Journal: The Journal of Biological Chemistry

Article Title: An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

doi: 10.1074/jbc.M117.801936

Figure Lengend Snippet: Role of the di-proline motif in degradation of Δ9-desaturase. N-terminal amino acid sequences of Δ9-desaturases were compared (D. melanogaster DESAT1 (NP_652731), Mus musculus SCD1 (NP_033153), M. musculus SCD3 (NP_077770), Homo sapiens SCD1 (NP_005054), Danio rerio stearoyl-CoA desaturase (AAO25582), and S. cerevisiae Ole1 (CAA96757)) (A). Schematic illustration of DESAT1, mouse SCD1, and constructed mutants is shown (B). S2 cells expressing mouse SCD1-FLAGC, chimera-FLAGC, chimera (AA)-FLAGC, and SCD1 (di-Pro)-FLAGC were treated with C18:1 (100 μm) for 6 h (C) or DESAT1 inhibitor 37c (1 μm) for 16 h (D), and the amounts of endogenous DESAT1, FLAG-tagged exogenous Δ9-desaturases, and α-tubulin protein were detected with anti-DESAT1 antibody, anti-FLAG antibody, and anti-α-tubulin antibody, respectively. Band intensities were determined by ImageJ software, and levels of SCD1 proteins are shown relative to the amount of SCD1 protein in vehicle-treated cells (C and D). Mean ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001; n.s., not significant.

Article Snippet: DESAT1 coding sequence was isolated from a cDNA library of strain Canton-S. cDNA encoding mouse SCD1 was purchased from ORIGENE.

Techniques: Construct, Expressing, Software

Journal: Cells

Article Title: Therapeutic Potential of Stearoyl-CoA Desaturase1 (SCD1) in Modulating the Effects of Fatty Acids on Osteoporosis

doi: 10.3390/cells13211781

Figure Lengend Snippet: Schematic diagram illustrating the research concept. In osteoporosis models, fatty acids such as SFA and MUFA are secreted from expanding MAT. This study suggests that SCD1 can promote bone regeneration by converting SFA, which is known to have a negative effect on osteoblast differentiation and MSC proliferation, into MUFA, which can have a positive effect on bone metabolism. This highlights the therapeutic potential of MSCs through SCD1 overexpression in bone regeneration in osteoporosis models.

Article Snippet: After blocking with 5% bovine serum albumin (BSA) in TBST (Tris-buffered saline with Tween 20) for 1 h, the membranes were washed and incubated sequentially with a primary anti-SCD1 antibody (#2794, Cell Signaling Technology, Beverly, MA, USA) and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies.

Techniques: Over Expression

Journal: Cells

Article Title: Therapeutic Potential of Stearoyl-CoA Desaturase1 (SCD1) in Modulating the Effects of Fatty Acids on Osteoporosis

doi: 10.3390/cells13211781

Figure Lengend Snippet: Effect of SCD1 overexpression on osteogenic differentiation reduced by palmitic acid (PA). ( A ) Production of adenovirus-based SCD1 vector. ( B ) Confirmation of cell viability of BM-MSCs according to virus MOI. ( C ) Verification of MOI-dependent overexpression of Ad-SCD1. ( D ) Confirmation of normal virus infection and GFP expression in BM-MSCs. ( E ) Measurement of cell viability with the virus. ( F – I ) Confirmation of the effect of Ad-SCD1 on osteogenic differentiation of PA-treated BM-MSCs through comparison of ALP and ARS staining, ALP activity, and calcium deposition. (*** p < 0.001).

Article Snippet: After blocking with 5% bovine serum albumin (BSA) in TBST (Tris-buffered saline with Tween 20) for 1 h, the membranes were washed and incubated sequentially with a primary anti-SCD1 antibody (#2794, Cell Signaling Technology, Beverly, MA, USA) and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies.

Techniques: Over Expression, Plasmid Preparation, Virus, Infection, Expressing, Comparison, Staining, Activity Assay

Journal: Cells

Article Title: Therapeutic Potential of Stearoyl-CoA Desaturase1 (SCD1) in Modulating the Effects of Fatty Acids on Osteoporosis

doi: 10.3390/cells13211781

Figure Lengend Snippet: In vivo experiments to verify the effect of SCD1 on bone defect recovery in an osteoporosis model. ( A ) Schematic diagram of the animal modeling experiment. ( B ) X-ray scan images of the left distal femur at 1, 3, 5, and 7 weeks after cell transplantation into the defect. ( C ) BMD measurements by DEXA imaging at 1, 3, 5, and 7 weeks (* p < 0.05, *** p < 0.001).

