylated α-tubulin ( E ) in NCI-H929 and JJN3 cells treated with oleacein for 24 h; GAPDH was used as loading control.
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    Effect of RBE on expression of MSC markers after rMSCs were cultured for 3 days. Expression levels of MSC markers were determined by real-time RT-RCR and normalized to the abundance of <t>GAPDH</t> . The culture conditions were: α-MEM containing
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    Effect of RBE on expression of MSC markers after rMSCs were cultured for 3 days. Expression levels of MSC markers were determined by real-time RT-RCR and normalized to the abundance of <t>GAPDH</t> . The culture conditions were: α-MEM containing
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    Effect of RBE on expression of MSC markers after rMSCs were cultured for 3 days. Expression levels of MSC markers were determined by real-time RT-RCR and normalized to the abundance of <t>GAPDH</t> . The culture conditions were: α-MEM containing
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    Effect of RBE on expression of MSC markers after rMSCs were cultured for 3 days. Expression levels of MSC markers were determined by real-time RT-RCR and normalized to the abundance of <t>GAPDH</t> . The culture conditions were: α-MEM containing
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    Effect of RBE on expression of MSC markers after rMSCs were cultured for 3 days. Expression levels of MSC markers were determined by real-time RT-RCR and normalized to the abundance of <t>GAPDH</t> . The culture conditions were: α-MEM containing
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    Effect of RBE on expression of MSC markers after rMSCs were cultured for 3 days. Expression levels of MSC markers were determined by real-time RT-RCR and normalized to the abundance of <t>GAPDH</t> . The culture conditions were: α-MEM containing
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    Treatment of high-fat-diet–fed mice with GalNAc- Stk25 ASO markedly reduced hepatic STK25 levels. Mice were treated with GalNAc- Stk25 ASO (12.5 mg/kg/wk), Stk25 ASO (50 mg/kg/wk), GalNAc-control ASO (12.5 mg/kg/wk), or placebo (PBS) for 6 weeks. ( A ) Hepatic Stk25 mRNA expression. ( B–D ) STK25 protein abundance in the ( B ) liver, ( C ) skeletal muscle, and ( D ) WAT. Protein levels were analyzed by densitometry; representative Western blots are shown with pan-actin, <t>glyceraldehyde-3-phosphate</t> dehydrogenase (GAPDH), or β-actin used as loading controls. ( E ) Representative liver sections processed for immunofluorescence with anti-STK25 antibody (red); nuclei were stained with DAPI (blue). Scale bars : 20 μm. ( A–D ) Data are means ± SD (n = 6–8 mice per group) compared with a control group of mice dosed with PBS. Cntr, control. * P
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    Treatment of high-fat-diet–fed mice with GalNAc- Stk25 ASO markedly reduced hepatic STK25 levels. Mice were treated with GalNAc- Stk25 ASO (12.5 mg/kg/wk), Stk25 ASO (50 mg/kg/wk), GalNAc-control ASO (12.5 mg/kg/wk), or placebo (PBS) for 6 weeks. ( A ) Hepatic Stk25 mRNA expression. ( B–D ) STK25 protein abundance in the ( B ) liver, ( C ) skeletal muscle, and ( D ) WAT. Protein levels were analyzed by densitometry; representative Western blots are shown with pan-actin, <t>glyceraldehyde-3-phosphate</t> dehydrogenase (GAPDH), or β-actin used as loading controls. ( E ) Representative liver sections processed for immunofluorescence with anti-STK25 antibody (red); nuclei were stained with DAPI (blue). Scale bars : 20 μm. ( A–D ) Data are means ± SD (n = 6–8 mice per group) compared with a control group of mice dosed with PBS. Cntr, control. * P
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    Treatment of high-fat-diet–fed mice with GalNAc- Stk25 ASO markedly reduced hepatic STK25 levels. Mice were treated with GalNAc- Stk25 ASO (12.5 mg/kg/wk), Stk25 ASO (50 mg/kg/wk), GalNAc-control ASO (12.5 mg/kg/wk), or placebo (PBS) for 6 weeks. ( A ) Hepatic Stk25 mRNA expression. ( B–D ) STK25 protein abundance in the ( B ) liver, ( C ) skeletal muscle, and ( D ) WAT. Protein levels were analyzed by densitometry; representative Western blots are shown with pan-actin, <t>glyceraldehyde-3-phosphate</t> dehydrogenase (GAPDH), or β-actin used as loading controls. ( E ) Representative liver sections processed for immunofluorescence with anti-STK25 antibody (red); nuclei were stained with DAPI (blue). Scale bars : 20 μm. ( A–D ) Data are means ± SD (n = 6–8 mice per group) compared with a control group of mice dosed with PBS. Cntr, control. * P
