Fig 2: Establishment of BETi-resistant IMR-32 (IMR-32-JQ1R) cells(A) Dose-response curve for cell viability using WST-8 assay of IMR-32 and IMR-32-JQ1R cells treated with different concentrations (0, 0.031, 0.063, 0.13, 0.25, 0.50, 1.0, and 2.0 μmol/L) of JQ1 or OTX015 for 48 h. Data represent means and SD of two independent experiments performed in triplicate. (B) Phase-contrast images of IMR-32 and IMR-32-JQ1R cells after treatment with DMSO or JQ1 or OTX015 at 0.5 μmol/L for 48 h. (C) Western blot analysis of MYCN expression in IMR-32 and IMR-32-JQ1R cells 48 h after treatment with JQ1 (upper) or OTX015 (lower) at 0, 0.25, 0.50, or 1.0 μmol/L. (D) Relative expression of MYCN mRNA assessed by qRT-PCR in IMR-32 and IMR-32-JQ1R after treatment with DMSO or JQ1 or OTX015 at 0.50 μmol/L for 48 h. Bar, SD for triplicate experiments. ∗p < 0.05. (E) Western blot analysis (right) and cell growth assay (left) of IMR32 and IMR32-JQ1R cells after transfection with si-NC, si-MYCN #1, or si-MYCN #2 (each 20 nmol/L). The cell growth rate was assessed by WST-8 assay using a relative ratio compared with day 0. Bar, SD for triplicate experiments. ∗p < 0.05. (F) In vitro cell growth of IMR-32 and IMR-32-JQ1R cells. The cell growth rate was assessed by WST-8 assay using a relative ratio compared with day 0. Bar, SD for triplicate experiments. ∗p < 0.05. (G) Western blot analysis of the indicated proteins in IMR-32 and IMR-32-JQ1R cells.

Fig 3: Blocking TBX2 in an in vivo mouse model of PCa progression and metastasis leads to increased miR-200c-3p and reduced SOX2 and N-MYC expression. (A) Western blots showing SOX2 and N-MYC expression in TBX2-modulated PCa cells; (B) Western blots showing SOX2 expression in LNCaP or 22Rv1 cells treated with exosomes (20 μg/mL, added a total of two times on alternate days) isolated from TBX2-modulated human prostate cancer (PCa) cells. Densitometric analysis of the Western blots is provided in Figure S4; (C) a schematic of the orthotopic xenograft experiment; (D) immunohistochemical (IHC) staining showing SOX2 and N-MYC expression in the orthotopic xenografts of PC3Neo or PC3TBX2DN human PCa cells. Densitometric analysis of the IHC images is provided in Figure S5; (E) miR-200c-3p expression in the orthotopic xenografts of PC3Neo or PC3TBX2DN cells using quantitative real-time RT-PCR analysis (qRT-PCR). Data represent the average of triplicates values ± S.D.; Student’s unpaired 2-tailed t-tests were performed to compare the two groups, *, p ≤ 0.05; **, p < 0.01, and ***, p ≤ 0.001, ****, p ≤ 0.0001. The uncropped Western blot images can be found in Figures S8–S10.

Fig 4: Schematic representation of the molecular mechanism of raltitrexed (RTX)‐induced DNA damage and cell death. Overexpression of N‐Myc promotes DNA replication, which in turn causes high levels of DNA replication stress and DNA damage. DNA replication stress is further enhanced by reduced dTTP concentration after RTX treatment, which leads to apoptotic cell death in the MYCN‐amplified neuroblastoma cells. TS, thymidylate synthase

Fig 5: Effects of N-Myc and TEM8 on the proliferative ability and tubule formation of PCa cells. A The mRNA and protein expressions of TEM8 were verified after LNCaP/N-Myc and C4-2/N-Myc cells were treated with TEM8-shRNA. B Compared with the control group, the overexpression of N-Myc and TEM8 affected the proliferative ability of LNCaP and C4-2 cells. C HUVEC tube formation assay was determined using the supernatant from LNCaP cells