Journal: International Journal of Molecular Sciences

Article Title: NGF Modulates Cholesterol Metabolism and Stimulates ApoE Secretion in Glial Cells Conferring Neuroprotection against Oxidative Stress

doi: 10.3390/ijms23094842

Figure Lengend Snippet: NGF modulates the expression of the main proteins involved in cholesterol homeostasis in U373 cells and enhances ApoE secretion in U373 culture medium. ( A – G ) Representative Western blot and densitometric analysis of SREBP-1, SREBP-2, HMGCR, LDLr, NPC1, ABCA1, and ApoE in U373 cells treated with vehicle (Ctrl) and NGF (100 ng/mL) for 48 h. n = 6 different experiments. Actin and vinculin were used as loading controls. ( H ) Quantification of ApoE levels (µg/mL) by ELISA assay in vehicle- and NGF-treated U373 culture medium. n = 3 different experiments. Data represent means ± SD. Statistical analysis was assessed by using unpaired Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: ApoE mRNA silencing was performed on U373 cells using ApoE siRNA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-29708) according to the manufacturer’s instructions.

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

Journal: International Journal of Molecular Sciences

Article Title: NGF Modulates Cholesterol Metabolism and Stimulates ApoE Secretion in Glial Cells Conferring Neuroprotection against Oxidative Stress

doi: 10.3390/ijms23094842

Figure Lengend Snippet: Effects of p75NTR modulation by LM11A-31 on cholesterol metabolism and secretion. ( A – D ) Representative Western blot and densitometric analysis of HMGCR, ABCA1, NPC1, and ApoE in U373 cells treated with vehicle (Ctrl) and LM11A-31 (0.1 µM) for 48 h. n = 4 different experiments. Vinculin served as a housekeeping protein to normalize protein loading. ( E ) Quantification of ApoE amount (µg/mL) by ELISA assay in culture medium from vehicle- and LM11A-31-treated U373 cells. n = 3 different experiments. ( F ) Cholesterol quantification (total cholesterol, free cholesterol, and cholesteryl esters) in the culture medium of U373 cells treated with vehicle (Ctrl) and LM11A-31 (0.1 µM) for 48 h. n = 3 different experiments. Data represent means ± SD. Statistical analysis was carried out by using unpaired Student’s t test. *** p < 0.001.

Article Snippet: ApoE mRNA silencing was performed on U373 cells using ApoE siRNA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-29708) according to the manufacturer’s instructions.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: NGF Modulates Cholesterol Metabolism and Stimulates ApoE Secretion in Glial Cells Conferring Neuroprotection against Oxidative Stress

doi: 10.3390/ijms23094842

Figure Lengend Snippet: Effect of U373 conditioned medium on neuronal differentiation. ( A ) Representative images in bright field of N1E-115 cells cultured in fresh DMEM (Ctrl), in conditioned medium derived from control U373 (U373-Ctrl), NGF-pre-treated U373 (U373-NGF), and NGF-pre-treated U373 silenced for ApoE (U373-NGF ApoE siRNA) for 48 h. ( B ) Morphological analysis of neurite-bearing cells, neurite length and number of neurites of N1E-115 cells in the presence of fresh DMEM (Ctrl), in conditioned medium derived from control U373 (U373-Ctrl), NGF-pre-treated U373 (U373-NGF), and NGF-pre-treated U373 silenced for ApoE (U373-NGF ApoE siRNA) for 48 h. n = 5 different experiments. Data represent means ± SD. Statistical analysis was performed by using one-way ANOVA, followed by Tukey’s post hoc test.

Article Snippet: ApoE mRNA silencing was performed on U373 cells using ApoE siRNA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-29708) according to the manufacturer’s instructions.