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    HTT mRNA knock-down induced by AAV5-miHTT-451 and AAV5-miSNP50T-451 vectors in the striatum of HD rats. ( a ) The structure and sequence of the hsa-pre-miR-451a precursor used in this study with the miRBase-predicted guide strand highlighted in pink ( www.mirbase.org ). ( b ) Schematic representation of the AAV5-miHTT-451 and AAV5-miSNP50T-451 expression cassettes; and LV-mtHTT-50C and LV-mtHTT-50T encoding a chimeric mutant HTT sequence with either C or T isoform of SNP rs362331. ( c ) Bilateral co-injections in the striatum (STR) of rats with LV-mtHTT-50C or LV-mtHTT-50T, and AAV5-miHTT-451 or AAV5-miSNP50T-451 vectors. The experimental groups and injection sites are outlined. ( d ) qPCR to determine AAV5 genome copies (gc) in the striatum of AAV5-miHTT-451 and AAV5-miSNP50T-451 injected rats ( n =3), two months post-injection. Primers directed to the CAG promoter were used and the gc values were calculated based on the standard curve and considering the background signal from the negative control. ( e ) TaqMan qPCR assay shows HTT mRNA knock-down in the striatum ( n =2-3) induced by the AAV5-miHTT-451 and AAV5-miSNP50T-451 expression products. Human HTT-specific exon-spanning primers were used and HTT values were subsequently normalized to <t>GAPDH,</t> an internal control set at 100%. All data were analyzed using one-way ANOVA. * P ⩽0.05; ** P ⩽0.01; *** P ⩽0.001; **** P ⩽0.0001. The values were calculated as a mean±s.d.
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    HTT mRNA knock-down induced by AAV5-miHTT-451 and AAV5-miSNP50T-451 vectors in the striatum of HD rats. ( a ) The structure and sequence of the hsa-pre-miR-451a precursor used in this study with the miRBase-predicted guide strand highlighted in pink ( www.mirbase.org ). ( b ) Schematic representation of the AAV5-miHTT-451 and AAV5-miSNP50T-451 expression cassettes; and LV-mtHTT-50C and LV-mtHTT-50T encoding a chimeric mutant HTT sequence with either C or T isoform of SNP rs362331. ( c ) Bilateral co-injections in the striatum (STR) of rats with LV-mtHTT-50C or LV-mtHTT-50T, and AAV5-miHTT-451 or AAV5-miSNP50T-451 vectors. The experimental groups and injection sites are outlined. ( d ) qPCR to determine AAV5 genome copies (gc) in the striatum of AAV5-miHTT-451 and AAV5-miSNP50T-451 injected rats ( n =3), two months post-injection. Primers directed to the CAG promoter were used and the gc values were calculated based on the standard curve and considering the background signal from the negative control. ( e ) TaqMan qPCR assay shows HTT mRNA knock-down in the striatum ( n =2-3) induced by the AAV5-miHTT-451 and AAV5-miSNP50T-451 expression products. Human HTT-specific exon-spanning primers were used and HTT values were subsequently normalized to <t>GAPDH,</t> an internal control set at 100%. All data were analyzed using one-way ANOVA. * P ⩽0.05; ** P ⩽0.01; *** P ⩽0.001; **** P ⩽0.0001. The values were calculated as a mean±s.d.
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    Effects of immunosuppression on the expression of different immune response markers in LPMC. LPMC where isolated from the caecal mucosa of control (ctrl) and immunosuppressed (IS) rabbits. Expression of selected mRNAs was determined by rt-qPCR relative to <t>GAPDH</t> as housekeeping gene. Bars represent Mean ± SEM from two pooled experiments. n = 2–4 animals per group. *P
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    Effects of immunosuppression on the expression of different immune response markers in LPMC. LPMC where isolated from the caecal mucosa of control (ctrl) and immunosuppressed (IS) rabbits. Expression of selected mRNAs was determined by rt-qPCR relative to <t>GAPDH</t> as housekeeping gene. Bars represent Mean ± SEM from two pooled experiments. n = 2–4 animals per group. *P
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    Effects of immunosuppression on the expression of different immune response markers in LPMC. LPMC where isolated from the caecal mucosa of control (ctrl) and immunosuppressed (IS) rabbits. Expression of selected mRNAs was determined by rt-qPCR relative to <t>GAPDH</t> as housekeeping gene. Bars represent Mean ± SEM from two pooled experiments. n = 2–4 animals per group. *P
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    BNIP-2 knockdown suppresses RhoA activity and retards cell polarization during early cell spreading. ( A ) Endogenous RhoA activity of MDA-MB-231 cells was examined in the presence of different amounts of BNIP-2. Lysates of MDA-MB-231 cells transfected with BNIP-2–targeting siRNA (siBNIP-2) or transiently expressing gradually increasing amount of GFP-BNIP-2 (illustrated by blue triangle) were used for pulldown with immobilized glutathione S -transferase (GST)–RBD (GST-RBD) of rhotekin and then Western blotted with RhoA, BNIP-2, and <t>glyceraldehyde-3-phosphate</t> dehydrogenase (GAPDH) antibodies. Blue arrow denotes the GFP-BNIP-2, while purple arrow denotes the endogenous BNIP-2. The ratio of active RhoA to total RhoA is normalized to untransfected in lane 2 and labeled at the bottom. ( B ) Snapshots of shVector control and BNIP-2 knockdown cells during spreading on collagen I–coated plastic-bottom plates. Representative cells spreading at 0, 20, 40, and 54 min are displayed. ( C ) Cell aspect ratios are quantified and plotted for control and BNIP-2 knockdown cells during 60-min spreading. Data are means ± SEM of two independent experiments.
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    Image Search Results


