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1.
Fig. 8

Fig. 8. HerGa-mediated superoxide generation disrupts the cytoskeleton. From: A Mechanistic Study of Tumor-Targeted Corrole Toxicity.

Where indicated, MDA-MB-435 cells were incubated with the indicated concentrations of Tiron for 1h before treatment with 1 uM HerGa or Mock (PBS) treatment for 24h, followed by fixation and immunofluorescence processing to label actin (red), tubulin (green), and nuclei (blue). Cells were imaged as described in Fig. 6. Bar, ~20 microns.

Jae Youn Hwang, et al. Mol Pharm. ;8(6):2233-2243.
2.
Fig. 6

Fig. 6. HerGa disrupts the cytoskeleton. From: A Mechanistic Study of Tumor-Targeted Corrole Toxicity.

A, MDA-MB-435 cells treated with S2Ga or HerGa at 1uM final corrole concentration, or HerPBK10 alone at equivalent protein concentration to HerGa, were assessed for cytoskeletal changes by 24h of treatment by fluorescence labeling of actin (red) or tubulin (green). Blue, nucleus. Epi-fluorescence imaging was performed using filter cubes to detect DAPI (ex: 380nm and em: 400nm), FITC (ex: 488nm and em: 530nm), CY3 (ex: 550nm and em: 580nm), and corrole (ex: 425nm and em: 620nm), and images collected by a 40x objective (Nikon Planfluor, NA: 0.75). To increase image contrast, background subtraction from each acquired image was performed using ImageJ. Bar, ~20 microns.

Jae Youn Hwang, et al. Mol Pharm. ;8(6):2233-2243.
3.
Fig. 7

Fig. 7. Microtubule stabilization abrogates events downstream of HerGa uptake. From: A Mechanistic Study of Tumor-Targeted Corrole Toxicity.

MDA-MB-435 cells were incubated in media containing 5 uM taxol for 15 min before exposure to HerGa (see Methods). At 24h after exposure, cells received (A) 20nM TMRM and were monitored for mitochondrial TMRM accumulation as described previously; or (B) luminol and assayed for superoxide levels as described in the Methods. *, P<0.0001 compared to HerGa treatment. **, P<0.0001. Statistical significances determined by two-tailed unpaired t-tests. C–D, Effect of taxol on HerGa uptake. C, Confocal images were acquired of live MDA-MB-435 cells after treatment with 5uM HerGa (−/+ pretreatment with 5 uM taxol; see Methods). D, Cytosolic fluorescence from the same cell region per time point was measured and average fluorescence variations plotted over time, comparing HerGa −/+ taxol.

Jae Youn Hwang, et al. Mol Pharm. ;8(6):2233-2243.
4.
Fig. 1

Fig. 1. HerPBK10 is required for enhanced internalization & cell death. From: A Mechanistic Study of Tumor-Targeted Corrole Toxicity.

A, MDA-MB-435 cells were exposed to either HerGa or S2Ga (1 uM final corrole concentration) and imaged live by fluorescence confocal microscopy. Micrographs show fluorescence and brightfield overlays at key time points of uptake. B shows a comparison of fluorescence images acquired at 1h after HerGa or S2Ga uptake. C, Quantification of uptake in A. The cytosolic accumulation of fluorescence in each cell was quantified by selecting cytosolic regions and averaging fluorescence intensity using Image J. D, Cell death dose curve. MDA-MB-435 cells were incubated with HerGa or S2Ga at the indicated doses for 24h before cell survival was assayed by crystal violet (CV) stain. Cell survival is expressed as CV absorbance of each HerGa-treated sample normalized by mock (PBS) treated samples, or CV abs of experimental/control. Error bars represent 1 SD of triplicate treatments.

Jae Youn Hwang, et al. Mol Pharm. ;8(6):2233-2243.
5.
Fig. 2

Fig. 2. HerGa impacts mitochondrial membrane permeability. From: A Mechanistic Study of Tumor-Targeted Corrole Toxicity.

MDA-MB-435 cells exposed to HerGa or S2Ga (at 1 uM corrole concentration), HerPBK10, or PBS received 20nM TMRM in PBS at 24h after treatment, followed by two-photon excited confocal fluorescence imaging after TMRM uptake. A, Cells displaying respective fluorescences as the z-stacked maximum intensity projection of each acquired fluorescence image. B (upper), Mitochondria/cytoplasm ratio of TMRM fluorescence averaged from three independent experiments represented by A (TMRM uptake). For each experiment, the average fluorescence intensity for 10 different mitochondria regions and one mitochondria-free region (cytoplasm) within the same cells were measured respectively. B (lower), Timecourse of membrane collapse. TMRM uptake during HerGa treatment was assessed as described in the Methods with mitochondrial/cytoplasm TMRM fluorescence ratios obtained at the indicated timepoints after HerGa treatment. *, P<0.05 compared to (B, upper) mock (PBS) treatment, or (B, lower) time point 0, as determined by 2-tailed unpaired t-test.

Jae Youn Hwang, et al. Mol Pharm. ;8(6):2233-2243.
6.
Fig. 3

Fig. 3. ΔΨ(m) disruption requires endosome escape. From: A Mechanistic Study of Tumor-Targeted Corrole Toxicity.

