UCD38B does not induce calcium-dependent necrosis.

<p>(<b>A</b>) LDH cell death assays were carried out with MDA-MB-231 cells that were treated with UCD38B and ionomycin (IM) for 24 hours with and without 1 hour pre-treatment with 10 µM BAPTA-AM. (<b>B</b>) Cell viability after 24 hours was measured in the absence and presence of 25 µM RIP1 necroptosis inhibitor necrostatin-1 using the MTT assay. (<b>C</b>) LDH cell death assays were carried out with cells treated with 8 µM shikonin or 250 µM UCD38B in the presence and absence of 25 µM necrostatin-1. Results depicted in each panel are representative of three independent experiments. *, <i>P<0.05</i>; **, <i>P</i><0.001.</p

UCD38 does not significantly increase Annexin V staining.

<p>MDA-MB-231 cells were treated as indicated with DMSO control, 50 µM cisplatin (CP) as an inducer of caspase-mediated apoptosis, or 250 µM UCD38B, and then analyzed by FACS for annexin V binding and propidium iodide uptake. The accumulation of cells in quadrant (Q2) is characteristic of apoptosis while the accumulation of cells in quadrant 1 (Q1) is characteristic of necrosis.</p

Differential cell permeability of amiloride derivatives.

<p>(<b>A</b>) The structures of amiloride and the two C(5) derivatives employed in these studies are depicted. The Bn in the UCD38B structure refers to a benzyl group. (<b>B</b>) MDA-MB-231 cells were incubated with vehicle control or 250 µM amiloride, UCD74A or UCD38B for 10 minutes and rinsed with PBS. Treated cells were then imaged by brightfield (upper panels) or fluorescence (lower panels) microscopy, the fluorescence intensities of 20 randomly selected cells were quantified, and averages with standard error are indicated.</p

UCD38B does not cause cell death via autophagy.

<p>(<b>A,B</b>) MDA-MB-231 cells were treated for 24 hours with increasing concentrations of UCD38B in the presence of DMSO control, 10 µM Compound C, or 25 µM chloroquine (CQ). (<b>A</b>) Lysates were immunoblotted for LC3 and actin, and (<b>B</b>) viability was determined using the MTT cell death assay. (<b>C,D</b>) Cells were transfected with scrambled control or Beclin1-directed siRNA oligonucleotides, and (<b>C</b>) lysates were immunoblotted for beclin1, LC3 and actin, and (<b>D</b>) viability was determined using the MTT cell death assay.</p

UCD38B-induced cell death occurs independently of the cell cycle and proliferation.

<p>MDA-MB-231 cells were left untreated (None) or were treated with scrambled (Scr) or cyclin D1 knockdown (KD) siRNA oligonucleotides for 72 hours. (<b>A</b>) Lysates were immunoblotted for cyclin D1 and actin proteins. (<b>B</b>) Cell proliferation after 24 hours was measured by MTT assay. (<b>C</b>) Cells treated with either scrambled control (squares) or knockdown (circles) oligonucleotides were exposed to increasing concentrations of UCD38B for 24 hours, and the fraction cell viability was determined using the MTT assay. Results depicted in each panel are representative of at least three independent experiments. *, <i>P</i><0.05.</p

Amiloride and UCD38B do not induce breast tumor cell death through caspase-dependent apoptosis.

<p>MDA-MB-231 cells were treated for 24 hours with various concentrations of (<b>A,B</b>) amiloride or (<b>C,D</b>) UCD38B. (<b>A,C</b>) Cell lysates were immunoblotted with antibodies to PARP to assess cleaved PARP (arrow) formation, and with antibodies specific for cleaved caspase-7. CP indicates treatment with 50 µM cisplatin. (<b>B,D</b>) The viability of MDA-MB-231 cells after 24 hours was measured by MTT after treatment with various concentrations of amiloride or UCD38B in the presence and absence of 1 µM z-VAD-fmk. (<b>E</b>) Cells were treated without and with 50 µM CP for 24 hours in the presence of either DMSO control or 1 µM unlabled z-VAD-fmk to block caspase labeling. Cells were then treated with FITC-z-VAD-fmk, and stained cells determined and quantified by fluorescence microscopy. (<b>F</b>) Cells were treated with 5 µM tamoxifen in the presence and absence of 1 µM z-VAD-fmk, as indicated, and cell viability was measured using an MTT assay. Results depicted in each panel are representative of at least three independent experiments.</p

AIF translocates to the nucleus upon treatment of cells with UCD38B.

<p>(<b>A</b>) MDA-MB-231 cells were treated with 500 µM amiloride, or 250 µM UCD38B for 2 hours, and cells were fractionated into nuclear (N) and cytosolic (C) components. Fractions were immunoblotted for CREB and histone H3 as nuclear markers, GAPDH as a cytosolic marker, and AIF. (<b>B</b>) Bands from the blot depicted in (A) were digitally quantified, and the nuclear AIF as a fraction of the total AIF was plotted for each condition. (<b>C</b>) Cells were treated for 2 hours with DMSO control or 250 µM amiloride, UCD74A or UCD38B. Cells were then fixed and stained with DAPI to visualize the nucleus (blue) and antibodies to AIF (green), and examined by confocal fluorescence microscopy. Results depicted in each panel are representative of 2–3 independent experiments.</p

AIF is required for efficient UCD38B-induced cell death.

<p>(<b>A</b>) MDA-MB-231 cells were left untreated, or were treated with scrambled control (scr) or AIF-directed (KD) siRNA oligonucleotides for the indicated number of days. Cell lysates were immunoblotted for actin and AIF. (<b>B</b>) The blots from (A) were quantified and the relative amount of AIF (AIF/actin ratio) plotted. (<b>C</b>) Cells were treated with control or knockdown oligonucleotides for 3 days, then treated without or with 500 µM amiloride, 250 µM UCD74A or 250 µM UCD38B for another 24 hours, and the percent of cells that exhibit trypan blue uptake was evaluated. Results depicted in each panel are representative of three independent experiments. **, <i>P</i><0.001.</p

Induction of breast tumor cell death by the amiloride derivative UCD38B.

<p>(<b>A</b>) MDA-MB-231, (<b>B</b>) MCF7, and (<b>C</b>) SKBR3 breast cancer cells were exposed for 24 hours to various concentrations of amiloride, UCD74A, or UCD38B, as indicated. Cell viability was measured using the MTT assay. Results depicted in each panel are representative of three independent experiments.</p

UCD38B treatment results in rapid swelling of the endoplasmic reticulum.

<p>MDA-MB-231 cells were treated with (<b>A</b>) DMSO control (x11357), (<b>B</b>) amiloride (x15455), (<b>C</b>) UCD74A (x15455) or (<b>D–F</b>) UCD38B (x15455, x20500, x38518) for two hours, and fixed cells were imaged by transmission electron microscopy. Examples of endoplasmic reticulum (ER) and mitochondrial (M) structures are indicated.</p