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. 2023 Aug 9;30(10):1414–1425. doi: 10.1038/s41417-023-00648-5

Fig. 3. Co-treatment of 3-BP and cetuximab inhibits system Xc− activity by activating FOXO3a/AMPKα/pBeclin1 pathway.

Fig. 3

A DLD-1(KRASG13D/-), HT29(BRAFV600E), or Caco-2-CR cells were treated with 5 μM 3-BP and/or 10 μg/ml cetuximab for four days, and immunoblotting assay analyzed the protein level of FOXO3a, AMPKα, phosphorylated Beclin1 and total Beclin1. B–F DLD-1(KRASG13D/-), HT29(BRAFV600E), or Caco-2-CR cells with or without AMPKα knockdown were treated with 5 μM 3-BP and 10 μg/ml cetuximab for four days. Immunoblotting assay analyzed the protein level of AMPKα, phosphorylated AMPKα, total Beclin1, and phosphorylated Beclin1 (B). The effect of AMPKα knockdown on glutamate released (C), GSH level (D), cell viability (E), and MDA production (F) was evaluated. G–K DLD-1(KRASG13D/-), HT29(BRAFV600E), or Caco-2-CR cells with or without Compound C (1 μM) treatment were treated with 5 μM 3-BP and 10 μg/ml cetuximab for four days. Immunoblotting assay analyzed the protein level of total AMPKα, phosphorylated AMPKα, total Beclin1, and phosphorylated AMPKα (G). The effect of Compound C (1 μM) treatment on glutamate released (H), GSH level (I), cell viability (J), and MDA production (K) was evaluated. L DLD-1(KRASG13D/-), HT29(BRAFV600E), or Caco-2-CR cells with or without FOXO3a knockdown were treated with 5 μM 3-BP and 10 μg/ml cetuximab for four days. Immunoblotting assay analyzed the protein level of FOXO3a, AMPKα, and phosphorylated Beclin1. M The DLD-1(KRASG13D/-), HT29(BRAFV600E), or Caco-2-CR cells with or without FOXO3a knockdown were treated with 5 μM 3-BP and 10 μg/ml cetuximab for 48 h. The effect of FOXO3a knockdown on the transcriptional activity of the AMPKα gene was evaluated. Data are expressed as mean ± SD, n = 3 biological replicates.