    Oleacein affects the acetylome but not the methylome of MM cells. ( A ) Global DNA methylation was measured in MM cells treated for 24 h with oleacein, as reported in materials and methods. Quantitative Real Time PCR (QRT-PCR) ( B ) and WB analysis ( C ) of DNMT1, DNMT3A and DNMT3B in JJN3 cells treated for 24 h with oleacein; GAPDH was used as loading control. WB analysis of acetylated histone H3, histone H3, acetylated histone H4, histone H4 ( D ) and acetylated α-tubulin ( E ) in NCI-H929 and JJN3 cells treated with oleacein for 24 h; GAPDH was used as loading control.

    Journal: Cancers

    Article Title: Anti-tumor Activity and Epigenetic Impact of the Polyphenol Oleacein in Multiple Myeloma

    doi: 10.3390/cancers11070990

    Figure Lengend Snippet: Oleacein affects the acetylome but not the methylome of MM cells. ( A ) Global DNA methylation was measured in MM cells treated for 24 h with oleacein, as reported in materials and methods. Quantitative Real Time PCR (QRT-PCR) ( B ) and WB analysis ( C ) of DNMT1, DNMT3A and DNMT3B in JJN3 cells treated for 24 h with oleacein; GAPDH was used as loading control. WB analysis of acetylated histone H3, histone H3, acetylated histone H4, histone H4 ( D ) and acetylated α-tubulin ( E ) in NCI-H929 and JJN3 cells treated with oleacein for 24 h; GAPDH was used as loading control.

    Article Snippet: The following single-tube TaqMan assays (Applied Biosystems, Carlsbad, CA, USA) were used to detect and quantify genes using the Viia7 DX real time PCR instrument (Life Technologies, Waltham, MA, USA): DNMT1 (Hs00154749_m1), DNMT3a (Hs01027166_m1), DNMT3b (Hs00171876_m1), HDAC1 (Hs02621185_s1), HDAC2 (Hs00231032_m1), HDAC3 (Hs00187320_m1), HDAC4 (Hs01041638_m1), HDAC6 (Hs00195869_m1), and GAPDH (Hs02786624 g1). miRNA expression levels were determined by TaqMan RT-PCR, using the single-tube TaqMan miRNA assays (hsa-miR-29b, assay ID 000413; hsa-miR-22, assay ID 000398, Applied Biosystems) to quantify mature miRNAs, by the use of the StepOne Thermocycler (Thermo Fisher Scientific, Waltham, MA, USA) and the sequence detection system, as previously reported [ ]; miRNAs expression levels were normalized on RNU44 (assay ID 001094).

