Fig 2: PHF20 regulates apoptosis and improves cisplatin sensitivity via the OCT4-p-STAT3-MCL1 signaling pathway. (A) Western blotting was used to determine the expression levels of PHF20, OCT4, MCL1, PARP1, STAT3 and p-STAT3 in FaDu cells co-transfected with lentivirus-mediated PHF20 shRNA and OCT4 overexpression plasmid. OCT4 overexpression restored the PHF20 knockdown-mediated reduction in the levels of p-STAT3, MCL1 and cleaved PARP1. (B) Quantitative analysis of the western blot results in A. Compared with those in the shPHF + pCDNA3.1 group, the relative levels of p-STAT3 and MCL1 increased, whereas those of cleaved PARP1 decreased in the shPHF + OCT4 group. Mixed model ANOVA was used for analysis. (C) Reverse transcription-quantitative PCR assay demonstrated that the mRNA expression levels of OCT4A and OCT4B1 were decreased, whereas the expression levels of OCT4B were not affected by PHF20 knockdown compared with those in the Scr group. (D) Western blot analysis of OCT4A and OCT4B1 expression levels in the shPHF and control groups. (E) Quantitative analysis of the western blot results in D. Compared with those in the control cells, the relative protein expression levels of OCT4A and OCT4B1 were decreased in the shPHF group. All experiments were performed thrice. **P<0.01 and ***P<0.001. PHF20, plant homeodomain finger protein 20; shPHF, lentivirus-mediated shRNA targeting PHF20; Scr, scramble shRNA; PARP1, poly(ADP-ribose) polymerase 1; MCL1, myeloid cell leukemia-1; STAT3, phosphorylated signal transducer and activator of transcription 3; p-, phosphorylated; OCT4, octamer-binding transcription factor 4.

Fig 3: PHF20 inhibition suppresses tumorigenicity via apoptosis in the HSCC cell xenograft model. (A) Representative images of tumors isolated from mice in the shPHF20 and control groups. (B) The tumor growth curves in the two groups. (C) Quantitative analysis of positive cells in (D) the immunohistochemical staining of PHF20, Ki67, TUNEL and p-STAT3 in tumor samples from mice in the two groups. Magnification, ×400. Scale bar, 100 µm. Compared with those in the Scr group, the percentage of apoptotic cells indicated by TUNEL staining was significantly increased, the number of p-STAT3+ cells decreased, and the number of Ki67+ cells did not obviously differ in the shPHF group. (E) Schematic representation illustrating how the knockdown of PHF20 may synergize with cisplatin to promote apoptosis via the OCT4-p-STAT3-MCL1 signaling pathway. PHF20, plant homeodomain finger protein 20; shPHF, lentivirus-mediated shRNA targeting PHF20; Scr, scramble shRNA; PARP1, poly(ADP-ribose) polymerase 1; MCL1, myeloid cell leukemia-1; casp-3, caspase-3; STAT3, phosphorylated signal transducer and activator of transcription 3; p-STAT3, phosphorylated signal transducer and activator of transcription 3; OCT4, octamer-binding transcription factor 4; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.