Figure 1.
Effect on HL-60 cells and RBCs after α-HL. (A) Intensity of the nucleus (in blue color) staining with DAPI dye (λexcitation= 405 nm, λemission= 480–510 nm) increased for the first couple of minutes, where the three nuclear lobes (white arrowhead) became clearly visible due to the uptake of the dye from the outside medium. After some time (5 min), the intensity of the nucleus started decreasing, and three distinct lobes of the nucleus started disappearing. Nile red intensity (λexcitation= 540 nm, λemission= 565–610 nm) corresponding to the membrane (in red color) started decreasing rapidly just after toxin treatment at a 100 nM toxin concentration. (B) Toxin treatment showed membrane protrusion (in red color) from HL-60 cells at 100 nM toxin concentration. (C) Flow cytometry analysis of 10 nM, 100 nM, and 1 µM toxin-treated HL-60 cells stained with PI. Compared with the untreated cells, the toxin-treated cells started becoming necrotic, as evident by the increase in PI-positive cells. Data are shown as the mean ± SD of triplicate measurements (n = 3 biological triplicates). (D) Concentration-dependent hemolysis of rabbit erythrocytes (10%) was monitored after adding different concentrations of purified monomeric α-HL at 37°C. (E) Different stages of RBC damage were captured using NS-TEM after adding the toxin: i. untreated control RBC; ii. damaged membrane of RBCs after α-HL addition; iii. zoomed view of the membrane area showed circular-shaped particles (yellow encircled); iv. 2D class averages of the particles confirmed the circular shape of the toxin (scale bar: 10 nm); v. lysed cell after completion of hemolysis; and vi. a zoomed view of the lysed-membrane area showed circular-shaped particles (yellow encircled). (F) Toxin-incubated RBC samples were imaged under a confocal microscope at different time points: (a) just after toxin addition; (b) starting point of membrane damage 2 mins post-toxin addition; (c) lysis initiation state after 5 mins; and (d) mostly lysed cells after 10 min. The cell membranes (in red color) were stained using Nile red dye (λexcitation= 540 nm, λemission= 565–610 nm). (G) Correlation of toxin-mediated damage in RBCs using high-resolution cryo-EM imaging with the confocal data (shown in top left). The protruded portions’ size from cells under confocal and cryo-EM in the left side panel was around 1 µm. The ratio of width to length of cells under confocal and cryo-EM was 1.2 in the middle panel. The ratio of width to length of cells under confocal and cryo-EM was 0.74 in the right side panel. (H) SDS-PAGE of RBC treated with α-HL (from left side to right side, control RBC, RBC pellet after toxin incubation, supernatant from the toxin-incubated sample, protein marker, supernatant sample after heating). RBCs, red blood cells. Source data are available for this figure: SourceData F1.

Effect on HL-60 cells and RBCs after α-HL. (A) Intensity of the nucleus (in blue color) staining with DAPI dye (λexcitation= 405 nm, λemission= 480–510 nm) increased for the first couple of minutes, where the three nuclear lobes (white arrowhead) became clearly visible due to the uptake of the dye from the outside medium. After some time (5 min), the intensity of the nucleus started decreasing, and three distinct lobes of the nucleus started disappearing. Nile red intensity (λexcitation= 540 nm, λemission= 565–610 nm) corresponding to the membrane (in red color) started decreasing rapidly just after toxin treatment at a 100 nM toxin concentration. (B) Toxin treatment showed membrane protrusion (in red color) from HL-60 cells at 100 nM toxin concentration. (C) Flow cytometry analysis of 10 nM, 100 nM, and 1 µM toxin-treated HL-60 cells stained with PI. Compared with the untreated cells, the toxin-treated cells started becoming necrotic, as evident by the increase in PI-positive cells. Data are shown as the mean ± SD of triplicate measurements (n = 3 biological triplicates). (D) Concentration-dependent hemolysis of rabbit erythrocytes (10%) was monitored after adding different concentrations of purified monomeric α-HL at 37°C. (E) Different stages of RBC damage were captured using NS-TEM after adding the toxin: i. untreated control RBC; ii. damaged membrane of RBCs after α-HL addition; iii. zoomed view of the membrane area showed circular-shaped particles (yellow encircled); iv. 2D class averages of the particles confirmed the circular shape of the toxin (scale bar: 10 nm); v. lysed cell after completion of hemolysis; and vi. a zoomed view of the lysed-membrane area showed circular-shaped particles (yellow encircled). (F) Toxin-incubated RBC samples were imaged under a confocal microscope at different time points: (a) just after toxin addition; (b) starting point of membrane damage 2 mins post-toxin addition; (c) lysis initiation state after 5 mins; and (d) mostly lysed cells after 10 min. The cell membranes (in red color) were stained using Nile red dye (λexcitation= 540 nm, λemission= 565–610 nm). (G) Correlation of toxin-mediated damage in RBCs using high-resolution cryo-EM imaging with the confocal data (shown in top left). The protruded portions’ size from cells under confocal and cryo-EM in the left side panel was around 1 µm. The ratio of width to length of cells under confocal and cryo-EM was 1.2 in the middle panel. The ratio of width to length of cells under confocal and cryo-EM was 0.74 in the right side panel. (H) SDS-PAGE of RBC treated with α-HL (from left side to right side, control RBC, RBC pellet after toxin incubation, supernatant from the toxin-incubated sample, protein marker, supernatant sample after heating). RBCs, red blood cells. Source data are available for this figure: SourceData F1.

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