topfluor pi4p  (Avanti Polar)

Journal: Nature Cell Biology

Article Title: Lipid droplet-associated lncRNA LIPTER preserves cardiac lipid metabolism

doi: 10.1038/s41556-023-01162-4

Figure Lengend Snippet: a , RT-qPCR analysis of LIPTER , U6 (nuclear RNA) and GAPDH (cytosolic RNA) expression levels in fractionated nuclei and cytoplasm of hiPSC-CMs. Bars represent mean values ± s.e.m. (n = 3 independent experiments). b , Representative images showing no detectable interactions of giant lipid vesicles generated by TopFluor-PI with Alexa594-labeled LIPTER or Antisense -LIPTER (AS- LIPTER ). The experiment was carried out 3 times with similar outcomes. c , A specific protein band only pulled down by LIPTER- MS2 in HEK293T cells and visualized in SDS PAGE gel by silver staining. MS2 binding protein was FLAG-tagged. The experiment was carried out 2 times with similar outcomes. d , Western blotting confirming MYH10 pulldown by LIPTER in HEK293T cells. The experiment was carried out 2 times with similar outcomes. e , 3D confocal immunofluorescent images showing colocalization of LIPTER YFP-MS2 , MYH10, and LD in WT hiPSC-CMs (also see Supplementary Video ). The lower right graph indicates the view of merged images. The experiment was carried out 3 times with similar outcomes. f , Immunofluorescent images showing MYH10-ACTIN cytoskeleton in WT hiPSC-CMs. The experiment was carried out 3 times with similar outcomes. Source numerical data and unprocessed blots are available in source data.

Article Snippet: TopFluor TMR PA (Avanti Polar Lipids, 810240) and TopFluor PI4P (Avanti Polar Lipids, 810185) were dissolved in RNA–lipid binding buffer (100 nM), and a final concentration of 25 nM of each was used.

Techniques: Quantitative RT-PCR, RNA Expression, Generated, Labeling, SDS Page, Silver Staining, Binding Assay, Western Blot

Journal: Nature Cell Biology

Article Title: Lipid droplet-associated lncRNA LIPTER preserves cardiac lipid metabolism

doi: 10.1038/s41556-023-01162-4

Figure Lengend Snippet: a , RNA FISH detecting cytosolic co-localization of LIPTER with LDs stained by Oil Red O staining. WT and LIPTER KO hiPSC-CMs were treated with palmitic acid (200 μM) for 6 h to induce LD formation. b , Quantification of LIPTER co-localization with LDs in WT hiPSC-CMs ( n = 6 independent experiments). c , RT–qPCR detection of LIPTER and ACTB RNA enrichments in total lipids isolated from WT hiPSC-CMs ( n = 3 independent experiments). d , RNA–lipid overlay assay showing selective interaction of LIPTER with PA and PI4P. AS- LIPTER is control. PS, phosphatidylserine; PE, phosphatidylethanolamine; DAG, diacylglycerol; cholesterol; PC, phosphatidylcholine; sphingomyelin; PG, phosphatidylglycerol. e , Interactions between giant lipid vesicles formed with TopFluor-labelled PA/PI4P and Alexa594-labelled LIPTER /AS- LIPTER . f , g , MST quantifying PA ( f ) and PI4P ( g ) interactions with LIPTER or AS- LIPTER . h , Schematic of the MS2-BioTRAP system for LIPTER binding protein pulldown and live cell LIPTER tracing. i , Top: MS2 YFP protein binds MS2-tagged LIPTER to form particles (purple arrows), co-localizing with Rhodamine B-palmitic acid-labelled LDs (red arrows) in WT hiPSC-CMs (yellow arrows). Bottom: WT hiPSC-CMs expressing MS2 YFP protein and empty MS2-tag vector show evenly distributed YFP without formation of particles. j , Western blot showing MYH10 pulldown by anti-GFP antibody in hiPSC-CMs expressing MS2- LIPTER /-AS- LIPTER and MS2 YFP , representative of three independent experiments. k , LIPTER enrichment by anti-MYH10 antibody in WT hiPSC-CMs ( n = 3 independent experiments). l , Schematic of truncated LIPTER fragments. m , RNA–lipid overlay assay detecting interactions of truncated LIPTER with PA and PI4P. n , o , Images showing interactions of giant lipid vesicles formed by TopFluor-PA ( n ) and TopFluor-PI4P ( o ) with Alexa594-labelled exon 1 + 2 and exon 3 of LIPTER . p , Western blot of MYH10 pulldown in HEK293T cells transfected with MS2-tagged LIPTER fragments and MS2 FLAG . q , Confocal fluorescence images showing co-localizations of LIPTER- MS2 YFP , MYH10 and LDs in WT hiPSC-CMs. r , Model of human intramyocyte LD transport system via LIPTER and MYH10-ACTIN cytoskeleton. In b , c and k , bars are presented as mean ± s.e.m. Unpaired two-tailed t -test is used for comparison. Source numerical data and unprocessed blots are available in source data.

Article Snippet: TopFluor TMR PA (Avanti Polar Lipids, 810240) and TopFluor PI4P (Avanti Polar Lipids, 810185) were dissolved in RNA–lipid binding buffer (100 nM), and a final concentration of 25 nM of each was used.

Techniques: Staining, Quantitative RT-PCR, Isolation, Overlay Assay, Control, Binding Assay, Expressing, Plasmid Preparation, Western Blot, Transfection, Fluorescence, Two Tailed Test, Comparison