Article Snippet: After blocking with 5% bovine serum albumin (BSA) in TBST (Tris-buffered saline with Tween 20) for 1 h, the membranes were washed and incubated sequentially with a primary anti-SCD1 antibody (#2794, Cell Signaling Technology, Beverly, MA, USA) and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies.

Techniques: In Vivo, Transplantation Assay, Imaging

Journal: PLoS ONE

Article Title: Ontogeny of Hepatic Energy Metabolism Genes in Mice as Revealed by RNA-Sequencing

doi: 10.1371/journal.pone.0104560

Figure Lengend Snippet: A) One-way hierarchical cluster map of lipid metabolism genes that changed significantly in at least one age compared to Day 60. mRNA expression of genes that were statistically different from those in mice 60 days of age (control) have the percent indicated within the heatmap square (control = 100%). B) Examples of FPKM (fragments per kilobase of exon per million reads mapped) values of individual genes of lipid metabolism throughout development. C) Western blot analysis of Scd1 protein expression in liver of pre-weaning and post-weaning mice, α-tubulin was used as a loading control. For gene function information see .

Article Snippet: Membranes were then probed with antibodies specific for Scd1 (1∶1000 dilution; 2438S; Cell Signaling Technology, Inc., Danvers, MA) or α-tubulin (1∶5000 dilution; CP06; EMD-Millipore Corp, Billerica, MA).

Techniques: Expressing, Control, Western Blot

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

Journal: PLoS ONE

Article Title: A proteomics approach for the identification of cullin-9 (CUL9) related signaling pathways in induced pluripotent stem cell models

doi: 10.1371/journal.pone.0248000

Figure Lengend Snippet: CUL9 KO express normal levels of key fatty acid metabolism enzymes SCD1 ( A ) and DHCR24 ( B ) n-3; mean +/- SEM; Analysis done using student’s t-test, α = 0.05. CUL9 KO hPSCs have the same oxygen consumption rate (OCR) ( C ), ATP production ( D ), spare respiratory capacity ( E ), and levels of proton leak ( F ) as parental and isogenic WT hPSCs. CUL9 KO hNPCs also display no abnormalities ( G-J ). n = 3 independent experiments done in triplicate, error bars are +/- SEM. (OCR) was measured using the Seahorse Biosciences Mito Stress Test on an XFe96 analyzer. ATP production was calculated from the corresponding OCR traces in panels A and C for each condition. Spare respiratory capacity is the difference between maximal respiration or basal respiration. Oligomycin inhibits ATP synthase interrupting the electron transport chain ultimately disrupting mitochondrial respiration and ATP production. FCCP is an uncoupler, uncoupling ATP production from the electron transport chain. Rotenone (complex I inhibitor) and antimycin A (complex III inhibitor) completely inhibit mitochondrial respiration, only permitting nonmitochondrial respiration to persist.

Article Snippet: We used antibodies against OCT4 (Cell Signaling Technology, Cat. 75463S), NANOG (Cell Signaling Technology, Cat. 4903S), SOX2 (Cell Signaling Technology, Cat. 5049S), Cleaved Caspase-3 (Cell Signaling Technology, Cat. 9661S), CUL9 (Bethyl Laboratories, Cat. A300-98A), CUL7 (Bethyl Laboratories, Cat. A300-223A), ANAPC7 (Bethyl Laboratories, Cat. A302-551), FZR1 (Abcam, Cat. ab3242), and Cyclin-A2 (Cell Signaling Technology, Cat. 4656T), PAX6 (Cell Signaling Technology Cat. 60433), Nestin (STEMCELL Technologies, Cat. 60091), TUBB3 (Cell Signaling Technology, Cat. 4466S), MAP2 (Thermo Fisher Scientific, Cat. 131500), beta-Actin (Sigma, Cat. A1978), alpha-Tubulin (Sigma, Cat. T9026), and GAPDH (Cell Signaling Technology, Cat. 5147S), SOX3 (Thermo Fisher, Cat. PA5-35983), TM7SF2 (Thermo Scientific, Cat. 12033-1-AP), CUX1 (Abcam, Cat. ab54583), and SCD1 (Bethyl, Laboratories, Cat. A305-259A-T).

Techniques: