Page 64 - Read Online

Page 8 of 11 Jiang et al. Hepatoma Res 2019;5:5 I http://dx.doi.org/10.20517/2394-5079.2018.97

Figure 4. Functional characterization of dimethylbromobenzene-cysteine p53-MDM2/MDMX inhibitor. A-C: dose-dependent growth
+/+
+/+
inhibition of HCT116 p53 (A), HCT116 p53 (B) and SK-hep-1 (C) cells upon various treatments as determined by the MTT assay to
monitor the pesticide effects. Three cell lines were plated in 96-well plates at a density of 2,500 cells/well (100 μL). After 24 h, cells
were treated with drug sample at the indicated concentrations and times in FBS-free mediums, respectively. The in vitro cytotoxicity was
then measured by using a standard MTT (Thermo Fisher scientific) assay after 72 h drug treatment. (mean ± SD, n = 4); D-F: apoptosis
levels measured by FACS in three cell lines treated with SP0, SP1 SP2 and SP3 for 48 h incubation at concentration of 50 μmol/L; G-I: the
average means of the apoptosis calculated three independent experiments like D-F. P values were calculated by t-test (*P < 0.05; **P < 0.01;
***P < 0.001)

cellular uptakes of FITC-labled SP0, SP1, SP2 and SP3 in HCT116 cells after 6 h incubation in 37 °C at a
concentration of 100 μmol/L. As shown in Supplementary Figure 1, SP3 showed the strongest ability of
cellular internalization (> 75%), whereas neither SP1 nor SP2 showed exceed 15% cellular internalization.
Moreover, there exists no cellular uptakes for the three stapled peptides at 4 °C incubation [Supplementary
Figure 1], suggesting that the cellular uptakes of the stapled peptide most likely result from the ATP-
dependent endocytosis. Furthermore, it is necessary to verify the biological activity of SPx to induce the