The image contains phasor plots illustrating the changes in normalized fluorescence intensity and F R E T efficiency at the plasma membrane (P M) post cholesterol supplementation using W S C in t s A 201 cells and rat D R G neurons. The plots are divided into two main sections: (A) and (B). Section (A) shows data for naive D R G neurons at different time points before and after W S C application, with F R E T efficiency values of 0.17, 0.18, 0.18, and 0.18 at respective times. Section (B) shows data for S N I D R G neurons with F R E T efficiency values of 0.05, 0.13, 0.14, and 0.16. Each plot includes axes labeled G and S, with time points marked in nanoseconds. The data points are color-coded and clustered around specific regions, indicating changes in P M properties over time. The plots also include regression lines and reference points for better visualization of trends. All values are approximated.
Distinct modes of PM modulation by cholesterol enrichment. (A) Strategies implemented to report the PM properties after WSC-mediated cholesterol supplementation. An mCherry-labeled OlyA was used to recognize outer leaflet cholesterol/SM complexes, whereas GRAM-W-eGFP was used to label free cholesterol levels in the inner leaflet of the PM. The OMD size was probed using FRET-based reporters, either CTxB-conjugated or peptide (L10)-based FRET pairs. (B) Time course of the change in normalized fluorescence intensity and the FLIM-FRET efficiency at the PM after 0.5 mg/ml WSC application in tsA201 cells, using four different probes as illustrated in panel A. Data were collected every 5 min to minimize photobleaching and shown as mean ± SEM, n = 4–10 cells. (C and D) Summary time course of the change in normalized GRAM-W fluorescence intensity at the PM and the FLIM efficiency of CTxB-AF488/AF647 FRET after 0.5 mg/ml WSC application for naïve rat DRG neurons (C) and for nociceptor rat DRG neurons of the SNI model (D). Data shown are mean ± SEM, n = 4–6 cells. The CTxB-based FRET level showed a significant increase after WSC for SNI DRG neurons (*P = 0.02, 0.02, and 0.03 at 5, 10, and 15 min time points using a two-sided paired t test) but no change for naïve neurons (n.s., no statistical significance).