Fig. 3
FBXL6 activates the oncogenic KRAS/MEK/ERK axis and promotes mTOR activation. a HE staining was utilized to determine the cancer tissue structure, and IHC was used to measure the p-ERK, p-mTOR, and p-S6 protein signals in WT, LC, KC and KLC mice. Representative consecutive IHC staining images are presented. Scale bars = 200 or 50 μm. b Western blotting was utilized to determine the protein levels of p-mTOR, p-4EBP1, p-70S6K, p-S6, p-Akt and p-ERK in WT (X112, 115 and 116), LC (X136, 178 and 182), KC (X151, 154 and 156) and KLC (X148, 171 and 190) mice. c After treatment with pharmacological inhibitors of PI3K (GDC-0326, 1 μmol/L), MEK (trametinib, 100 nmol/L), or mTOR (everolimus, 100 nmol/L); a PI3K inhibitor combined with an mTOR inhibitor; or an MEK inhibitor combined with an mTOR inhibitor for 48 h, KLC primary cells were lysed to extract total proteins. Thereafter, Western blotting was employed to measure the protein levels of p-mTOR, p-S6, p-4EBP1, p-70S6K, p-Akt and p-ERK. d After treatment with pharmacological inhibitors of PI3K (GDC-0326, 1 μmol/L), MEK (trametinib, 100 nmol/L), and mTOR (everolimus, 100 nmol/L) alone; a PI3K inhibitor combined with an mTOR inhibitor; or an MEK inhibitor combined with an mTOR inhibitor for 24, 48, and 72 h, the proliferation of KLC primary cells was analyzed by CCK-8 assay. Two-way ANOVA with the Bonferroni correction for multiple comparisons was used. ***P < 0.001. Fbxl6 F-box and leucine-rich repeat 6, Kras kirsten rat sarcoma, MEK mitogen-activated protein kinase kinase, ERK extracellular signal-regulated kinase, IHC immunohistochemistry, WT wild-type, LC LSL-Fbxl6KI/+;Alb-Cre, KC LSL-KrasG12D/+;Alb-Cre, KLC LSL-KrasG12D/+;LSL-Fbxl6KI/+;Alb-Cre, PI3K phosphatidylinositol 3‑kinase, mTOR mammalian target of rapamycin, S6 ribosomal protein S6, 70S6K ribosomal protein S6 kinase B1, 4EBP1 eukaryotic translation initiation factor 4E binding protein 1, PI3K-i inhibitor of PI3K, MEK-i inhibitor of MEK, mTOR-i inhibitor of mTOR