Fig 2: CBX inhibits FOXO3-mediated SESN3 expression and associated chemoprotection in high-stage NB cells. a Clonogenic survival of NB4/FOXO3 and NB8/FOXO3 cells was analyzed by CFA. The cells were treated for 72 hours with 50 nM 4OHT and 80 µM CBX alone and for further 72 hours with etoposide and doxorubicin in combination. A concentration of 0.1 µg/ml etoposide or 0.01 µg/ml doxorubicin were added to NB4/FOXO3 cells and 0.8 µg/ml etoposide or 0.08 µg/ml doxorubicin to NB8/FOXO3 cells. Colonies were stained with crystal violet. b Quantification of the CFA was performed by photometric measurement after discoloration with 0.5% SDS in 50% ethanol. Shown are means ± s.e.m. of at least three independent experiments; *P < 0.05, **P < 0.025, ***P < 0.01. c NB8/FOXO3 cells were treated with 100 nM 4OHT alone or in combination with 120 µM CBX for six hours and quantitative RT-PCR of SESN3 expression was performed. Shown are mean values ± s.e.m. of three independent experiments. Statistical analyses were done with the Student’s unpaired t test; *P < 0.05, **P < 0.025 compared with controls. d Immunoblot analyses of SESN3, P27KIP1, and FOXP1 expression in NB8/FOXO3 cells treated with 50 nM 4OHT and the indicated concentrations of CBX for 24 hours. GAPDH served as loading control

Fig 3: Effect of Yiqichutan on SUDHL-6 cells. (A) SUDHL-6 cells were treated with different concentrations of Yiqichutan. The viability of SUDHL-6 cells was measured with an MTS assay at 24, 48 and 72 h. (B) Cell apoptosis was detected by flow cytometry. (C) Genes associated with proliferation, such as PCNA, and apoptosis, including Bax, Bcl2 and PARP, signaling pathways were measured by RT-qPCR. (D) Expressions levels of miR-34a and Foxp1 in SUDHL-6 cells treated with Yiqichutan were detected by RT-qPCR. (E) RT-qPCR results indicated that the expression levels of Bax, Bcl2, PCNA and PARP, were significantly altered after Yiqichutan treatment. IC50=14.65 mg/ml. Data are presented as the mean ± SD of three separate experiments. *P<0.05, **P<0.01. L, low concentration of Yiqichutan 1/3 IC50; M, middle concentration, 1/2 IC50; H, high concentration, IC50. RT-qPCR, reverse transcription-quantitative PCR; miR, microRNA; Foxp1, forkhead box 1; PCNA, proliferating cell nuclear antigen; PARP, poly (ADP-ribose) polymerase.

Fig 4: FOXP1 and miR-21 form a double-negative feedback loop. a Volcano plot showing the differential expression of miRNAs at 96 h into TGF-β-induced EMT in A549 cells. The red dots represent miRNAs with a differential expression FDR < 0.05 and absolute log2-fold change > 1. The horizontal dotted line represents the log2(CPM) corresponding to 100 copies/cell. b Graph showing the sequence alignment of FOXP1 3’UTR with miR-21-5p. c The results of the luciferase reporter assay were quantified (bar charts). d Immunoblotting analysis of the protein abundance of indicated genes in A549 cells during TGF-β-induced EMT after a specific antagomiR was used to silence miR-21 expression. e Same as (d) for the qRT-PCR assay. f qRT-PCR analysis of the indicated genes in A549 cells during TGF-β-induced EMT after a specific antagomiR was used to silence miR-21 expression, using A549 cells whose miR-21 binding site in FOXP1 has been mutated by CRISPR-Cas9. g A549 cells undergoing TGF-β-induced EMT were treated with a siRNA targeting FOXP1, and the impact on miR-21 expression was quantified using qRT-PCR. n = 3; error bars indicate the means ± s.d. *p < 0.01, determined using a two-tailed Student’s t test. Source data are provided as a Source Data file

Fig 5: FOXA1 and FOXP1 are highly mutated in primary and metastatic prostate adenocarcinoma. The mutation frequency of FOXA1 and FOXP1 in primary PCa was assessed in a TCGA data set containing 501 samples (Firehose Legacy). The mutation burden in metastatic prostate cancer was assessed in the SU2C/PCF Dream Team data set containing 444 samples. Data were generated from cBioPortal.org (accessed on 5 February 2020).