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. 2022 Jul 14;13:4102. doi: 10.1038/s41467-022-30917-0

Fig. 7. Cell cytotoxicity triggered by cis-GdAzo activation upon IR.

Fig. 7

a, b Confocal microscopy of cancer cell (PANC-1) in the presence of PI before (0 min) and 30 min after incubation with either inactive cis-GdAzo (a) or active trans-GdAzo (b) (4 repetitions, permeabilised cells in red). c, d Confocal microscopy of cancer cell (PANC-1) in the presence of PI at 2 min and 30 min after cis-GdAzo introduction followed by GR (d) or no (c) irradiation (permeabilised cells in red). e Quantification of cell permeabilisation (PANC-1) by confocal microscopy after treatment with cis-GdAzo upon GR (n = 6 biologically independent samples, processed by a custom script, t-values = 3.110, 2.941, 2.815 at 250, 500, 850 µM). The medians ± interquartile ranges are reported. f, g, Flow cytometry of cancer cells (CCRF-CEM-ARAC-8C) in the presence of PI and cis-GdAzo (absence (-) or 500 µM (+)) upon GR (g) or without (f) irradiation at 30 min post-treatment. h Quantification of cell permeabilisation (CCRF-CEM-ARAC-8C) by flow cytometry after treatment with cis-GdAzo upon GR at 30 min post-irradiation (n = 3 biologically independent samples, t-values = 5.254, 8.565 at 500, 850 µM). The relative increase in PI-positive events compared to medium without cis-GdAzo is represented. i, EELS-TEM on cancer cell (PANC-1) in the presence of trans-GdAzo. Gd was detected in localised cytoplasmic areas (circle, top EELS spectrum, keV axis unit) and not in the surrounding cytoplasm (bottom EELS spectrum), cell membrane or nucleus (arrow). j, k Cell viability of Gem-resistant cancer cells (CCRF-CEM-ARAC 8 C) 4 days after treatment with cis-GdAzo upon GR in the absence (j) or presence (k) of Gem (n = 3 biologically independent samples, t-values = 6.854, 8.258, 3.636, 3.557 at 0, 250, 500, 850 µM in the absence of Gem and 6.454, 4.927, 3.099 at 0, 250, 500 µM in the presence of Gem). l Representation of the impact of cis-GdAzo on cancer cells upon IR as assumed from the microscopy experiments. The means ± standard deviations are reported unless otherwise specified. Two-way Anova (Bonferroni post-test) was used for statistical analyses (NI vs GR). ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars = 125 µm.