Irvalec Inserts into the Plasma Membrane Causing Rapid Loss of Integrity and Necrotic Cell Death in Tumor Cells
Figure 6
Interactions between Irvalec and the plasma cell membrane.
For these experiments, A549 cells were grown on LabTek chambered slides; 2P-FLIM images are representative of different experiments. (A) Visualization of changes in the plasma cell membrane organization in A549 cells without (I and II) or after treatment (III and IV) with 2 µM Irvalec. A549 cells were labelled with 1 µM PA-DPH and excited with polarized light microscopy. The direction of the linear polarized excitation beam is shown in the figure with red arrows: Horizontal (I and III) and Vertical (II and IV); Emission filter: 483/32; I–II: (XY section, Z = 0), III–IV (XY section, Z = 5 µm) (B) Localization of Irvalec in the cell membrane; A549 cells were treated with Irv-OG488 or Irv-A555 and analyzed by two-photon time-resolved fluorescence microscopy; I: untreated cells (XY section, Z = 0); II: cells treated with 0.6 µM of Irv-OG488 (XY section, Z = 0); III: cells treated with 2.2 µM of a mix of Irv-OG488 and Irvalec (1∶4.5) (XY image, Z = 0); IV and V: cells treated with 0.4 µM of a mix of Irv-OG488 and Irvalec (1∶3) (IV: XY image, Z = 5 µm; V: XZ section, Y position defined by white arrow in IV); VI: cells treated with 2 µM of a mix of Irv-A555 and Irvalec (1∶4). (XY section, Z = 10 µm); emission bandpass filters; I–V: FF01-520/35; VI: FF01-607/36; Total intensity scale in arbitrary units: 0–1(I, IV–VI); 0–2(II); 0–50 (III). (C) Representative example of time-resolved fluorescence resonance energy-transfer (FLIM-FRET) experiments in A549 cell exposed to a mix of 0.6 µM Irv-OG488, 1.8 µM Irv-A555 and 2.4 µM Irvalec; I (XY section, Z = 0) and II (YZ section, X position defined by white arrow in I): donor channel image (OG488); III (XY section, Z = 0) and IV (YZ section, X position defined by white arrow in III): acceptor channel image (A555); donor filter: FF01 520/35; acceptor filter: FF01 685/40; dichroic beam splitter: FF560-Di01; total intensity scale in arbitrary units: 0–7 (I–II); 0–1 (III–IV). Scale bar: 10 µm.