Fig. 3: Phenotypic effects of PPARG over-expression in 3D culture regulated by the PPARG-AKT3 functional axis.

A Characterisation of the phenotypic effect of PPARG over-expression in 3D culture representative images of EV7 and OE19 cells grown in 3D, images representative of three independent experiments. B Quantification of the two different spheroid appearances, represents three independent experiments and eight technical replicates per experiment. error bars show SEM Statistical significance denoted by *p ≤ 0.05 determined by 2-way Anova and Sidak’s multiple comparisons test. C Immunoblot analysis from 3D spheroids of EV7 and OE19 for mitochondrial ETC complexes, AKT3, CRM1 and VDAC1. HSC70 was used as loading control. Values above each band indicate the densitometry value of the band as normalised to the loading control HSC70 and compared to the EV7 band. Complex I is labelled in blue and complex IV in red to allow the reader to identify the correct densitometry value for the corresponding bands, image representative of three independent experiments. D siRNA knockdown of AKT3 effect in 3D culture appearance on EV7 and OE19 cells grown in 3D either treated with non-targeting siRNA (siNTS) or AKT3 targeting siRNA (siAKT3). Images representative of three independent experiments. E Quantification of the two different spheroid appearances following AKT3 knockdown, represents three independent experiments and ten technical replicates per experiment, error bars show SEM, Statistical significance denoted by *p ≤ 0.05 determined by 2-way Anova and Sidak’s multiple comparisons test. F Immunoblot analysis from lysates derived from 3D siRNA knockdown of AKT3, showing the ETC complexes, VDAC1 and AKT3. HSC70 was used for loading control. Complex I is labelled in blue and complex IV in red to allow the reader to identify the correct densitometry value for the corresponding bands, image representative of three independent experiments. Values above each band indicate the densitometry value of the band as normalised to the loading control HSC70 and compared to EV7 siNTS. G PPARG inhibitor effect on 3D spheroid appearance. EV7 and OE19 cells grown in 3D either treated with DMSO or PPARG inhibitor GW9662 at 20 μM. Images representative of three independent experiments. H Quantification of the two different spheroid appearances following GW9662 treatment, represents three independent experiments and ten technical replicates per experiment, error bars show SEM, Statistical significance denoted by *p ≤ 0.05 determined by 2-way Anova and Sidak’s multiple comparisons test. I Immnuoblot analysis from lysates derived from 3D spheroid treatment with PPARG inhibitor GW9662 at 20 μM, showing the ETC complexes, VDAC1 and AKT3. HSC70 was used for loading control. Complex I is labelled in blue and complex IV in red to allow the reader to identify the correct densitometry value for the corresponding bands, image representative of three independent experiments. Values above each band indicate the densitometry value of the band as normalised to the loading control HSC70 and compared to EV7 DMSO.