Journal: Lipids in Health and Disease

Article Title: Overexpression of stearoyl-CoA desaturase 1 in bone marrow mesenchymal stem cells enhance the expression of induced endothelial cells

doi: 10.1186/1476-511X-13-53

Figure Lengend Snippet: Realtime PCR and activity results of SCD1. Realtime PCR and enzyme activity analysis results showed that the amount of SCD1 mRNA (a) and enzyme activity (b) of the SCD1 overexpressed group was higher than that of the EV group (p < 0.01).

Article Snippet: Using SCD1 gene purchased from OriGene Technologies (SC108809) as a template, fragments of the SCD1 gene were amplified (Figure a) using primers capped with BamHI and EcoRI recognition sequences (Table SCD1*).

Techniques: Activity Assay

Journal: Lipids in Health and Disease

Article Title: Overexpression of stearoyl-CoA desaturase 1 in bone marrow mesenchymal stem cells enhance the expression of induced endothelial cells

doi: 10.1186/1476-511X-13-53

Figure Lengend Snippet: Vector construction of SCD1 overexpression. (a) An electrophoresis map of the PCR products of SCD1. (b) An electrophoresis map of the PCR products of connected vector pCDH-SCD1. (c) Western blot of SCD1 showed that the concentration of SCD1 in the SCD1 overexpressed group is higher than that in the EV group.

Article Snippet: Using SCD1 gene purchased from OriGene Technologies (SC108809) as a template, fragments of the SCD1 gene were amplified (Figure a) using primers capped with BamHI and EcoRI recognition sequences (Table SCD1*).

Techniques: Plasmid Preparation, Over Expression, Electrophoresis, Western Blot, Concentration Assay

Journal: Lipids in Health and Disease

Article Title: Overexpression of stearoyl-CoA desaturase 1 in bone marrow mesenchymal stem cells enhance the expression of induced endothelial cells

doi: 10.1186/1476-511X-13-53

Figure Lengend Snippet: Results of fluorescent quantitative PCR (mean ± SD)

Article Snippet: Using SCD1 gene purchased from OriGene Technologies (SC108809) as a template, fragments of the SCD1 gene were amplified (Figure a) using primers capped with BamHI and EcoRI recognition sequences (Table SCD1*).

Techniques: Real-time Polymerase Chain Reaction

Journal: Lipids in Health and Disease

Article Title: Overexpression of stearoyl-CoA desaturase 1 in bone marrow mesenchymal stem cells enhance the expression of induced endothelial cells

doi: 10.1186/1476-511X-13-53

Figure Lengend Snippet: Realtime PCR results of CD31, vWF and VE-cad. Realtime PCR results showed that the mRNA amount of CD31, vWF and VE-cad of the SCD1 overexpressed group was higher than that of the EV group and that of the normal group after 1 week (a) and 2 weeks (b) (p < 0.05).

Article Snippet: Using SCD1 gene purchased from OriGene Technologies (SC108809) as a template, fragments of the SCD1 gene were amplified (Figure a) using primers capped with BamHI and EcoRI recognition sequences (Table SCD1*).

Techniques:

Journal: Lipids in Health and Disease

Article Title: Overexpression of stearoyl-CoA desaturase 1 in bone marrow mesenchymal stem cells enhance the expression of induced endothelial cells

doi: 10.1186/1476-511X-13-53

Figure Lengend Snippet: Immunostaining of CD31 and vWF. The MSCs was induced into endothelial cells which were also transfected with/without SCD1 gene or empty-vector. Immunocytochemical staining was performed using CD31 and vWF antibodies. Positive staining is visualized by red color for vWF (Figure a, b, c) or CD31 (Figure d, e, f) . a/d was referred to SCD1 overexpressed group, b/e was normal group, c/f was empty-vector group. Microscopic image showed that SCD1 overexpressed group (g) had more endothelial cells than normal group (h) and empty-vector group (i) .

