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. 2021 Oct 1;125(11):1523–1532. doi: 10.1038/s41416-021-01561-0

Fig. 2. APR-246-induced apoptosis was further enhanced by its combination with 5-FU in ESCC with p53 missense mutation.

Fig. 2

a Proliferation assay. ESCC cell lines (KYSE410, TE1, TE8, and TE14) were treated with PBS, APR-246 (10 μmol/L), 5-FU (10 μmol/L), or the combination of APR-246 (10 μmol/L) + 5-FU (10 μmol/L) for 72 h. b Apoptosis assay. Percentage of cells with positive annexin V staining after treatment with PBS, APR-246 (20 μmol/L), 5-FU (20 μmol/L), or the combination of APR-246 (20 μmol/L) + 5-FU (20 μmol/L) in ESCC cell lines (KYSE410, TE1, TE8, and TE14). c ROS activities under the same conditions as in b. d Western blotting to assess relevant protein levels (cleaved PARP, p53, p73, Noxa, BAX, Bcl-2, PUMA, p53AIP1, p21, and E2F-1) under the same condition as in b. e PCR results assessing TAp73 and ΔNp73 mRNA levels under the same condition as in e (ΔΔCt method). f Comparison of ROS activities after treatment with control, combination treatment (APR-246 20 μmol/L + 5-FU 20 μmol/L), or combination treatment plus N-acetylcysteine as assessed by flow cytometry in ESCC cell lines (KYSE410, TE1, TE8, and TE14). g Western blotting to assess relevant protein levels under the same conditions as in d. *P < 0.05. Wt wild type, Mut missense mutation, Null frameshift or nonsense mutation.