    Techniques: DNA Methylation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

    Oleacein targets HDACs. QRT-PCR ( A ) and WB analysis ( B ) of HDAC1, HDAC2, HDAC3, HDAC4, HDAC6 in JJN3 cells treated with oleacein for 24 h; GAPDH was used as loading control. ( C ) WB analysis of HDAC4 and HDAC6 in nuclear (N) and cytoplasmic (C) protein fractions from JJN3 cells treated for 24 h with oleacein; histone H1 and GAPDH were used as nuclear and cytoplasmic marker, respectively. ( D ) WB analysis of Sp1 in JJN3 cells treated with oleacein for 24 h. ( E ) WB analysis of Sp1 in JJN3 cells treated with 5.0 µM oleacein with or without 20.0 µM Z-ITED-FMK; GAPDH was used as loading control. * p

    Journal: Cancers

    Article Title: Anti-tumor Activity and Epigenetic Impact of the Polyphenol Oleacein in Multiple Myeloma

    doi: 10.3390/cancers11070990

    Figure Lengend Snippet: Oleacein targets HDACs. QRT-PCR ( A ) and WB analysis ( B ) of HDAC1, HDAC2, HDAC3, HDAC4, HDAC6 in JJN3 cells treated with oleacein for 24 h; GAPDH was used as loading control. ( C ) WB analysis of HDAC4 and HDAC6 in nuclear (N) and cytoplasmic (C) protein fractions from JJN3 cells treated for 24 h with oleacein; histone H1 and GAPDH were used as nuclear and cytoplasmic marker, respectively. ( D ) WB analysis of Sp1 in JJN3 cells treated with oleacein for 24 h. ( E ) WB analysis of Sp1 in JJN3 cells treated with 5.0 µM oleacein with or without 20.0 µM Z-ITED-FMK; GAPDH was used as loading control. * p

    Article Snippet: The following single-tube TaqMan assays (Applied Biosystems, Carlsbad, CA, USA) were used to detect and quantify genes using the Viia7 DX real time PCR instrument (Life Technologies, Waltham, MA, USA): DNMT1 (Hs00154749_m1), DNMT3a (Hs01027166_m1), DNMT3b (Hs00171876_m1), HDAC1 (Hs02621185_s1), HDAC2 (Hs00231032_m1), HDAC3 (Hs00187320_m1), HDAC4 (Hs01041638_m1), HDAC6 (Hs00195869_m1), and GAPDH (Hs02786624 g1). miRNA expression levels were determined by TaqMan RT-PCR, using the single-tube TaqMan miRNA assays (hsa-miR-29b, assay ID 000413; hsa-miR-22, assay ID 000398, Applied Biosystems) to quantify mature miRNAs, by the use of the StepOne Thermocycler (Thermo Fisher Scientific, Waltham, MA, USA) and the sequence detection system, as previously reported [ ]; miRNAs expression levels were normalized on RNU44 (assay ID 001094).

    Techniques: Quantitative RT-PCR, Western Blot, Marker

    Oleacein enhances the anti-MM activity of carfilzomib. ( A ) CTG assay was performed on NCI-H929 cells treated with oleacein (2.5, 5.0 or 10.0 µM) and carfilzomib (0.1, 0.5 and 1.0 nM). Results are expressed as percentage of the viability of vehicle-treated cells. The right panel reports values of fraction affected (Fa) and combination indexes (CI) in a triplicate experiment, as calculated by the Calcusyn software. ( B ) Annexin V/7-AAD staining of NCI-H929 cells after treatment with vehicle (DMSO), 5.0 µM oleacein and 1.0 nM carfilzomib for 24 h; a representative FACS experiment is reported. ( C ). WB analysis of pro-Caspase 3, cleaved caspase 3, SP1, HDAC2, HDAC3, HDAC4, HDAC6, and acetylated histone H4 in NCI-H929 cells treated with carfilzomib (1.0 nM), oleacein (5.0 µM) or a combination of the two; α-tubulin or GAPDH were used as loading controls.

