Supplementary Figure 2: Determination of LINK-A-PIP₃ and LINK-A-Ins (1,3,4,5)P₄ interactions by giant unilamellar vesicles and MS2-TRAP.

(a and b) Fluorescence imaging of interaction between DOPC lipid vesicles (visualized by Nile Red) and indicated lncRNA (visualized by YOYO-1) (a) or YOYO-1 dye only (b). Left panel: Representative images. Scale bars, 50 nm. Middle and right panels: fluorescence intensities along the diagonal bars (middle panel) and intensity correlation between two channels (right panel) were shown. (c) Overlap coefficient between channel 1 (Nile Red) and channel 2 (YOYO-1) was calculated based on the number of giant lipid vesicles, LINK-A (n = 34 lipid vesicles), BCAR4 (n = 19 lipid vesicles), Lnc-131 (n = 21 lipid vesicles), and H19 (n = 7 lipid vesicles) (median, one-way ANOVA, ***P < 0.001). (d and g) Relative expression level of MS2-tagged full-length LINK-A (d) or ΔPIP₃ deletion mutant (g) in MDA-MB-231 cells was detected by RT–qPCR. (e and h) ELISA assay detecting the conversion of PIP₂ to PIP₃ by immunoprecipitated PI3K p110α from cells pretreated with DMSO, PI(1,4,5,6)P₄ or PI(1,3,4,5,6)P₅ (100 μM, 2 h) (e) or transfected with MS2-tagged full-length LINK-A or ΔPIP₃ deletion mutant (h). (f and i) Immunoblotting detection of immunoprecipitated PI3K p110α from cells pretreated with DMSO, PI(1,4,5,6)P₄ or PI(1,3,4,5,6)P₅ (100 μM, 2 h) (f) or transfected with MS2-tagged full-length LINK-A or ΔPIP₃ deletion mutant (i). For d, e, g and h, mean ± s.e.m. were derived from n = 3 independent experiments (n.s. P > 0.05 and ***P < 0.001, two-tailed paired Student’s t-test). Statistics source data for a are in Supplementary Table 6. Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.