Article Snippet: Using SCD1 gene purchased from OriGene Technologies (SC108809) as a template, fragments of the SCD1 gene were amplified (Figure a) using primers capped with BamHI and EcoRI recognition sequences (Table SCD1*).

Techniques: Immunostaining, Transfection, Plasmid Preparation, Staining

Journal: Lipids in Health and Disease

Article Title: Overexpression of stearoyl-CoA desaturase 1 in bone marrow mesenchymal stem cells enhance the expression of induced endothelial cells

doi: 10.1186/1476-511X-13-53

Figure Lengend Snippet: Primer sequences of SCD1*, β-actin and SCD1**

Article Snippet: Using SCD1 gene purchased from OriGene Technologies (SC108809) as a template, fragments of the SCD1 gene were amplified (Figure a) using primers capped with BamHI and EcoRI recognition sequences (Table SCD1*).

Techniques: Sequencing

Journal: Oncogene

Article Title: The anticancer gene ORCTL3 targets stearoyl-CoA desaturase-1 for tumour-specific apoptosis

doi: 10.1038/onc.2014.93

Figure Lengend Snippet: ( a ) H-ras and p53mut transformed CV-1 cells were transiently cotransfected with GFP and luciferase, ORCTL3, or caspase 2 in a 1:3 ratio and cultivated with either 100 μM BSA-conjugated oleic acid or BSA for 24 hours. Luciferase-control background was subtracted. Data are means ± SD (n=4). ( b ) The SCD1 inhibitor CAY10566 or equal volumes of DMSO were applied to WT as well as H-ras and p53mut transformed CV-1 cells and apoptosis was quantified after 48 hours by fluorescein diacetate (FDA) staining. ( c ) ORCTL3-HA was transfected into 293T cells (right, input). Cell lysates were immunoprecipitated with an α-HA antibody (left panels) that revealed multiple, probably glycosylated, forms of ORCTL3 or with an antibody against the endogenous SCD1 (middle panels). The immunoprecipitates were analysed using appropriate antibodies. ( d ) The desaturation index (18:1n-9/18:0) upon transfection of the indicated expression constructs into normal (top panel) and H-ras transfected (bottom panel) CV1 cells. ( e ) 293T cells were transfected with ORCTL3, RIP1 and ANT1 with either β-Gal or a plasmid expressing SCD1 in a 3:1 ratio. 48 hours post-transfection, apoptosis was assessed by staining the cells with DiOC 6 /PI and analysing by flow cytometry. Data represent the means ± SD (n=3). Raw data were normalised to transfection efficiency estimated by GFP. ( f ) Endogenous SCD1 mRNA level was semi-quantitatively determined in dublicates by RT-PCR in WT-, H-ras-, and P53-mut-transformed CV-1 cells.

Article Snippet: Human ORCTL3 and SCD1 cDNAs were obtained from OriGene Technologies, Inc.

Techniques: Transformation Assay, Luciferase, Control, Staining, Transfection, Immunoprecipitation, Expressing, Construct, Plasmid Preparation, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction

Journal: Oncogene

Article Title: The anticancer gene ORCTL3 targets stearoyl-CoA desaturase-1 for tumour-specific apoptosis.

