Fig. 7: Membrane-tethered intralumenal siRNA-LNP colocalize with recruited membrane damage sensors.

HeLa cells expressing GFP-CHMP2A and mScarlet-EEA1 or mScarlet-Rab5, or YFP-galectin-9 and mScarlet-Rab5 were incubated with 100 nM AF647¼-siRNA-LNPs for 90 min. Cells were fixed with PFA and imaged using Airyscan confocal microscopy. a, b Representative image details showing the spatial relationship of CHMP2A or galectin-9 recruitment to single or multiple membrane-adjacent intralumenal AF647-siRNA-LNP (indicated by color-coded stars). Brightness and contrast were adjusted individually for all images. Scale bars are 500 nm. c–e EEA1⁺ or Rab5⁺ endosomes with membrane-adjacent intralumenal siRNA-LNP and membrane-associated CHMP2A or galectin-9 were identified, and a vector from the center of the endosome to the respective focus was computed. The vector magnitude difference and angular difference between the LNP and galectin-9 or CHMP2A vectors were calculated, in endosomes with one or two pairs of siRNA-LNP and CHMP2A or galectin-9 maxima. d Calculated angular difference between LNP and galectin-9 or CHMP2A vectors, in endosomes with one or two paired siRNA-LNP and CHMP2A or galectin-9 maxima. Circles are paired vectors (pooled), N = 30, 33 or 30 single paired maxima and 12, 16 or 10 dual paired maxima, for CHMP2A vs. EEA1, CHMP2A vs. Rab5 and galectin-9 vs. Rab5, respectively. Bars are median ± i.q.r. e Calculated vector magnitude difference between CHMP2A or galectin-9 and siRNA-LNP vectors. Values > 0 indicate greater distance from center of endosome to CHMP2A or galectin-9 focus than to siRNA-LNP focus. Bars are median ± i.q.r. Circles are paired vectors (pooled), N = 42, 49 or 40 paired maxima for CHMP2A vs. EEA1, CHMP2A vs. Rab5 and galectin-9 vs. Rab5, respectively. a–e All data and images are from 2 (CHMP2A vs. EEA1) or 3 (CHMP2A vs. Rab5 and galectin-9 vs. Rab5) independent experiments, with 15–20 cells analyzed for each condition. f HeLa cells expressing mCherry-galectin-9 were treated with 100 nM AF647-siRNA BODIPY-MC3 LNPs for 1.5–4 h and imaged continuously using VT-iSIM. BODIPY-MC3 (free lipid) was acquired at 488/525 nm excitation/emission. Images are representative of 2 independent experiments. Brightness and contrast were adjusted separately for all images. Scale bar is 500 nm.