Fig 2: The up-regulation of MCM2 enhanced the stemness of HCC cells.a Representative images of tumor-spheres in the MCM2 over-expression and control groups of the indicated cells, corresponding histogram and corresponding sphere morphology percentage chart. b Representative images of tumor-spheres formation in the limiting dilution assays of the indicated cells and the log-dose slope of the MCM2 over-expression and control groups. c Representative images of colony in the MCM2 over-expression and control groups of the indicated cells and corresponding histogram; d The immunoblotting for Bmi1, AKT1, CD133, ALDH1A1 and Epcam expression in the Vector and MCM2 groups of the indicated cells, determined by Western Blot assay; Data are shown as the mean ± SD from independent experiments. *P < 0.05.

Fig 3: Western blotting of downstream regulated proteins of CUDC-907 and immunohistochemistry staining. (A) The expression of c-Myc is regulated by the HDAC and PI3K/AKT/mTOR pathway, and both can be inhibited by CUDC-907. (B) Expression levels of proteins [c-Myc, p-AKT, p-S6, p-4EBP1, and acetyl histone H3K9 (H3K9ac)] in HCC cell lines affected by CUDC-907 were detected by western blotting. GAPDH was set as the internal reference. (C) Expression levels of proteins (same as figure 5B) from xenografted tumor with or without CUDC-907 treatment were detected. (D) Immunohistochemistry staining. Representative images of Ki67, c-Myc, and TUNEL staining of xenografts treated with CUDC-907 or vehicle. (E) Quantification of immunohistochemistry staining was calculated by the immunohistochemistry score method. The percentage of apoptotic area in tumor slides of TUNEL staining was calculated by IMAGE J software. *p < 0.05, **p < 0.001 (result compared with Vehicle group).

Fig 4: miR-19a mediates the PI3K/AKT signaling pathway in the hypoxia-exposed CRC cells: (a) downstream mRNAs of miR-19a predicted using the StarBase system; (b) transfection efficacy of miR-19a mimic examined using RT-qPCR (*p < 0.05, two-way ANOVA); (c) activation of PI3K/AKT and Wnt/ß-catenin signaling pathways in HCT116 and SW480 cells after miR-19a mimic transfection examined by western blot analysis (*p < 0.05, two-way ANOVA); (d, e) expression of miR-19a and STAT3 in HCT116 and SW480 cells after si-STAT3 or si-STAT3+miR-19a mimic transfection examined by RT-qPCR (*#p < 0.05, two-way ANOVA; *compared to hypoxia+STAT3-NC; #compared to si-STAT3+miR-19a control group); (f) phosphorylation of PI3K and AKT in cells examined by western blot analysis (*#p < 0.05, two-way ANOVA; *compared to hypoxia+STAT3-NC; #compared to si-STAT3+miR-19a control group). Data were presented as the mean ± SD from three independent experiments.

Fig 5: CDEL, as well as CAMP overexpression can induce caspase-3 mediated apoptosis. (A) Expression analysis of apoptosis-related proteins, including cleaved-caspase-3, caspase-3, cleaved-PARP, p53, BAX, Bcl-2 and Bcl-xL using western blot analysis. (B) Expression analysis of cell cycle-related proteins, including Cyclin B1 and PERK using western blot analysis. (C) Expression analysis of phosphorylated and total Akt and ERK. The ratio of p-Akt/total Akt and p-ERKs/total ERKs was quantified. Representative blots of independent experiments are shown. The bands were quantified relative to ß-actin. *P<0.05, **P<0.01 vs. control. CDEL, LL-37 C-terminal deletion mutant; PARP, poly(ADP-ribose) polymerase; PERK, PKR-like ER kinase; p-, phospho.