doi: 10.1038/onc.2014.93

Figure Lengend Snippet: Figure 4. ORCTL3 targets stearoyl-CoA desaturase. (a) H-ras and p53mut- transformed CV-1 cells were transiently cotransfected with GFP and luciferase, ORCTL3 or caspase 2 in a 1:3 ratio and cultivated with either 100 μM BSA-conjugated oleic acid or BSA for 24 h. Luciferase-control background was subtracted. Data are means ± s.d. (n = 4). (b) The SCD1 inhibitor CAY10566 or equal volumes of dimethyl sulfoxide were applied to WT as well as H-ras and p53mut-transformed CV-1 cells and apoptosis was quantified after 48 h by fluorescein diacetate staining. (c) ORCTL3-HA was transfected into 293T cells (right, input). Cell lysates were immunoprecipitated with an α-HA antibody (left panels) that revealed multiple, probably glycosylated, forms of ORCTL3 or with an antibody against the endogenous SCD1 (middle panels). The immunoprecipitates were analysed using appropriate antibodies. (d) The desaturation index (18:1n-9/18:0) upon transfection of the indicated expression constructs into normal (top panel) and H-ras-transfected (bottom panel) CV-1 cells. (e) 293T cells were transfected with ORCTL3, RIP1 and ANT1 with either β-Gal or a plasmid-expressing SCD1 in a 3:1 ratio. 48 h post-transfection, apoptosis was assessed by staining the cells with DiOC6/PI and analysing by flow cytometry. Data represent the means ± s.d. (n = 3). Raw data were normalised to transfection efficiency estimated by GFP. (f) Endogenous SCD1 mRNA level was semi-quantitatively determined in dublicates by reverse transcription–PCR in WT-, H-ras-, and P53-mut-transformed CV-1 cells. *Po0.05, **Po0.005, ***Po0.0005.

Article Snippet: Human ORCTL3 and SCD1 complementary DNAs were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Transformation Assay, Luciferase, Control, Staining, Transfection, Immunoprecipitation, Expressing, Construct, Plasmid Preparation, Cytometry, Reverse Transcription

Journal: Molecular Cancer Therapeutics

Article Title: Rapamycin Regulates Stearoyl CoA Desaturase 1 Expression in Breast Cancer

doi: 10.1158/1535-7163.mct-09-0980

Figure Lengend Snippet: Figure 1. Rapamycin decreases SCD1 expression. A, MDA-MB-468, MCF7, and BT-474 cells were treated with 100 nmol/L rapamycin or 0.01% DMSO for 24 hours. Polysomal RNA was separated by sucrose gradient centrifugation. Total polysomal RNA was extracted and hybridized to Affymetrix Human Genome U133 Plus 2.0 chips. The RNA expression in the rapamycin-treated samples was compared with that of untreated total and polysomal RNA samples using Student's t-test. The gene of interest was considered significant in each cell line if it met a false discovery rate of 20%. All comparisons that met this cutoff are demarcated by an asterisk (*). Data are means ± SE. B, Q-PCR analysis was done to quantitatively assess total RNA, and monosomal and polysomal fractions in MDA-MB-468 and MCF7 cells treated with rapamycin versus vehicle for 24 hours. Actin was used as the endogenous control. RNA expression in rapamycin-treated and untreated samples was compared by using Student's t-test. Data are means ± range (min–max). C, Northern blot analysis for SCD1 and actin was done on total RNA isolated from MDA-MB-468 and MCF7 cells grown in either rapamycin or vehicle for 24 and 96 hours. D, to study the effect of rapamycin on SCD1 in vivo, MDA-MB-468 or MCF7 xenografts were treated with either rapamycin or vehicle for 1 day or 3 weeks. Tumor volumes at day 22 are shown as means ± SE. Vehicle versus rapamycin groups were compared using Student's t-test (top left). Protein lysates prepared from three xenografts were printed on RPPA slides and probed with P-S6RP (Ser 240/244) antibody. Relative P-S6RP expression in rapamycin-treated and untreated groups were compared by using Student's t-test (top right). Data are means ± SE. Total RNA from three tumor samples from each group was evaluated using Q-PCR to assess SCD1 and actin expression (bottom). Data are means ± SE.

Article Snippet: Chemical structures of ycin (22), LY294002 (23), and BEZ235 (24) are n in Supplementary Fig. S1. nucleotides and plasmids gonucleotides were synthesized by Sigma-Aldrich. uman SCD1 cDNA, the human β-actin cDNA, he myc/DDK-tagged human SCD1 ORF and its l were all obtained from Origene Technologies, he promoter-reporter gene plasmid hSCD-Luc was a gift from Dr. James M. Ntambi (University sconsin, Madison, WI; ref. 25).

