Fig. 5: PI3Kδ activity discriminates EMT from stemness acquisition based on different TGFβ signaling.

a Immunofluorescence staining for ZO-1 (green), actin microfilaments using phalloidin (red) and nuclei (blue) of 3D culture in organoids of Huh7 treated or not with PI3Kδ specific inhibitor idelalisib (CAL-101) at different concentrations after 6 days of 3D and quantification of the canaliculus and inverted polarity phenotype percentage in the different conditions (n > 49 for each condition). b RT-qPCR analysis of different genes expression performed in organoids of Huh7, Huh7+CAL-101 (5 µM) and Huh7 + siPI3Kδ after 6 days of 3D culture in two independent experiments performed in duplicate for Huh7 + 5CAL and in triplicate for HuH7 + siPI3Kδ. Data are presented as log₁₀ mRNA fold change in Huh7+CAL-101 or Huh7 + siPI3Kδ compared to Huh7. c Immunofluorescence staining for the different markers indicated in the panel in organoids of Huh7, Huh7 + PI3Kδ, Huh7+CAL-101 (5 µM) and Huh7 + siPI3Kδ. Scale bar: 10 µm. The right panels present E-cadherin (green) and actin microfilaments using phalloidin (red) line profile blots of the white lines. d Quantification of relative intensity of these markers. Each dot of the graph corresponds to an organoid. e RT-qPCR analysis of different genes expression performed in organoids of HuH7, Huh7 + PI3Kδ, Huh7+CAL-101 (5 µM) and Huh7 + siPI3Kδ after 6 days of 3D culture in two independent experiments performed in duplicate. f Smad3 transcriptional activity, measured using CAGA-luciferase reporter, in HuH7, Huh7+ CAL-101 (5 µM) and Huh7 + PI3Kδ treated with TGFβ (2 ng/ml) and Huh7 treated with TGFβ and SB431542 (2 µM). Data represent a typical experiment performed in triplicate. g Schematic representation of the PI3Kδ-dependant plasticity with the EMT and stemness routes. All values are expressed as mean ± S.E.M.