    Journal: Cancers

    Article Title: Anti-tumor Activity and Epigenetic Impact of the Polyphenol Oleacein in Multiple Myeloma

    doi: 10.3390/cancers11070990

    Figure Lengend Snippet: Oleacein enhances the anti-MM activity of carfilzomib. ( A ) CTG assay was performed on NCI-H929 cells treated with oleacein (2.5, 5.0 or 10.0 µM) and carfilzomib (0.1, 0.5 and 1.0 nM). Results are expressed as percentage of the viability of vehicle-treated cells. The right panel reports values of fraction affected (Fa) and combination indexes (CI) in a triplicate experiment, as calculated by the Calcusyn software. ( B ) Annexin V/7-AAD staining of NCI-H929 cells after treatment with vehicle (DMSO), 5.0 µM oleacein and 1.0 nM carfilzomib for 24 h; a representative FACS experiment is reported. ( C ). WB analysis of pro-Caspase 3, cleaved caspase 3, SP1, HDAC2, HDAC3, HDAC4, HDAC6, and acetylated histone H4 in NCI-H929 cells treated with carfilzomib (1.0 nM), oleacein (5.0 µM) or a combination of the two; α-tubulin or GAPDH were used as loading controls.

    Article Snippet: The following single-tube TaqMan assays (Applied Biosystems, Carlsbad, CA, USA) were used to detect and quantify genes using the Viia7 DX real time PCR instrument (Life Technologies, Waltham, MA, USA): DNMT1 (Hs00154749_m1), DNMT3a (Hs01027166_m1), DNMT3b (Hs00171876_m1), HDAC1 (Hs02621185_s1), HDAC2 (Hs00231032_m1), HDAC3 (Hs00187320_m1), HDAC4 (Hs01041638_m1), HDAC6 (Hs00195869_m1), and GAPDH (Hs02786624 g1). miRNA expression levels were determined by TaqMan RT-PCR, using the single-tube TaqMan miRNA assays (hsa-miR-29b, assay ID 000413; hsa-miR-22, assay ID 000398, Applied Biosystems) to quantify mature miRNAs, by the use of the StepOne Thermocycler (Thermo Fisher Scientific, Waltham, MA, USA) and the sequence detection system, as previously reported [ ]; miRNAs expression levels were normalized on RNU44 (assay ID 001094).

    Techniques: Activity Assay, CTG Assay, Software, Staining, FACS, Western Blot

    Oleacein triggers cell cycle blockade and apoptosis. ( A ) Cell cycle analysis was performed on NCI-H929 cells by PI staining, 24 h after treatment with oleacein or vehicle (DMSO). ( B )Western Blot (WB) analysis of p27 KIP1 and p21 CIP1 in whole cell lysates from MM cells after treatment with oleacein for 24 h; actin was used as loading control. ( C ) Annexin V/7-AAD staining of MM cells after treatment with oleacein for 48 h; a representative experiment on NCI-H929 cells is shown on the left side. ( D ) WB of PARP1, cleaved caspase-3 and cleaved caspase-8 in NCI-H929 and JJN3 cell lines after 24 h of oleacein treatment; GAPDH was used as loading control. * p

    Journal: Cancers

    Article Title: Anti-tumor Activity and Epigenetic Impact of the Polyphenol Oleacein in Multiple Myeloma

    doi: 10.3390/cancers11070990

    Figure Lengend Snippet: Oleacein triggers cell cycle blockade and apoptosis. ( A ) Cell cycle analysis was performed on NCI-H929 cells by PI staining, 24 h after treatment with oleacein or vehicle (DMSO). ( B )Western Blot (WB) analysis of p27 KIP1 and p21 CIP1 in whole cell lysates from MM cells after treatment with oleacein for 24 h; actin was used as loading control. ( C ) Annexin V/7-AAD staining of MM cells after treatment with oleacein for 48 h; a representative experiment on NCI-H929 cells is shown on the left side. ( D ) WB of PARP1, cleaved caspase-3 and cleaved caspase-8 in NCI-H929 and JJN3 cell lines after 24 h of oleacein treatment; GAPDH was used as loading control. * p

    Article Snippet: The following single-tube TaqMan assays (Applied Biosystems, Carlsbad, CA, USA) were used to detect and quantify genes using the Viia7 DX real time PCR instrument (Life Technologies, Waltham, MA, USA): DNMT1 (Hs00154749_m1), DNMT3a (Hs01027166_m1), DNMT3b (Hs00171876_m1), HDAC1 (Hs02621185_s1), HDAC2 (Hs00231032_m1), HDAC3 (Hs00187320_m1), HDAC4 (Hs01041638_m1), HDAC6 (Hs00195869_m1), and GAPDH (Hs02786624 g1). miRNA expression levels were determined by TaqMan RT-PCR, using the single-tube TaqMan miRNA assays (hsa-miR-29b, assay ID 000413; hsa-miR-22, assay ID 000398, Applied Biosystems) to quantify mature miRNAs, by the use of the StepOne Thermocycler (Thermo Fisher Scientific, Waltham, MA, USA) and the sequence detection system, as previously reported [ ]; miRNAs expression levels were normalized on RNU44 (assay ID 001094).