Techniques: Expressing, Gradient Centrifugation, RNA Expression, Control, Northern Blot, Isolation, In Vivo

Journal: Molecular Cancer Therapeutics

Article Title: Rapamycin Regulates Stearoyl CoA Desaturase 1 Expression in Breast Cancer

doi: 10.1158/1535-7163.mct-09-0980

Figure Lengend Snippet: Figure 2. SCD1 expression is inhibited by PI3K/mTOR inhibitors and increased by insulin signaling. A, MDA-MB-468, MCF7, and BT-474 cells were treated with 100 nmol/L rapamycin or DMSO for 24, 48, 72, and 96 hours, and 10% SDS-PAGE and Western blotting using SCD1 and actin antibody were done (top). MCF7 cells were treated with various concentrations of rapamycin for 96 hours. Western blotting was done using SCD1 and actin antibody (bottom). B, MCF7 cells were incubated overnight in serum-free media. Cells were then cultured for 8 hours in one of the following conditions: no treatment, medium containing 10 μg/mL of insulin, or 100 ng/mL of IGF-I. SDS-PAGE (10%) and Western blotting using SCD1 antibody and actin were done (left). The experiments were replicated in triplicate and quantified according to a relative expression of SCD1/β-actin. Top band was used for quantification. Relative SCD1 expression in the treatment groups was compared with that of the no treatment group using Student's t-test (middle). Data are means ± SE. After overnight serum starvation, cells were cultured for 8 hours in one of the following conditions: no treatment or medium containing 25 or 100 ng/mL of IGF-I in the absence or presence of pretreatment with 100 nmol/L rapamycin (right). SDS-PAGE (10%) and Western blotting using SCD1 antibody and actin were done. C, MCF7 cells were transfected with control and constitutively active Akt (CA-Akt) plasmids. Sixty hours later, serum-free media were added and cells were incubated for an additional 36 hours. Western blotting was done for SCD1, Akt, P-S6RP (Ser 240/244), and actin (right). D, left, MDA-MB-468 cells were cultured for 24 hours with no treatment, DMSO, 100 nmol/L rapamycin, and 50 μmol/L LY294002. Western blotting was done using SCD1 and actin antibody. S6K1 and phospho-S6K1 (Thr 389) were used to confirm inhibition of the mTOR pathway. Middle, MDA-MB-468 cells were cultured with DMSO, or 1, 10, or 100 nmol/L BEZ235. Lysates were collected at 6 hours (for P-Akt and Akt) or 24 hours (for SCD1 and actin). Western blotting was done using P-Akt (Thr 308), Akt, SCD1, and actin antibody. Right, MDA-MB-468 cells were cultured with DMSO or 1, 10, or 100 nmol/L BEZ235. Lysates were collected at 6 hours and Western blotting was done using P-Akt (Ser 473), Akt, and actin antibody.

Article Snippet: Chemical structures of ycin (22), LY294002 (23), and BEZ235 (24) are n in Supplementary Fig. S1. nucleotides and plasmids gonucleotides were synthesized by Sigma-Aldrich. uman SCD1 cDNA, the human β-actin cDNA, he myc/DDK-tagged human SCD1 ORF and its l were all obtained from Origene Technologies, he promoter-reporter gene plasmid hSCD-Luc was a gift from Dr. James M. Ntambi (University sconsin, Madison, WI; ref. 25).

Techniques: Expressing, SDS Page, Western Blot, Incubation, Cell Culture, Transfection, Control, Inhibition

