Fig 2: Effect of selumetinib combined with KRT-232 on cell cycle, apoptosis, and p53 and MAPK signaling. (A) Cell cycle analysis of two CRC cell lines (HCT116 and B1011CL) and one PTC cell line (PDX.008CL) after treatment for 24 h with vehicle, selumetinib (1 µM), KRT-232 (1 µM), or a combination of both. Percentages of cells in Sub-G1, G0-G1, S and G2/M phases of cell cycle are depicted. (B) Flow cytometry analysis of annexin-V positive cells in HCT116 colorectal cell line after treatment for 72 h by vehicle, selumetinib (1 µM), KRT-232 (1 µM) or combination of both. (C) Total percentage of annexin-V positive cell population shown in the graph after treatment for 72 h vehicle, selumetinib (1 µM), KRT-232 (1 µM) or combination of both. Values represent mean + /- SD. (*control vs combination, p = 0.046). (D) HCT116 CRC cells were treated with vehicle control (C), KRT-232 (1 µM, K), selumetinib (1 µM, S), or a combination of both (K + S) for 24 h. Cells were lysed and immunoblotting was performed using antibodies to identify changes in MDM2/p53 and MAPK pathways. Each blot had its loading control, in this panel one representative loading control (ß-actin) was shown. The full-length blots are shown in Supplementary Fig. S2. (E) This illustration summaries the relation of antigens that were probed in (D).

Fig 3: miR-3928 induces the expression and activation of p53 in GBM cells. (a) Band intensities from immunoblot replicates are quantified and graphed for A172, T98G, U87, and U251 cells transfected with either miRNA scrambled control (-) or miR-3928 (+). Representative immunoblot images for p53 and associated GAPDH are shown below the bar graph. Exogenously overexpressed miR-3928 leads to the upregulation of wild-type p53 in GBM cell lines A172 and U87 that have wild-type p53 (*) but not in T98G and U251 cell lines, which have mutant p53. (b) With immunoblots showing that miR-3928 overexpression induces p53 phosphorylation in A172 cells, UV treated cells are used as positive control for p53 phosphorylation/activation.

Fig 4: Mutant p53 proteins enhance ROS production in cancer cells. a The indicated cell lines were seeded in 96-well plates, incubated overnight, and transfected with the pRSuper-p53 vector and with plasmids for overexpression of mutant p53 (o.e. R175H; o.e. R273H), or their relative negative mock control (CTRL). DCF fluorescence intensity was analyzed by a multimode plate reader. Western blot of p53 was performed to test the effective knockdown of WT or mutant p53 and the overexpression of mutant p53 in the various cell lines indicated. b MDA-MB-231 cells stably silenced for p53 by lentiviral transduction with two different short hairpin sequences, were treated for 15 min with the ROS probe CM-H2DCFDA and then analyzed by FACS. Bars represent average (± SEM) of the percentage of DCF positive (+) cells measured by FACS from two independent biological experiments performed in technical duplicate. Statistically significant differences are indicated: *p < 0.05, ** p < 0.001. Western blot of p53 was performed to test the effective knockdown of mutant p53. c The intracellular oxidized and reduced glutathione were determined in MCF7 and Panc1 mutR273H-p53 cell lines after transient transfection with the pRsuper-p53 vector. d The indicated cell lines were seeded in 96-well plates, incubated overnight, and treated with 20 µM CP-31398 or 40 µM RITA for 48 h. DCF fluorescence intensity was measured by a multimode plate reader. e Live cell imaging: 48 h after transfection with plasmid coding for R175H or R273H mutant p53, or with the mock vector (CTRL), cells were incubated for 30 min with MitoSox probe (red) and Mitotracker Green (green). The RGB profile plotted along the dashed line drawn in the merge image is also shown. Merge and single channel images come from a single z-plane. Scale bar 10 µm. f Mitochondrial superoxide evaluation by MitoSox Red probe was analyzed with a multimode plate reader. Cells were seeded in 96-well plates, incubated overnight, and transfected with plasmid coding for R175H or R273H mutant p53, or the mock vector. All the experiments presented in this figure are representative of three biological replicates. P values were calculated with two-tailed t test. Statistical analysis: *p < 0.05

Fig 5: NXD30001 reduces activation of DNA damage response in GSCs after radiation. (A) Representative western blotting bands showing the expression level of p-ATR, p-CHK2, p-p53, p-H2AX in T4302 CD133+ cell cultures treated with different concentrations of NXD30001 with or without radiation. The result was quantified from the average of three different samples. (B) Quantification of western blotting bands. Error bars represent the standard error. * P < 0.05.