lpl sirna  (Santa Cruz Biotechnology)

Journal: Journal of Lipid Research

Article Title: Endocytosis of very low-density lipoproteins: an unexpected mechanism for lipid acquisition by breast cancer cells [S]

doi: 10.1194/jlr.RA119000327

Figure Lengend Snippet: LPL and VLDLR are involved in VLDL uptake in MDA-MB-231 BC cells. A: qRT-PCR verification of LPL and VLDLR knockdown in MDA-MB-231 BC cells treated with LPL or VLDLR siRNA. LPL and VLDLR gene expression of siRNA cells is presented relative to that of the Scr siRNA control. LPL siRNAs knocked down LPL mRNA, while VLDLR siRNAs significantly reduced both VLDLR and LPL mRNAs. B: Western blot shows VLDLR protein knockdown by siRNAs. C: siRNA knockdown of LPL or VLDLR reduces DiI-VLDL uptake in MDA-MB-231 BC cells. The greatest decreases were observed in VLDLR siRNA 1 cells, which had the highest efficiency knockdown of both VLDLR and LPL mRNAs. DiI-VLDL uptake was reduced by heparin by all treatments except VLDLR siRNA, where the reduction from VLDL alone was not significant. P ≥ 0.05 (ns), *P < 0.05, **P < 0.01, and ***P < 0.001. D: RAP (1 µM) inhibits DiI-VLDL binding and uptake at 37°C by MDA-MB-231 cells, as visualized by confocal microscopy. DiI-VLDL (red), DAPI (blue).

Article Snippet: MDA-MB-231 siRNA cells. siRNAs targeting LPL or VLDLR (Sigma-Aldrich) were transfected into MDA-MB-231 BC cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s guidelines. siRNAs used were LPL siRNA 1 (SASI_Hs01_00208454), LPL siRNA 2 (SASI_Hs01_00208455); VLDLR siRNA 1 (SASI_Hs02_00335553), VLDLR siRNA 2 (SASI_Hs01_00219062); and MISSION siRNA Universal Negative Control #1, SIC001 (Sigma-Aldrich).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Binding Assay, Confocal Microscopy

Journal: Diabetologia

Article Title: Chylomicrons stimulate incretin secretion in mouse and human cells

doi: 10.1007/s00125-017-4420-2

Figure Lengend Snippet: Chylomicron-mediated GLP-1 release in GLUTag cells is dependent on LPL. ( a ) Lpl expression was examined by RNA sequencing FACS-sorted primary L cells (GLU-Venus-positive, L+) and negative cells (GLU-Venus-negative, L−) collected in parallel from murine duodenum and GLUTag cells. FPKM, fragments per kilobase per million reads. ( b ) GLP-1 secretion from GLUTag cells treated with chylomicrons (CM; 10 μg/ml) in the presence or absence of orlistat (1 μg/ml, following 30 min pre-treatment). Data represent means ± SEM, n = 9 wells from three independent experiments; one-way ANOVA, *** p < 0.001. ( c ) GLUTag cells were transfected with 30 nmol/l Lpl siRNA or negative control (Ctrl) siRNA, and knockdown was validated by qRT-PCR. Data are presented as means ± SEM, n = 5–6 from three independent experiments; unpaired t test, ** p < 0.01. ( d ) GLP-1 secretion from negative control (white bars) or Lpl siRNA (grey bars) transfected GLUTag cells treated with chylomicrons (CM; 10 μg/ml) or forskolin/IBMX (F/I; 10 μmol/l each) in the presence of glucose (10 mmol/l). Data represent means ± SEM, n = 8–9 wells from three independent experiments; one-way ANOVA, * p < 0.05, *** p < 0.001

Article Snippet: GLUTag cells in 24-well plates were transfected with 30 nmol/l AllStars negative control siRNA, Ffar1 siRNA (Mm_Gpr40_2, target sequence: 5′-TGCGCTGGGCTTTCCATTGAA-3′) or Lpl siRNA (Mm_Lpl_5, target sequence: 5′-CAGCTCTATCTTGTTAGTTAA-3′) (Qiagen, Manchester, UK) using Lipofectamine 2000 (Thermo Fisher Scientific, Loughborough, UK) as per the manufacturer’s protocol.

Techniques: Expressing, RNA Sequencing, Transfection, Negative Control, Knockdown, Quantitative RT-PCR

Journal: Diabetologia

Article Title: Chylomicrons stimulate incretin secretion in mouse and human cells

doi: 10.1007/s00125-017-4420-2

Figure Lengend Snippet: Chylomicrons stimulate GLP-1 release via FFA1 in GLUTag cells. ( a ) FFA1 ( Ffar1 ) expression was examined by RNA sequencing FACS-sorted primary L cells (GLU-Venus-positive, L+) and negative cells (GLU-Venus-negative, L−) collected in parallel from murine duodenum and GLUTag cells. FPKM, fragments per kilobase per million reads. ( b ) GLP-1 secretion from GLUTag cells treated with chylomicrons (CM; 10 μg/ml) or the selective FFA1 agonist AM-1638 (1 μmol/l) in the presence or absence of the FFA1 antagonist GW1100 (1 μmol/l, with 30 min pre-treatment). Data represent means ± SEM, n = 9 wells from three independent experiments; one-way ANOVA, *** p < 0.001. ( c ) GLUTag cells were transfected with 30 nmol/l Ffar1 siRNA or negative control (Ctrl) siRNA, and knockdown was validated by qRT-PCR. Data are presented as means ± SEM, n = 7 from three independent experiments; unpaired t test, * p < 0.05. ( d ) GLP-1 secretion from negative control (white bars) or Ffar1 siRNA (grey bars) transfected GLUTag cells treated with chylomicrons (CM; 10 μg/ml), AM-1638 (1 μmol/l) or forskolin/IBMX (F/I; 10 μmol/l each) in the presence of glucose (10 mmol/l). Data represent means ± SEM, n = 9 wells from three independent experiments; one-way ANOVA, ** p < 0.01, *** p < 0.001

Article Snippet: GLUTag cells in 24-well plates were transfected with 30 nmol/l AllStars negative control siRNA, Ffar1 siRNA (Mm_Gpr40_2, target sequence: 5′-TGCGCTGGGCTTTCCATTGAA-3′) or Lpl siRNA (Mm_Lpl_5, target sequence: 5′-CAGCTCTATCTTGTTAGTTAA-3′) (Qiagen, Manchester, UK) using Lipofectamine 2000 (Thermo Fisher Scientific, Loughborough, UK) as per the manufacturer’s protocol.

Techniques: Expressing, RNA Sequencing, Transfection, Negative Control, Knockdown, Quantitative RT-PCR