Journal: Molecular Cancer Therapeutics

Article Title: Rapamycin Regulates Stearoyl CoA Desaturase 1 Expression in Breast Cancer

doi: 10.1158/1535-7163.mct-09-0980

Figure Lengend Snippet: Figure 4. Rapamycin regulates expression of mature SREBP1. A, MCF7 and MDA-MB-468 cells were treated with 100 nmol/L rapamycin or vehicle for 24 and 96 hours. SDS-PAGE (8%) and Western blotting using SREBP1 and actin antibodies were done. Each SREBP1 blot had a precursor band (P) and a smaller mature band (M). These results were confirmed in triplicate experiments. Nuclear protein extracts from MCF7 (B) and MDA-MB-468 (C) cells treated with 100 nmol/L rapamycin or vehicle for 1 and 4 days were assayed for specific transcription factor-DNA binding activity. All experiments were replicated in triplicate and quantified in comparison with vehicle using Student's t-test. Data are means ± SE. D, MDA-MB-468 cells were treated with 100 nmol/L rapamycin for 96 hours. SDS-PAGE and Western blotting using ACC, FAS, SCD1, SREBP1, and actin antibodies were done.

Article Snippet: Chemical structures of ycin (22), LY294002 (23), and BEZ235 (24) are n in Supplementary Fig. S1. nucleotides and plasmids gonucleotides were synthesized by Sigma-Aldrich. uman SCD1 cDNA, the human β-actin cDNA, he myc/DDK-tagged human SCD1 ORF and its l were all obtained from Origene Technologies, he promoter-reporter gene plasmid hSCD-Luc was a gift from Dr. James M. Ntambi (University sconsin, Madison, WI; ref. 25).

Techniques: Expressing, SDS Page, Western Blot, Binding Assay, Activity Assay, Comparison

Journal: Molecular Cancer Therapeutics

Article Title: Rapamycin Regulates Stearoyl CoA Desaturase 1 Expression in Breast Cancer

doi: 10.1158/1535-7163.mct-09-0980

Figure Lengend Snippet: Figure 5. eIF4E knockdown decreases SCD1 and SREBP1 expression and SCD1 promoter activity. A, MDA-MB-468 cells were transfected with siRNA for mTOR and eIF4E. After 72 hours, Western blotting with SCD1, mTOR, and eIF4E and actin was done. These results were confirmed in triplicate experiments. B, MDA-MB-468 cells were transfected with control siRNA or two separate sequences of eIF4E siRNA. After 72 hours, Western blotting with SCD1, eIF4E, and actin was done. C, MDA-MB-468 cells were transfected with single or pool siRNA for S6K1, and 72 hours later, Western blotting was done using S6K1, Akt, P-Akt (Thr 308), P-Akt (Ser 473), SCD1, SREBP1, and actin antibody (left). MDA-MB-468 cells were transfected with siRNA for eIF4E and S6K1. After 72 hours, Western blotting with SCD1, SREBP1, eIF4E, and S6K1 was done (right). These results were confirmed in triplicate experiments. D, MCF7 cells were first transfected with siRNA for eIF4E. Dual luciferase assays were then done after cotransfecting with SCD1 promoter reporter (hSCD1-Luc pGL3) and control (pRL) plasmids. Treatment with and without rapamycin served as control. These results reflect an average of three independent experiments done in triplicate (left). MDA-MB-468, MCF7, and BT-474 cells were transfected with vector, 4E-BP1 WT, or 4E-BP1 5A plasmids. SCD1 promoter reporter (hSCD1-Luc pGL3) and control (pRL-TK) plasmids were cotransfected, and 96 hours later, dual luciferase assays were carried out. Vector transfection served as control. Analysis was done by using one-way ANOVA and Tukey post hoc test (right). This experiment was repeated three times in triplicate. Bars, SE.

Article Snippet: Chemical structures of ycin (22), LY294002 (23), and BEZ235 (24) are n in Supplementary Fig. S1. nucleotides and plasmids gonucleotides were synthesized by Sigma-Aldrich. uman SCD1 cDNA, the human β-actin cDNA, he myc/DDK-tagged human SCD1 ORF and its l were all obtained from Origene Technologies, he promoter-reporter gene plasmid hSCD-Luc was a gift from Dr. James M. Ntambi (University sconsin, Madison, WI; ref. 25).

Techniques: Knockdown, Expressing, Activity Assay, Transfection, Western Blot, Control, Luciferase, Plasmid Preparation