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: 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:

Journal: JHEP Reports

Article Title: Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis

doi: 10.1016/j.jhepr.2021.100237

Figure Lengend Snippet: Aramchol significantly downregulates SCD1 mRNA and fibrogenic genes, and upregulates PPARG mRNA in HSCs. (A) LX-2 cells were treated with 5 μM and 10 μM Aramchol or DMSO (vehicle) for 24 or 48 h. Gene expression of SCD1 , PPARG , and fibrotic markers were evaluated by RT-qPCR. Dose-dependent inactivation of COL1A1 , ACTA2 , bPDGFR , and MMP2 along with SCD1 and simultaneous activation of PPARG mRNA are shown. (B) Primary human hepatic stellate cells isolated from 3 donors (Patients 1, 2, and 3) were treated with Aramchol as described in panel A for LX-2 cells. Dose-dependent downregulation of COL1A1 and ACTA2 along with SCD1 expression and activation of PPARG are shown. Data were normalised to GAPDH used as internal control. Results are reported as means ± SEM (n = 3). ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001, ∗∗∗∗ p <0.0001 using 2-tailed Studentàs t test with a 95% confidence interval. HSCs, hepatic stellate cells; PPARG, peroxisome proliferator-activated receptor gamma; SCD1, stearoyl CoA-desaturase 1.

Article Snippet: C-terminal Myc-DDK Flag tagged pCMV6-Entry empty vector (CAT# PS100001) and 1077 bp human SCD1 encoded cloned gene (accession# NM_005063) in the same expression vector (CAT# RC209148) were commercially purchased from Origene (MD, USA) and overexpressed in LX-2 cells.

Techniques: Gene Expression, Quantitative RT-PCR, Activation Assay, Isolation, Expressing, Control

Journal: JHEP Reports

Article Title: Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis

doi: 10.1016/j.jhepr.2021.100237

Figure Lengend Snippet: Aramchol inhibits Col1α1, αSMA, SCD1 and upregulates PPARG proteins in HSCs. (A) LX-2 cells were treated with 5 μM and 10 μM Aramchol or DMSO (vehicle) for 24 or 48 h. Protein expression was quantified by densitometry of αSMA, PPARG, and SCD1 bands in Western blots in Fig. S4A . Dose-dependent downregulation of αSMA and SCD1 and upregulation of PPARG were assessed. (B) Primary human hepatic stellate cells isolated from 3 donor livers were treated with Aramchol as described in panel A for LX-2 cells. Expression of αSMA, PPARG, and SCD1 proteins was analysed by densitometry of Western blots shown in Fig. S4B . Downregulation of αSMA, SCD1 and upregulation of PPARG protein expression by Aramchol treatment were evident. Data were normalised to GAPDH. (C) Secreted Col1α1 in culture media (both vehicle and Aramchol treated) from LX-2 cells was measured by ELISA using human pro-collagen 1a1 as a standard. Dose-dependent reduction of Col1α1 in culture media is evident. (D) Soluble Col1α1 protein in culture media was reduced by Aramchol in 3 isolates of phHSCs. Results are reported as means ± SEM (n = 3). ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001, ∗∗∗∗ p <0.0001 using 2-tailed Student's t test with a 95% confidence interval. HSCs, hepatic stellate cells; PPARG, peroxisome proliferator-activated receptor gamma; SCD1, stearoyl CoA-desaturase 1.

Article Snippet: C-terminal Myc-DDK Flag tagged pCMV6-Entry empty vector (CAT# PS100001) and 1077 bp human SCD1 encoded cloned gene (accession# NM_005063) in the same expression vector (CAT# RC209148) were commercially purchased from Origene (MD, USA) and overexpressed in LX-2 cells.

Techniques: Expressing, Western Blot, Isolation, Enzyme-linked Immunosorbent Assay

Journal: JHEP Reports

Article Title: Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis

doi: 10.1016/j.jhepr.2021.100237

Figure Lengend Snippet: Global effects of Aramchol on HSC gene expression by RNA-seq. RNA was extracted from LX-2 cells treated with 10 μM Aramchol or DMSO (vehicle) for 24 (A) or 48 (B) h, followed by RNAseq. Fibrogenic genes as well as SCD1 mRNA expression were significantly downregulated by Aramchol treatment at 48 h, consistent with findings from RT-qPCR ( Fig. 1 ). In contrast, SCD5 mRNA expression was not significantly affected by Aramchol. PPARG mRNA expression was upregulated compared with vehicle-treated cells. Results are reported as transcripts per million (TPM), n = 2. ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001. The p values were calculated using DeSeq2 differential gene expression analysis and adjusted for multiple testing. (C) After 48 h of 10 μM Aramchol treatment in LX-2 cells the cholesterol efflux regulatory genes ABCA1 and ABCG1 were upregulated along with PPARG. SCD1 was downregulated (but not SCD5 ) along with other fibrogenesis-related genes including IGFBP5 , OLFML2A , and CTFG . Volcano plot of Log2 fold change plotted against -Log10 adjusted p value. Each dot represents 1 gene. Differentially expressed genes with a -Log10 adjusted p value greater than 1.3 and a Log2 fold change greater than 1 or less than -1 are highlighted in purple. All other genes are represented as grey. HSC, hepatic stellate cell; PPARG, peroxisome proliferator-activated receptor gamma; SCD1, stearoyl CoA-desaturase 1.