    Techniques: Cell Cycle Assay, Staining, Western Blot

    Effect of RBE on expression of MSC markers after rMSCs were cultured for 3 days. Expression levels of MSC markers were determined by real-time RT-RCR and normalized to the abundance of GAPDH . The culture conditions were: α-MEM containing

    Journal: Cytotechnology

    Article Title: Rice bran extract affects differentiation of mesenchymal stem cells potency into osteogenic cells

    doi: 10.1007/s10616-013-9570-6

    Figure Lengend Snippet: Effect of RBE on expression of MSC markers after rMSCs were cultured for 3 days. Expression levels of MSC markers were determined by real-time RT-RCR and normalized to the abundance of GAPDH . The culture conditions were: α-MEM containing

    Article Snippet: Real time PCR was performed using the following primers and probes: CD44 Rn 00681157_m1, CD105 Rn0143378772_m1, CD166 Rn 00582112_m1, GAPDH Rn 99999916_sl (Applied Biosystems, Calrsbad, CA, USA) (for more details, see supplementary table 1).

    Techniques: Expressing, Cell Culture

    Treatment of high-fat-diet–fed mice with GalNAc- Stk25 ASO markedly reduced hepatic STK25 levels. Mice were treated with GalNAc- Stk25 ASO (12.5 mg/kg/wk), Stk25 ASO (50 mg/kg/wk), GalNAc-control ASO (12.5 mg/kg/wk), or placebo (PBS) for 6 weeks. ( A ) Hepatic Stk25 mRNA expression. ( B–D ) STK25 protein abundance in the ( B ) liver, ( C ) skeletal muscle, and ( D ) WAT. Protein levels were analyzed by densitometry; representative Western blots are shown with pan-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or β-actin used as loading controls. ( E ) Representative liver sections processed for immunofluorescence with anti-STK25 antibody (red); nuclei were stained with DAPI (blue). Scale bars : 20 μm. ( A–D ) Data are means ± SD (n = 6–8 mice per group) compared with a control group of mice dosed with PBS. Cntr, control. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Targeted Delivery of Stk25 Antisense Oligonucleotides to Hepatocytes Protects Mice Against Nonalcoholic Fatty Liver Disease

    doi: 10.1016/j.jcmgh.2018.12.004

    Figure Lengend Snippet: Treatment of high-fat-diet–fed mice with GalNAc- Stk25 ASO markedly reduced hepatic STK25 levels. Mice were treated with GalNAc- Stk25 ASO (12.5 mg/kg/wk), Stk25 ASO (50 mg/kg/wk), GalNAc-control ASO (12.5 mg/kg/wk), or placebo (PBS) for 6 weeks. ( A ) Hepatic Stk25 mRNA expression. ( B–D ) STK25 protein abundance in the ( B ) liver, ( C ) skeletal muscle, and ( D ) WAT. Protein levels were analyzed by densitometry; representative Western blots are shown with pan-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or β-actin used as loading controls. ( E ) Representative liver sections processed for immunofluorescence with anti-STK25 antibody (red); nuclei were stained with DAPI (blue). Scale bars : 20 μm. ( A–D ) Data are means ± SD (n = 6–8 mice per group) compared with a control group of mice dosed with PBS. Cntr, control. * P

    Article Snippet: The detection of specific proteins used the following primary antibodies: anti-STK25, anti-PCNA, anti-Ki67, anti-ACC (#3662; Cell Signaling Technology, Boston, MA), anti–phospho-ACC (Ser79; #3661; Cell Signaling Technology), anti–phospho-threonine (13-9200; Invitrogen), anti–pan-actin (sc-8432; Santa Cruz Biotechnology), anti–glyceraldehyde-3-phosphate-dehydrogenase (MA5-15738; Invitrogen), anti–β-actin (ab8226; Abcam), and horseradish-peroxidase–conjugated secondary antibodies anti-rabbit IgG (#7074; Cell Signaling Technology), anti-mouse IgG (#7076; Cell Signaling Technology), anti-rat IgG (#7077; Cell signaling Technology), and VeriBlot for IP Detection Reagent (ab131366; Abcam).