Techniques: Cell Culture, Derivative Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: NGF Modulates Cholesterol Metabolism and Stimulates ApoE Secretion in Glial Cells Conferring Neuroprotection against Oxidative Stress

doi: 10.3390/ijms23094842

Figure Lengend Snippet: Effects of U373 conditioned medium on oxidative stress in neurons. Representative images in bright field and quantitative assessment of neuronal morphology of N1E-115. Cells were previously treated (+) or not (−) with rotenone (0.1 µM) for 16 h, and then cultured in fresh DMEM (Ctrl), in conditioned medium derived from control U373 (U373-Ctrl), NGF-pre-treated U373 (U373-NGF), and NGF-pre-treated U373 silenced for apoE (U373-NGF ApoE siRNA) for 48 h. n = 5 different experiments. Data represent means ± SD. Statistical analysis was assessed by using one-way ANOVA, followed by Tukey’s post hoc test. ** p < 0.01, *** p < 0.001.

Article Snippet: ApoE mRNA silencing was performed on U373 cells using ApoE siRNA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-29708) according to the manufacturer’s instructions.

Techniques: Cell Culture, Derivative Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: NGF Modulates Cholesterol Metabolism and Stimulates ApoE Secretion in Glial Cells Conferring Neuroprotection against Oxidative Stress

doi: 10.3390/ijms23094842

Figure Lengend Snippet: ApoE secretion by NGF-treated U373 cells provides neuroprotection from oxidative stress. ( A ) Representative scheme of astrocyte–neuron co-cultures set up as described in the Materials and Methods Section. Neurite-bearing cells, neurite length, and cell number were assessed 48 h after the establishment of co-culture. ( B ) Representative images in bright field and quantitative assessment of neuronal morphology of N1E-115 cells, previously treated (+) or not (−) with rotenone (0.1 µM) for 16 h. N1E-115 were then kept in fresh DMEM (Ctrl), or co-cultured with control U373 (U373-Ctrl), NGF-pre-treated U373 (U373-NGF), and NGF-pre-treated U373 silenced for ApoE (U373-NGF ApoE siRNA) for 48 h. n = 5 different experiments. Data represent means ± SD. Statistical analysis was carried out by using one-way ANOVA, followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: ApoE mRNA silencing was performed on U373 cells using ApoE siRNA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-29708) according to the manufacturer’s instructions.

Techniques: Co-Culture Assay, Cell Culture, Control

Journal: Cardiovascular research

Article Title: Leptin promotes neointima formation and smooth muscle cell proliferation via NADPH oxidase activation and signalling in caveolin-rich microdomains.

doi: 10.1093/cvr/cvt126

Figure Lengend Snippet: Figure 3 The effect of apoE down-regulation on leptin-induced signalling in HASMCs. ApoE expressionin HASMCs wastransiently reducedusing apoE- specific siRNAs. Representative examples of the apoE (A) mRNA and (B) protein expression 72 h after transfection with apoE or scrambled (scr) control siRNAand the summaryof threetofiveindependent experiments areshown.(C) Flowcytometry revealedthatapoE siRNA transfectiondid notalter ObR expression. The statistical analysis and representative histograms of four independent experiments are shown. Western blot analysis was performed to examine the effect of apoE down-regulation on the leptin-induced (100 ng/mL) increase in (D) p47phox phosphorylation (four to six independent experi- ments). **P , 0.01 and ***P , 0.001 vs. control-treated, src siRNA-transfected cells; ##P , 0.01 vs. leptin-stimulated, scr siRNA-transfected cells.

Article Snippet: HASMCs were transfected with siRNA against either human apoE, cav-1, or scrambled (scr) siRNA (all Santa Cruz Biotechnologies) using metafectene SI transfection reagent (Biontex) and analysed 72 h later.

Techniques: Expressing, Transfection, Control, Western Blot, Phospho-proteomics

Journal: Cardiovascular research

Article Title: Leptin promotes neointima formation and smooth muscle cell proliferation via NADPH oxidase activation and signalling in caveolin-rich microdomains.

doi: 10.1093/cvr/cvt126

Figure Lengend Snippet: Figure 4 Importance of apoE expression, secretionand cell surfacebinding in mediating the effects of leptin. HASMCs were transfectedwith apoE or scr control siRNA and the effects of leptin (100 ng/mL) on (A) cell proliferation, (B) cyclin D1 mRNA expression (after normalization for b-actin mRNA ex- pression)and(C)ROSformationexamined.Resultsareexpressed-foldincreaseofcontrol-treatedcells(setat1).Proliferationwasalsoexaminedafter(D) incubation with CdM from apoE siRNA-transfected HASMCs or (E) pre-treatment of HASMCs with heparinase. *P , 0.05, **P , 0.01 and ***P , 0.001 vs. control-treated cells; #P , 0.05 and ##P , 0.01 vs. leptin-stimulated, scr siRNA-transfected cells.