A, Linear protein domain map comparing HerPBK10 and endosomolytic-deficient derivatives. Each protein is shown from carboxy (left) to amino (right) –terminus, respectively. Complexes made from combining S2Ga with truncated proteins, HerK10 and Her, are designated HerGa-2 and HerGa-3, and are compared to parental HerGa for the effect of each on mitochondrial uptake of TMRM and cell survival. B, TMRM and complex uptake in MDA-MB-435 cells. Cells were treated with 1 uM (final corrole dose) of each complex for 24 hours before media exchange and imaging. Where indicated, the media was replaced with 20nM TMRM in PBS and imaged after mitochondrial TMRM accumulation reached equilibrium (~1h). Both TMRM and corrole uptake images were collected by two-photon excited confocal fluorescence microscopy. Fluorescence micrographs show z-stacked maximum intensity projections of acquired images. Fl/BF, Fluorescence-brightfield overlay. C, Quantification of TMRM uptake in B (see methods). D, Relative cell survival (determined by crystal violet stain; see Methods) after treatment in B. *, P<0.05 compared to HerGa (two-tailed unpaired t-test).

Jae Youn Hwang, et al. Mol Pharm. ;8(6):2233-2243.
7.
Fig. 5

Fig. 5. Cytosolic HerGa induces superoxide generation. From: A Mechanistic Study of Tumor-Targeted Corrole Toxicity.

MDA-MB-435 cells treated with 1 uM HerGa, S2Ga, HerPBK10, or PBS (mock) for 1h were analyzed for superoxide via chemiluminescence detection (see Methods). Graphs (A–C) displays relative luminescence unit (RLU) decay over time. A, Contribution of endosomolysis on superoxide generation. MDA-MB-435 cells were treated and assayed for chemiluminescence as described earlier. HerGa, HerGa-2, HerGa-3, and S2Ga were added to cells at 1 uM corrole concentration. Individual components were added at the equivalent protein concentration to each respective complex. B, Lack of superoxide generation in a cell-free system. HerGa (1 uM) and equivalent concentrations of S2Ga, HerPBK10, or PBS were added directly to superoxide anion assay medium and luminol oxidation was measured as described in the Methods. C, Reduction of HerGa-mediated superoxide generation in MDA-MB-435 cells by the superoxide scavenger, Tiron. Cells were incubated with 1 mM Tiron for 1h before treatment with 1 uM HerGa for 24h, followed by superoxide measurement as described earlier. D, Effect of Tiron on HerGa-mediated ΔΨ (m) disruption. Cells were incubated with the indicated concentrations of Tiron before treatment with 1 uM HerGa for 24h, followed by measurement of TMRM uptake as described earlier. *, P<0.02 compared to HerGa alone, as determined by two-tailed unpaired t test.

Jae Youn Hwang, et al. Mol Pharm. ;8(6):2233-2243.
8.
Fig. 4

Fig. 4. Effect of pH on corrole retention and fluorescence lifetime. From: A Mechanistic Study of Tumor-Targeted Corrole Toxicity.

A, Measurement of HerGa fluorescence lifetime in titrating pH buffers. Fluorescence lifetime of 25uM HerGa solutions in different pH (5.0, 5.5, 6.0, 6.5, 7.0, and 7.5) was measured at room temperature and 37°C respectively, in a cell-free system. During the measurements, the temperature was strictly (~0.1°C) controlled by a ΔT culture dish system. Before adding the corroles to the chamber, the pH of each corrole solution was confirmed with a pH meter. B, Monitoring fluorescence lifetime changes of HerGa during uptake into MDA-MB-435 cells. Fluorescence lifetime images of HerGa were acquired at different time points (5, 10, 20, 30, 50, 70, and 90min) after addition of 25 uM HerGa into the delta T chamber containing attached MDA-MB-435 cells. A total of 25 images were acquired (0–4800ps; Time step: 200ps; gate width: 600ps, Ex: 424nm, light pulse width: 100fs). The images were analyzed (lower graph) using the first order exponential decay fitting method. C, Monitoring HerGa fluorescence as a reflection of uptake kinetics. Two-photon fluorescence imaging-enabled acquisition and analysis of HerGa (25 uM) at various depths during uptake in MDA-MB-435 cells. Images were acquired at different time points of HerGa uptake (5, 15, 30, and 60 minutes after addition to cells) and at different z-depths with a step size of 350nm (Ex: 780nm; Em: 600–650nm). Micrographs show images at 6um depth at indicated time points during HerGa uptake. The graph shows the fluorescence intensity z-depth profile of the region selected by a dotted circle in the first micrograph. D, Evaluating corrole retention under decreasing pH conditions. The acidity of a HEPES-buffered saline solution was adjusted to the indicated pH levels and pre-assembled HerGa was added to each pH buffer and incubated for 30 min at room temp (in a cell-free system), followed by filtration through 10K mwco membranes to remove any released corrole. The absorbances at 424 nm (maximum absorbance wavelength of gallium corrole) were obtained from the preassembled complex before incubation with each pH buffer (“Input”) and the filtered complex recovered from the ultrafiltration device after incubation with each pH buffer (“Retentate”). Error bars represent SD of repeat experiments. N=2–3 samples/pH per experiment.

Jae Youn Hwang, et al. Mol Pharm. ;8(6):2233-2243.

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