    Techniques: Mouse Assay, Allele-specific Oligonucleotide, Expressing, Western Blot, Immunofluorescence, Staining

    HTT mRNA knock-down induced by AAV5-miHTT-451 and AAV5-miSNP50T-451 vectors in the striatum of HD rats. ( a ) The structure and sequence of the hsa-pre-miR-451a precursor used in this study with the miRBase-predicted guide strand highlighted in pink ( www.mirbase.org ). ( b ) Schematic representation of the AAV5-miHTT-451 and AAV5-miSNP50T-451 expression cassettes; and LV-mtHTT-50C and LV-mtHTT-50T encoding a chimeric mutant HTT sequence with either C or T isoform of SNP rs362331. ( c ) Bilateral co-injections in the striatum (STR) of rats with LV-mtHTT-50C or LV-mtHTT-50T, and AAV5-miHTT-451 or AAV5-miSNP50T-451 vectors. The experimental groups and injection sites are outlined. ( d ) qPCR to determine AAV5 genome copies (gc) in the striatum of AAV5-miHTT-451 and AAV5-miSNP50T-451 injected rats ( n =3), two months post-injection. Primers directed to the CAG promoter were used and the gc values were calculated based on the standard curve and considering the background signal from the negative control. ( e ) TaqMan qPCR assay shows HTT mRNA knock-down in the striatum ( n =2-3) induced by the AAV5-miHTT-451 and AAV5-miSNP50T-451 expression products. Human HTT-specific exon-spanning primers were used and HTT values were subsequently normalized to GAPDH, an internal control set at 100%. All data were analyzed using one-way ANOVA. * P ⩽0.05; ** P ⩽0.01; *** P ⩽0.001; **** P ⩽0.0001. The values were calculated as a mean±s.d.

    Journal: Gene Therapy

    Article Title: AAV5-miHTT gene therapy demonstrates suppression of mutant huntingtin aggregation and neuronal dysfunction in a rat model of Huntington’s disease

    doi: 10.1038/gt.2017.71

    Figure Lengend Snippet: HTT mRNA knock-down induced by AAV5-miHTT-451 and AAV5-miSNP50T-451 vectors in the striatum of HD rats. ( a ) The structure and sequence of the hsa-pre-miR-451a precursor used in this study with the miRBase-predicted guide strand highlighted in pink ( www.mirbase.org ). ( b ) Schematic representation of the AAV5-miHTT-451 and AAV5-miSNP50T-451 expression cassettes; and LV-mtHTT-50C and LV-mtHTT-50T encoding a chimeric mutant HTT sequence with either C or T isoform of SNP rs362331. ( c ) Bilateral co-injections in the striatum (STR) of rats with LV-mtHTT-50C or LV-mtHTT-50T, and AAV5-miHTT-451 or AAV5-miSNP50T-451 vectors. The experimental groups and injection sites are outlined. ( d ) qPCR to determine AAV5 genome copies (gc) in the striatum of AAV5-miHTT-451 and AAV5-miSNP50T-451 injected rats ( n =3), two months post-injection. Primers directed to the CAG promoter were used and the gc values were calculated based on the standard curve and considering the background signal from the negative control. ( e ) TaqMan qPCR assay shows HTT mRNA knock-down in the striatum ( n =2-3) induced by the AAV5-miHTT-451 and AAV5-miSNP50T-451 expression products. Human HTT-specific exon-spanning primers were used and HTT values were subsequently normalized to GAPDH, an internal control set at 100%. All data were analyzed using one-way ANOVA. * P ⩽0.05; ** P ⩽0.01; *** P ⩽0.001; **** P ⩽0.0001. The values were calculated as a mean±s.d.

    Article Snippet: Quantitative real-time PCR reaction was performed to detect HTT mRNA knock-down using TaqMan gene expression assays (Applied Biosystems, Foster City, CA, USA), human HTT (Hs00918134_m1), rat GAPDH (Rn01462662_g1); and miHTT expression levels using custom TaqMan microRNA expression assay or U6 snRNA (001973; Applied Biosystems).

