Fig. 4

Effects of 4-IPP and metformin on C2 organoids. a MIF/CD74 signaling in C2 organoids. Lysates from C2 organoids expressed MIF, CD74 and presented activated AKT and AMPK. b Hematoxylin eosin and immunohistochemical staining of C2 tumour sample with anti-MIF and anti-CD74 antibodies. Magnifications at 10X. c Micrographs of C2 organoids before and after drug treatments. 4-IPP and metformin treatments induced a marked morphological change in C2 organoids, resulting in a loss of their original spheroid organization. Magnifications: 10X. Scale bars = 50 μm. d C2 organoids were treated with 5 mM metformin for 120 h or 50 and 100 μM 4-IPP for 24 and 48 h. The percentage of cell death was determined by trypan blue exclusion assay. Data are expressed as the mean ± SD. *P < 0.01 compared with untreated control and 50 μM 4-IPP. **P < 0.01 compared with untreated control and 100 μM 4-IPP. We have not observed significant increase in the percentage of cell death after metformin treatments compared with untreated controls. All the experiments were replicated at least three times. e Immunoblots of the principal proteins involved in CD74/MIF pathway. C2 organoids were untreated or treated with 50 and 100 μM 4-IPP for 24 h; lysates were resolved by 4–12% SDS–PAGE and immunoblotted. f Immunoblots of the principal proteins involved in metformin pathway. C2 organoids were untreated or treated with 5 mM metformin for 120 h; lysates were resolved by 4–12% SDS–PAGE and immunoblotted. g Global tyrosine and threonine phosphorylation changes in C2 organoids after 4-IPP and metformin treatments. C2 organoids were untreated or treated with 50 and 100 μM 4-IPP for 24 h and with 5 mM metformin for 120 h; lysates were resolved by 4–12% SDS–PAGE and immunoblotted. h Immunoblots showing the expression and activity of the PP2A and PP1 proteins before and after 4-IPP or metformin treatments. Blots are representative of the results from multiple (at least two) experiments. i Observed consequences of the 4-IPP or metformin treatments on C2 organoids