Fig 2: Effect of TQ on PI3K/Akt/mTOR signaling expression in MV4-11 and K562 cells. RT-qPCR analyses were performed. The graph demonstrates the downregulation of PI3K, Akt, and mTOR genes and upregulation of PTEN in (A) MV4-11 cells and (B) K562 cells after treatment with TQ. The data represent three separate experiments and are presented as the median and interquartile range (error bars). Wilcoxon signed ranks test was conducted. Where * p < 0.05 and *** p < 0.001 are significant versus untreated control cells.

Fig 3: FoxM1 inhibitor induced RASSF1A and PTEN expression and YAP phosphorylation in mCRC cells. T84 and Colo 205 cells were treated with FoxM1 inhibitor, thiostrepton (Th) with different doses (0–8 µM) for 48 h. (A,B) mRNA was extracted after 24 h for detection of RASSF1A by qRT-PCR. Bar graphs show quantitative results normalized to GAPDH mRNA levels. Cell lysates were monitored by immunoblot for FoxM1(C), RASSF1A (D), p-YAP and total YAP (E), PTEN (F), and GAPDH as indicated. Immunoblots were quantified by scanning densitometry and normalized against GAPDH expression (lower panels of (C–E) for FoxM1, RASSF1A and p-YAP, respectively. Right panel of (F) represents the quantification of PTEN). (G) Patient derived organoids were treated with (8 µM) and without thiostrepton for 24 h and 48 h. Organoid cell lysates were analyzed by immunoblot for RASSF1A expression and quantified by densitometry ((G), lower panel). The results are from three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fig 4: GSK3ß-regulated MiR target proteins are increased following GSK3ß inhibition – mechanisms for enhanced MiR biogenesis by GSK3ß. (A) Luciferase activity in lysates of HEK293T cells transfected with pMiRTarget-ACLY-WT or pMiRTarget-ACLY-miR27a binding site (BS) mutant (for details see Supplementary Figure S9) and treated with 99021 (2 µM, 24 h). Luciferase was normalised for transfection efficiency (ß-galactosidase activity) and mean ± SEM of three independent experiments performed in duplicate is shown. (B) qRT-PCR analysis of (i) ACLY, (ii) ZEB1, (iii) PTEN and (iv) FOXO1 3?UTR levels in HEK293T cells treated ± 99021 (2 µM) for 72 h. L19 was used as a normalisation gene. Columns: mean ± SEM for three independent experiments performed in triplicate. (C, D, E and F) Western blot analysis of (C) ACLY, (D) ZEB1, (E) PTEN (F) and FOXO1 protein levels in lysates of HEK293T cells treated ± 99021 (2 µM) for 72 h. ß-Actin was used as a loading control and for normalisation. Representative blots of three independent experiments are shown (i). Densitometry was performed using Image J software (ii). Columns: mean ± SEM for three independent experiments. *P = 0.05. (G) A mechanism for enhanced miR biogenesis by GSK3ß. GSK3ß phosphorylates Drosha at S300 and S302. This does not alter levels of miR biogenesis proteins or modulate Drosha localisation but increases Drosha association with DGCR8, p72 and pri-miRs and enhances Drosha pri-miR cleavage activity, reducing pri-miR and increasing mature miR levels. GSK3ß achieves these effects as a component of the Microprocessor, binding to p72 and DGCR8 in an RNA-dependent manner.

Fig 5: Quantification and expression of key cell-function proteins after 48 h exposure to 50 µM NiCl2 or Ni-acetate in PDAC cell lines. These assays quantified the amount of PTEN (A), PARP (B), caspase 3/7 activity (C), and cleaved (active) caspase 3 expression (D). The major difference observed between groups was dependent on the cell line, with each of the PDAC cell lines expressing different protein levels. Treatment effects were evident in PTEN expression in the Panc-10.05 cell line, in which NiCl2 exposure resulted in a significant (p < 0.01) increase in PTEN expression. Caspase 3/7 activity was significantly (p < 0.01) reduced after exposure to Ni-acetate. Data are expressed as the mean ±SD of four (protein expression) or eight (caspase 3/7 activity) assays performed in duplicate (** p < 0.01; *** p < 0.001).