*3.1.* α*M Prevented CDDP-Induced Cell Death*

LLC-PK1 cells were incubated with various CDDP concentrations: 5, 10, 20, 30, and 40 μM (Figure 1A) and it was observed that at 30 μM CDDP, reduced approximately 50% of cell viability, as we previously reported [26]. Later, cells were incubated with αM concentrations between 1 and 5 μM. As we expected, αM was not toxic up to 5 μM (Figure 1B). To evaluate protection by αM, αM and 30 μM CDDP were co-incubated. We observed that αM protected the cells against CDDP-induced cytotoxicity in a concentration-dependent manner and the protection with the least concentration was found at 4 μM (Figure 1C). So, the subsequent experiments were carried out with 4 μM αM and 30 μM CDDP.

**Figure 1.** Protective effect of alpha mangostin (αM) against cis-dichlorodiammineplatinum II (CDDP) induced cell death. Lilly laboratory culture porcine kidney (LLC-PK1) cells were treated with (**A**) 0–40 μM cisplatin, (**B**) 0–5 μM αM or (**C**) co incubated with 30 μM CDDP and increasing concentrations of αM for 24 h. After treatment, cell viability was measured by the fluorescein diacetate (FDA) assay. The data are presented as mean ± SD, *n* = 5–7. \*\*\* *p* < 0.001, \*\* *p* < 0.01 and \* *p* < 0.05 vs. control (0). ### *p* < 0.001 and ## *p* < 0.01 vs. CDDP.

CDDP's toxicity is partially associated with oxidative stress. To evaluate the effect of CDDP-induced oxidative stress, we evaluated the lipid peroxidation markers 4HNE and MDA by Western blot (WB). As we show in Figure 2, CDDP increased the level of MDA (Figure 2A,B) but not those of 4HNE (Figure 2C,D). αM was unable to prevent CDDP-induced MDA increase.
