Journal: Journal of Lipid Research

Article Title: Slc43a3 is a regulator of free fatty acid flux ¹

doi: 10.1194/jlr.RA119000294

Figure Lengend Snippet: mRNA expression of membrane transporters in OP9 cells and tissues. A: Levels of membrane transporter mRNA in wild-type mouse liver, epididymal fat, subcutaneous fat, and brown adipose tissue, each with n = 4. B: Levels of membrane transporter mRNA in OP9 cells. OP9 cells were treated with 0.5 mM isobutyl-methylxanthine, 1 μM dexamethasone, and 1 μg/ml insulin for differentiation for 5 days before mRNA analysis. Results are representative of three independent experiments, each with n = 6. C: Levels of membrane transporter mRNA in differentiated primary mouse preadipocytes. D: Slc43a3 mRNA expression in adipocytes versus stromal vascular cells. Results are representative of three independent experiments, each with n = 6. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: OP9 cells were seeded into 12-well plates (Costar) and transfected with Slc43a3 Predesigned Silencer Select siRNA or scrambled siRNA control for knockdown, and Slc43a3 expression plasmid and pCDNA6/V5-HisB as blank control for overexpression.

Techniques: Expressing

Journal: Journal of Lipid Research

Article Title: Slc43a3 is a regulator of free fatty acid flux ¹

doi: 10.1194/jlr.RA119000294

Figure Lengend Snippet: Effects of Slc43a3 knockdown and overexpression on genes involved in lipid metabolism. A: Lipid accumulation in differentiated OP9 cells transfected with scrambled siRNA (control), Slc43a3 siRNA, pcDNA6 (control), or pEZ-M51-Slc43a3-His clone. OP9 cells were transfected with the scrambled siRNA (control), Slc43a3 siRNA, pcDNA6 (control), or pEZ-M51-Slc43a3-His clone and differentiated for 5 days. Bodipy 493/503 (1 nM) was added in the media before fluorescent microscopy. Results are representative of three independent experiments. Scale bar 200 microns. B: Western blot of Slc43a3 in OP9 cells transfected with scrambled siRNA (control), Slc43a3 siRNA, pcDNA6 (control), or pEZ-M51-Slc43a3-His clone. C: Effects of knockdown and overexpression of Slc43a3 in OP9 cells on genes involved in lipid metabolism. Results are representative of two to three independent experiments. D: Effects of knockdown and overexpression of Slc43a3 in OP9 cells on genes involved in esterification. Results are representative of two independent experiments, each with n = 4–6. *P < 0.05.

Article Snippet: OP9 cells were seeded into 12-well plates (Costar) and transfected with Slc43a3 Predesigned Silencer Select siRNA or scrambled siRNA control for knockdown, and Slc43a3 expression plasmid and pCDNA6/V5-HisB as blank control for overexpression.

Techniques: Over Expression, Transfection, Microscopy, Western Blot

Journal: Journal of Lipid Research

Article Title: Slc43a3 is a regulator of free fatty acid flux ¹

doi: 10.1194/jlr.RA119000294

Figure Lengend Snippet: Effects of Slc43a3 knockdown and overexpression on FA transport. A: Effects of knockdown and overexpression of Slc43a3 in OP9 cells on FA uptake. Results are representative of two to three independent experiments. Slc43a3 knockdown, n = 22; Slc43a3 overexpression, n = 12; Control combined, n = 36. *P < 0.05, **P < 0.01, ***P < 0.001. B: Effects of knockdown of Slc43a3 in OP9 cells on FA efflux. Transfected OP9 cells were loaded with fluorescent lipid BODIPY C1-C12 bound to 1% BSA, and FA efflux assayed following the quencher-based FA efflux assay. Results are representative of three to four independent experiments with replication of nine in each treatment. The cumulative FA efflux was calculated for the first 5 min of the reaction. *P < 0.05, **P < 0.01. C: Effects of knockdown of Slc43a3 in OP9 cells on FA efflux using 14C-palmitic acid. Transfected and differentiated OP9 cells were loaded with 20 μM 14C-palmitic acid. Cells were then stimulated either with 20 μM forskolin and 1 mM isobutyl-methylxanthine or 100 nM isoproterenol for lipolysis. Media were collected after 60 min for measurement of FA radioactivity and glycerol released (D). *P < 0.05, **P < 0.01.

Article Snippet: OP9 cells were seeded into 12-well plates (Costar) and transfected with Slc43a3 Predesigned Silencer Select siRNA or scrambled siRNA control for knockdown, and Slc43a3 expression plasmid and pCDNA6/V5-HisB as blank control for overexpression.