    Techniques: Sequencing, Expressing, Mutagenesis, Injection, Real-time Polymerase Chain Reaction, Negative Control

    Effects of immunosuppression on the expression of different immune response markers in LPMC. LPMC where isolated from the caecal mucosa of control (ctrl) and immunosuppressed (IS) rabbits. Expression of selected mRNAs was determined by rt-qPCR relative to GAPDH as housekeeping gene. Bars represent Mean ± SEM from two pooled experiments. n = 2–4 animals per group. *P

    Journal: Scientific Reports

    Article Title: Preventive Trichuris suis ova (TSO) treatment protects immunocompetent rabbits from DSS colitis but may be detrimental under conditions of immunosuppression

    doi: 10.1038/s41598-017-16287-4

    Figure Lengend Snippet: Effects of immunosuppression on the expression of different immune response markers in LPMC. LPMC where isolated from the caecal mucosa of control (ctrl) and immunosuppressed (IS) rabbits. Expression of selected mRNAs was determined by rt-qPCR relative to GAPDH as housekeeping gene. Bars represent Mean ± SEM from two pooled experiments. n = 2–4 animals per group. *P

    Article Snippet: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression was measured as endogenous control (#Oc03823402_g1, Applied Biosystems) and used for calculation of relative mRNA expression by the ΔΔCt method.

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    BNIP-2 knockdown suppresses RhoA activity and retards cell polarization during early cell spreading. ( A ) Endogenous RhoA activity of MDA-MB-231 cells was examined in the presence of different amounts of BNIP-2. Lysates of MDA-MB-231 cells transfected with BNIP-2–targeting siRNA (siBNIP-2) or transiently expressing gradually increasing amount of GFP-BNIP-2 (illustrated by blue triangle) were used for pulldown with immobilized glutathione S -transferase (GST)–RBD (GST-RBD) of rhotekin and then Western blotted with RhoA, BNIP-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. Blue arrow denotes the GFP-BNIP-2, while purple arrow denotes the endogenous BNIP-2. The ratio of active RhoA to total RhoA is normalized to untransfected in lane 2 and labeled at the bottom. ( B ) Snapshots of shVector control and BNIP-2 knockdown cells during spreading on collagen I–coated plastic-bottom plates. Representative cells spreading at 0, 20, 40, and 54 min are displayed. ( C ) Cell aspect ratios are quantified and plotted for control and BNIP-2 knockdown cells during 60-min spreading. Data are means ± SEM of two independent experiments.

    Journal: Science Advances

    Article Title: BNIP-2 retards breast cancer cell migration by coupling microtubule-mediated GEF-H1 and RhoA activation

    doi: 10.1126/sciadv.aaz1534

    Figure Lengend Snippet: BNIP-2 knockdown suppresses RhoA activity and retards cell polarization during early cell spreading. ( A ) Endogenous RhoA activity of MDA-MB-231 cells was examined in the presence of different amounts of BNIP-2. Lysates of MDA-MB-231 cells transfected with BNIP-2–targeting siRNA (siBNIP-2) or transiently expressing gradually increasing amount of GFP-BNIP-2 (illustrated by blue triangle) were used for pulldown with immobilized glutathione S -transferase (GST)–RBD (GST-RBD) of rhotekin and then Western blotted with RhoA, BNIP-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. Blue arrow denotes the GFP-BNIP-2, while purple arrow denotes the endogenous BNIP-2. The ratio of active RhoA to total RhoA is normalized to untransfected in lane 2 and labeled at the bottom. ( B ) Snapshots of shVector control and BNIP-2 knockdown cells during spreading on collagen I–coated plastic-bottom plates. Representative cells spreading at 0, 20, 40, and 54 min are displayed. ( C ) Cell aspect ratios are quantified and plotted for control and BNIP-2 knockdown cells during 60-min spreading. Data are means ± SEM of two independent experiments.

    Article Snippet: Antibodies and chemicals For Western blot, primary antibody catalog numbers and dilution factors are as follows: polyclonal anti–BNIP-2 antibodies were purchased from Sigma-Aldrich (HPA026843) and GeneTex (GTX114283) or self-purified from rabbit serum, as previously described ( ); monoclonal anti–GEF-H1 (ab155785) was purchased from Abcam; monoclonal anti-RhoA was from Santa Cruz Biotechnology (sc-418); polyclonal anti–Phospho-Myosin Light Chain 2 (Ser19) antibody was from Cell Signaling Technology (#3671); monoclonal anti–α-tubulin was from Sigma-Aldrich (T9026); anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Life Technologies (Invitrogen); rabbit immunoglobulin G (IgG; sc-2027); and mouse IgG (sc-2025) were from Santa Cruz Biotechnology.

    Techniques: Activity Assay, Multiple Displacement Amplification, Transfection, Expressing, Western Blot, Labeling