Article Snippet: C-terminal Myc-DDK Flag tagged pCMV6-Entry empty vector (CAT# PS100001) and 1077 bp human SCD1 encoded cloned gene (accession# NM_005063) in the same expression vector (CAT# RC209148) were commercially purchased from Origene (MD, USA) and overexpressed in LX-2 cells.

Techniques: Gene Expression, RNA Sequencing, Expressing, Quantitative RT-PCR

Journal: JHEP Reports

Article Title: Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis

doi: 10.1016/j.jhepr.2021.100237

Figure Lengend Snippet: SCD1 knockdown reduces HSC activation and upregulates PPARG. LX2 cells were transfected with either a SCD1 siRNA or non-targeting fluorescent red siRNA. At 48, 72, and 96 h after transfection the cells and culture media were harvested and total mRNA and protein were purified. (A) SCD1 mRNA expression was measured by RT-qPCR to establish the time of optimal knockdown. At 72 h after siRNA transfection SCD1 mRNA was significantly reduced. (B) At 72 h after SCD1 siRNA transfection profibrotic genes along with SCD1 were significantly downregulated and PPARG was upregulated. (C) Protein expression of αSMA and SCD1 quantification corresponds to Western blots in Fig. S5 . Human GAPDH was used as internal control in both RT-qPCR and densitometric assessments. (D) Secreted Col1α1 in culture media from SCD1 siRNA transfected LX-2 was measured with ELISA using human pro-collagen 1a1 as standard. Significant reduction of Col1α1 secretion from culture media at 72 h after siRNA transfection indicate downregulation of Col1α1 protein in cells. Results are reported as means ± SEM (n = 3). ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001 using 2-tailed Student's t test with a 95% confidence interval. HSC, hepatic stellate cell; ns, non-significant; PPARG, peroxisome proliferator-activated receptor gamma; SCD1, stearoyl CoA-desaturase 1.

Article Snippet: C-terminal Myc-DDK Flag tagged pCMV6-Entry empty vector (CAT# PS100001) and 1077 bp human SCD1 encoded cloned gene (accession# NM_005063) in the same expression vector (CAT# RC209148) were commercially purchased from Origene (MD, USA) and overexpressed in LX-2 cells.

Techniques: Knockdown, Activation Assay, Transfection, Purification, Expressing, Quantitative RT-PCR, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: JHEP Reports

Article Title: Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis

doi: 10.1016/j.jhepr.2021.100237

Figure Lengend Snippet: Aramchol does not further reduce HSC activation in SCD1-knockdown HSCs whereas Aramchol does not suppress fibrogenesis if SCD1 is overexpressed . (A) LX2 cells were transfected either with non-targeting siRNA or SCD1 siRNA for 24 h. After 24 h of transfection cells were maintained in either DMSO (vehicle) or 10 μM Aramchol for an additional 48 h. In the presence of SCD1 knockdown there was no additional downregulation of COL1A1 , ACTA2 , and SCD1 or upregulation of PPARG by Aramchol. (B) LX-2 cells transiently transfected with SCD1 or empty vector were incubated with or without 10 μM Aramchol for 48 h. COL1A1 , ACTA2 , and SCD1 mRNA expression were assessed by RT-qPCR using GAPDH as internal control. Expression of COL1A1 or ACTA2 mRNAs was not downregulated by Aramchol treatment when SCD1 was overexpressed. Results are shown as mean ± SEM (n = 3). ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001 using 2-tailed Student's t test with a 95% confidence interval. EV, empty vector; HSCs, hepatic stellate cells; ns, non-significant; NT siRNA, non-targeting siRNA; OE SCD1, overexpressed SCD1; PPARG, peroxisome proliferator-activated receptor gamma; SCD1, stearoyl CoA-desaturase 1.

Article Snippet: C-terminal Myc-DDK Flag tagged pCMV6-Entry empty vector (CAT# PS100001) and 1077 bp human SCD1 encoded cloned gene (accession# NM_005063) in the same expression vector (CAT# RC209148) were commercially purchased from Origene (MD, USA) and overexpressed in LX-2 cells.

Techniques: Activation Assay, Knockdown, Transfection, Plasmid Preparation, Incubation, Expressing, Quantitative RT-PCR, Control

Journal: JHEP Reports

Article Title: Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis

doi: 10.1016/j.jhepr.2021.100237

Figure Lengend Snippet: Aramchol downregulates SCD1 and upregulates PPARG in primary human hepatocytes. Primary human hepatocytes isolated from 3 donors were treated with 5 μM and 10 μM Aramchol or DMSO (vehicle) for 24 or 48 h. Dose-dependent downregulation of SCD1 , and upregulation of PPARG mRNA expression were quantified with RT-qPCR using GAPDH as an internal control. Results are reported as means ± SEM (n = 3). ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001 using 2-tailed Student's t test with a 95% confidence interval. PPARG, peroxisome proliferator-activated receptor gamma; SCD1, stearoyl CoA-desaturase 1.

Article Snippet: C-terminal Myc-DDK Flag tagged pCMV6-Entry empty vector (CAT# PS100001) and 1077 bp human SCD1 encoded cloned gene (accession# NM_005063) in the same expression vector (CAT# RC209148) were commercially purchased from Origene (MD, USA) and overexpressed in LX-2 cells.

Techniques: Isolation, Expressing, Quantitative RT-PCR, Control