*2.1. Treatment with Gliotoxin Suppresses Growth and Reduces Resistance in Paclitaxel-Resistant Ovarian Cancer Cells*

We first determined the 50% inhibitory concentration (IC*50*) value of paclitaxel in ovarian cancer cells using Cell Counting Kit-8 (CCK-8) assays as described in the Methods section. The IC50 values of paclitaxel in CaOV3 and SKOV3 cells were 2.71 ± 0.01 nM and 5.15 ± 0.02 nM, respectively, at 48 h. Established paclitaxel-resistant ovarian cancer cells showed profound morphological differences compared to parental cell lines under microscope observation (Supplemental Figure S1A). Additionally, paclitaxel-resistant CaOV3 cells (CaOV3/PTX\_R) and SKOV3 cells (SKOV3/PTX\_R) sustained their proliferation rates after exposure to a high dose (100 nM) of paclitaxel (Supplemental Figure S1B). We next investigated whether treating chemoresistant ovarian cancer cells with gliotoxin prevents cell growth and induces apoptosis. Chemoresistant ovarian cancer cells not only upregulated multidrug resistant-associated proteins (MDR1 and MRP1-3) but also induced the expression of

anti-apoptotic proteins, including X-linked inhibitor of apoptosis protein (XIAP), and cell survival (Figure 1A). Although exposure to gliotoxin slightly prevented the proliferation of CaOV3/PTX\_R and SKOV3/PTX\_R cells (Figure 1B), treating drug-resistant ovarian cancer cells with gliotoxin failed to induce cleaved caspase-9 (active p37) and caspase-3 (active p19/17) or the downstream target cleaved poly (ADP-ribose) polymerase (PARP) (Figure 1C). However, the levels of drug-resistant proteins were markedly decreased after treatment with gliotoxin (Figure 1D). These results suggest that gliotoxin renders the chemoresistant ovarian cancer cells vulnerable to cytotoxic agents, even though the drug alone could not induce the apoptotic death of cancer cells.

**Figure 1.** Treatment with gliotoxin (GTX) suppressed cell growth and reduced resistance in paclitaxel-resistant ovarian cancer cells. (**A**) The chemical structure of gliotoxin used in the whole experiment in this study. (**B**) Total protein from each group of PTX-sensitive cells (PTX\_S) and PTX-resistant cells (PTX\_R) was analyzed by Western blotting with the indicated antibodies. The expressions of multidrug resistant-associated proteins (MDR1-3), X-linked inhibitor of apoptosis protein (XIAP), and surviving were increased in CaOV3/PTX\_R and SKOV3/PTX\_R cells (**C**) Cells were treated with the indicated drug concentration for 24 h. Cell viability was measured using a Cell Counting Kit-8 assay. The absorbance at 450 nm is presented. n = 3. \**p* < 0.001 (GTX-treated PTX\_S ovarian cancer cells vs. DMSO-treated PTX\_S ovarian cancer cells); \*\**p* < 0.001 (GTX-treated PTX\_R ovarian cancer cells vs. DMSO-treated PTX\_R ovarian cancer cells). (**D**,**E**) Cells (1.5 <sup>×</sup> <sup>10</sup>5/well) were treated with 5 <sup>μ</sup>M GTX for 24 h. Total protein was subjected to Western blot analysis with the indicated antibodies. β-actin served as an internal control. Treatment with GTX of PTX\_R ovarian cancer cells reduced the expression of MDR1-3, XIAP, and surviving, but not the cleavage of caspase-9 (active p37/35) and caspase-3 (active p19/17). The results are representative of three independent experiments.