Techniques: Over Expression, Transfection, Radioactivity

Journal: Journal of Lipid Research

Article Title: Slc43a3 is a regulator of free fatty acid flux ¹

doi: 10.1194/jlr.RA119000294

Figure Lengend Snippet: Effects of ACSL inhibition on FA transport. A: Effects of triacsin on ACSL activity in control and Slc43a3 knockdown OP9 cells. OP9 cells were transfected with scrambled siRNA (control) or Slc43a3 siRNA and differentiated for 5 days. Cells were incubated with triacsin (5 nM) for 1 h prior to measurement of ACSL activity. OA, palmitic acid (PA), and AA were assayed separately as substrates. B: Effects of triacsin on FA uptake in control and Slc43a3 knockdown OP9 cells. OP9 cells were transfected with scrambled siRNA (control) or Slc43a3 siRNA and differentiated for 5 days. Cells were preincubated with triacsin (5 nM) for 1 h prior to incubation with fluorescent lipid BODIPY C1-C12 for measurement of FA uptake. C: Effects of triacsin on FA efflux in control and Slc43a3 knockdown OP9 cells. OP9 cells were transfected with scrambled siRNA (control) or Slc43a3 siRNA and differentiated for 5 days. Differentiated cells were loaded with 20 μM 14C-palmitic acid, and then preincubated with triacsin (5 nM) for 1 h prior to incubation with 100 nM isoproterenol. Media were collected after 1 h for measurement of FA radioactivity released. Results are representative of two independent experiments, each with n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: OP9 cells were seeded into 12-well plates (Costar) and transfected with Slc43a3 Predesigned Silencer Select siRNA or scrambled siRNA control for knockdown, and Slc43a3 expression plasmid and pCDNA6/V5-HisB as blank control for overexpression.

Techniques: Inhibition, Activity Assay, Transfection, Incubation, Radioactivity

Journal: Journal of Lipid Research

Article Title: Slc43a3 is a regulator of free fatty acid flux ¹

doi: 10.1194/jlr.RA119000294

Figure Lengend Snippet: Effects of Slc43a3 knockdown on intracellular free FA concentrations and FAO. OP9 cells were transfected with scrambled siRNA (control) or Slc43a3 siRNA and differentiated for 5 days. Differentiated cells were treated without or with 100 nM isoproterenol, and then intracellular protein bound (B) and unbound (A) free FA concentrations measured using an ADIFAB2 FA indicator. Results are representative of two independent experiments, each with n = 3. *P < 0.05, **P < 0.01. C: FAO. OP9 cells were transfected with scrambled siRNA (control) or Slc43a3 siRNA and differentiated for 5 days. Differentiated cells were treated without or with 100 nM isoproterenol in the presence of 3H-palmitate, and then FAO was measured as described in the Materials and Methods. Results are representative of three independent experiments, each with n = 5–6. *P < 0.05.

Article Snippet: OP9 cells were seeded into 12-well plates (Costar) and transfected with Slc43a3 Predesigned Silencer Select siRNA or scrambled siRNA control for knockdown, and Slc43a3 expression plasmid and pCDNA6/V5-HisB as blank control for overexpression.

Techniques: Transfection

Journal: Journal of Lipid Research

Article Title: Slc43a3 is a regulator of free fatty acid flux ¹

doi: 10.1194/jlr.RA119000294

Figure Lengend Snippet: Levels of Slc43a3 in adipose tissue of CD36 KO mice. A: Levels of Slc43a3 RNA in adipose tissue of wild-type and CD36 KO mice. Total RNA was prepared from subcutaneous and retroperitoneal adipose tissues from wild-type and CD36 KO mice. Levels of Slc43a3 were assayed using TaqMan RT-PCR as described in the Materials and Methods. Results are the summary of three independent assays with n = 3–4 in each group. B: Effects of Slc43a3, CD36, and double knockdown in OP9 cells on free FA uptake compared with control (scrambled siRNA). C: AUC of FA uptake for the first 6 min. Results are representative of three independent experiments, each with n = 8. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: OP9 cells were seeded into 12-well plates (Costar) and transfected with Slc43a3 Predesigned Silencer Select siRNA or scrambled siRNA control for knockdown, and Slc43a3 expression plasmid and pCDNA6/V5-HisB as blank control for overexpression.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Lipid Research

Article Title: Slc43a3 is a regulator of free fatty acid flux ¹

doi: 10.1194/jlr.RA119000294

Figure Lengend Snippet: Effects of Slc43a3 on cAMP and ATP levels. cAMP and ATP levels were measured by ELISA. Changes of extracellular (A) and intracellular (B) cAMP levels, and cellular ATP levels (C) after transfection with Slc43a3 siRNA and pEZ-M51-Slc43a3-His clone in OP9 cells and treatment with forskolin with isobutyl-methylxanthine, and isoproterenol for 5 and 15 min. Intracellular values were obtained at 15 min. Results are representative of three independent experiments, each with n = 3–6. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: OP9 cells were seeded into 12-well plates (Costar) and transfected with Slc43a3 Predesigned Silencer Select siRNA or scrambled siRNA control for knockdown, and Slc43a3 expression plasmid and pCDNA6/V5-HisB as blank control for overexpression.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection

Journal: Cellular & Molecular Biology Letters

Article Title: Slc25a5 regulates adipogenesis by modulating ERK signaling in OP9 cells

doi: 10.1186/s11658-022-00314-y

Figure Lengend Snippet: Slc25a5 is increased in adipogenic differentiation. RNA-seq data were obtained from OP9 cells and ASCs undergoing adipogenic differentiation and those that were not. A Upregulated and downregulated gene expression profiles were selected. B Venn diagram illustrating the upregulated genes in both OP9 and ACSs during adipogenic differentiation. C Ten GO terms representing the most upregulated genes during adipogenic differentiation. D , E Expression profiles of the Slc25 gene family during adipogenic differentiation. F The relative mRNA level of Slc25a5 was measured with RT–qPCR. The values are mean ± SEM ( n = 3). ** P < 0.01; *** P < 0.001 versus controls

Article Snippet: Mouse OP9 cells (CCTCC, Wuhan, China) were cultured in MEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO 2 .

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

Journal: Cellular & Molecular Biology Letters

Article Title: Slc25a5 regulates adipogenesis by modulating ERK signaling in OP9 cells

doi: 10.1186/s11658-022-00314-y

Figure Lengend Snippet: Inhibiting Slc25a5 prevents adipogenic differentiation. Before adipogenic differentiation, OP9 cells were transfected with Slc25a5 siRNA, and 24 h later, the cells were incubated with 1 mM rosiglitazone to induce adipogenic differentiation. A RT–qPCR verified the efficiency of Slc25a5 knockdown in OP9 cells. B Oil Red O staining; scale bar is 200 μm. C Cellular TG levels were measured. D RT–qPCR was performed to assess the expression of the adipogenic factors PPARγ, FABP4, FASN, and C/EBPα. E Western blot of selected subunits of the OXPHOS complexes. F Cellular levels of ATP. Values are the mean ± SEM ( n = 3). * P < 0.05; ** P < 0.01 versus control

Article Snippet: Mouse OP9 cells (CCTCC, Wuhan, China) were cultured in MEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO 2 .

Techniques: Transfection, Incubation, Quantitative RT-PCR, Knockdown, Staining, Expressing, Western Blot, Control

Journal: Cellular & Molecular Biology Letters

Article Title: Slc25a5 regulates adipogenesis by modulating ERK signaling in OP9 cells

doi: 10.1186/s11658-022-00314-y

Figure Lengend Snippet: Transcriptomics analysis implicates ERK pathway as a target of Slc25a5 . After adipogenic differentiation, OP9 cells with or without Slc25a5 knockdown were collected, and a transcriptome analysis was performed. A PCA score plot showing clear clusters in each group. B Scatterplot of DEGs between the control and Slc25a5 -knockdown cells (fold change ≥ 1.5, and P ≤ 0.06). C Heat map of the DEGs between the control and Slc25a5 -knockdown cells. D GO analysis of downregulated DEGs. E Protein levels of ERK, p-ERK, P38, p-P38, JNK, p-JNK, and PPARγ were measured by western blot analysis. The values are the mean ± SEM ( n = 3). * P < 0.05 versus control

Article Snippet: Mouse OP9 cells (CCTCC, Wuhan, China) were cultured in MEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO 2 .

Techniques: Knockdown, Control, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: Slc25a5 regulates adipogenesis by modulating ERK signaling in OP9 cells

doi: 10.1186/s11658-022-00314-y

Figure Lengend Snippet: ERK inhibitor reverses Slc25a5 knockdown-induced adipogenesis inhibition. Before adipogenic differentiation was induced, OP9 cells were transfected with Slc25a5 siRNA, and 24 h later, these cells were incubated with 1 mM rosiglitazone and PD98059 (50 μM). A Cellular triglyceride (TG) levels were measured. B Oil Red O staining was performed after adipogenic differentiation, and the results were visualized by light microscopy. The scale bar is 200 µm. C RT–qPCR was performed to assess the expression of the adipogenic factors PPARγ , FABP4 , FASN , and C/EBPα after adipogenic differentiation. D Protein levels of ERK, p-ERK, and PPARγ were measured by western blot analysis. The values are the mean ± SEM ( n = 3). a versus b represents differences between groups; b versus b represents no differences between groups

Article Snippet: Mouse OP9 cells (CCTCC, Wuhan, China) were cultured in MEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C and 5% CO 2 .

Techniques: Knockdown, Inhibition, Transfection, Incubation, Staining, Light Microscopy, Quantitative RT-PCR, Expressing, Western Blot