Fig. 5: LIPTER selectively binds PA and PI4P on LDs and MYH10 protein.
From: Lipid droplet-associated lncRNA LIPTER preserves cardiac lipid metabolism
a, RNA FISH detecting cytosolic co-localization of LIPTER with LDs stained by Oil Red O staining. WT and LIPTERKO 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: MS2YFP 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 MS2YFP 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 MS2YFP, 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 MS2FLAG. q, Confocal fluorescence images showing co-localizations of LIPTER-MS2YFP, 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.