Article Snippet: HASMCs were transfected with siRNA against either human apoE, cav-1, or scrambled (scr) siRNA (all Santa Cruz Biotechnologies) using metafectene SI transfection reagent (Biontex) and analysed 72 h later.

Techniques: Expressing, Control, Incubation, Transfection

Journal: Cardiovascular research

Article Title: Leptin promotes neointima formation and smooth muscle cell proliferation via NADPH oxidase activation and signalling in caveolin-rich microdomains.

doi: 10.1093/cvr/cvt126

Figure Lengend Snippet: Figure 5 Effects of leptin on the activation and subcellular localization of cav-1 in HASMCs. (A) Stimulation of HASMCs with leptin (100 ng/mL) time dependently increased the phosphorylation of cav-1. Summarized results (eight independent experiments) and representative western blots are shown. *P , 0.05 and **P , 0.01 vs. unstimulated cells set at 1. (B) Leptin enhanced the membrane localization of cav-1 (green signal). Compare findings in leptin- vs. control-stimulated HASMCs (lower vs. middle row). Results after omission of the first antibody are also shown (upper row). Size bars denote 25 mm. (C) Fractionation of HASMCs followed by western blot detection of cav-1 confirmed membrane accumulation of cav-1 (upper row) in response to leptin. Membranes were stripped and restained using antibodies against pan-cadherin, HSP70, or histone H1 as markers for the cell membrane, cytosol or nucleus, respectively. (D) Cav-1 immunoprecipitation followed by western blot detection of ObR, Src kinase, or cav-1 in scr or apoE siRNA-treated HASMCs after stimulation with leptin (100 ng/mL) or saline (control). (E) Transfection of HASMCs with apoE siRNA abolished the leptin-induced phos- phorylation of cav-1. Summarized results (four independent experiments) as well as representative western blots are shown. **P , 0.01 vs. unstimulated cells, #P , 0.05 vs. leptin-stimulated, scr siRNA-transfected cells.

Article Snippet: HASMCs were transfected with siRNA against either human apoE, cav-1, or scrambled (scr) siRNA (all Santa Cruz Biotechnologies) using metafectene SI transfection reagent (Biontex) and analysed 72 h later.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Membrane, Control, Fractionation, Immunoprecipitation, Saline, Transfection

Journal: Cardiovascular research

Article Title: Leptin promotes neointima formation and smooth muscle cell proliferation via NADPH oxidase activation and signalling in caveolin-rich microdomains.

doi: 10.1093/cvr/cvt126

Figure Lengend Snippet: Figure 6 Effects of leptin in cav-1 siRNA-treated HASMCs or mice lacking cav-1. (A) HASMCs were transfected with siRNAs to transiently reducecav-1 expression.Summarizedresults(n ¼ 4;***P , 0.001vs.scrsiRNA-transfectedcellssetat1)andrepresentativefindingsareshown.(B)Cav-1siRNAtrans- fectionabolishedtheeffectsofleptinoncyclinD1mRNAexpression(sixindependentexperiments).**P , 0.01vs.control-treated,scrsiRNA-transfected cells, ###P , 0.001 vs. leptin-treated, scr siRNA-transfected cells. (C) Morphometric analysis of lumen stenosis after vascular injury in control- or leptin- treated (via osmotic pumps) cav-1-deficient mice (n ¼ 4 per group). Size bars denote 50 mm.

Article Snippet: HASMCs were transfected with siRNA against either human apoE, cav-1, or scrambled (scr) siRNA (all Santa Cruz Biotechnologies) using metafectene SI transfection reagent (Biontex) and analysed 72 h later.

Techniques: Transfection, Expressing, Control