Fig. 5: Promotion of ferroptosis by butyrate via cAMP-PKA-mediated AKT-GSK3 activation.

A–C FFAR2-knockdown HT29 cells were treated with the indicated concentrations of NaB for 6 h, the levels of pS473-AKT, p-GSK3β and the indicated proteins were evaluated by WB (A). The knockdown efficiency was measured by qRT-PCR (B) and quantitative data for pS473-AKT/AKT is presented (C). D HCT116 cells were treated with the NaB for 6 h, and the cAMP level was detected. HT29 cells were treated with the indicated concentrations of NaB or in combination with H89 for 6 h, the levels of pS473-AKT, p-GSK3β and indicated the proteins were evaluated by WB (E) and quantitative data for pS473-AKT/AKT is presented (F). G–I FFAR2-knockdown HT29 cells were treated with the indicated concentrations of NaB for 6 h, and then WB was used to evaluate the levels of NRF2 (G). The knockdown efficiency was measured by qRT-PCR (H) and quantitative data for NRF2 protein levels are presented (I). After pretreatment treated with NaB or in combination with H89 for 6 h, HT29 cells were treated with CHX for the indicated time, and then WB was used to evaluate the levels of NRF2 (J), quantitative data for NRF2 protein levels were presented (K). HT29 cells were treated with the indicated concentrations of NaB or in combination with Forskolin for 6 h, and then WB was used to evaluate the levels of NRF2 (L), quantitative data for NRF2 protein levels were presented (M). FFAR2-knockdown HT29 cells were treated with the indicated concentrations of NaB for 6 h, and the expression of SLC7A11 was measured using qRT-PCR (N), the knockdown efficiency was measured by qRT-PCR (O). HT29 cells were treated with the indicated concentrations of NaB and in combination with H89 (P) or Forskolin (Q) for 6 h, and the expression of SLC7A11 was measured using qRT-PCR. R FFAR2-knockdown HT29 cells were treated with different concentrations of RSL3 or in combination with NaB, and the cell viability was examined using CCK-8. The knockdown efficiency was shown in (O). HT29 cells were treated with different concentrations of RSL3, NaB and in combination with H89 (S) or Forskolin (T) for 6 h, the cell viability was examined using CCK-8.