Fig. 5
ApoA1 downregulates KRT14 transcription by promoting the nuclear translocation of FOXO3a. (a) The level of nuclear FOXO3a (nFOXO3a) in ApoA1 stable-expressed cells (4T1ApoA1 and HCC1937ApoA1) was assayed using western blotting. (b) The level of nFOXO3a in 4T1ApoA1 cells transfected with the indicated dose of FOXO3a-overexpressing plasmid (pFOXO3a). (c) The nuclear translocation of FOXO3a was detected using immunofluorescence staining. 4T1 cells were transfected with pApoA1 for the indicated time, then fixed and subjected to immunofluorescence staining as described in the Methods section. (d) The expression of KRT14 in 4T1 cells transfected with the indicated dose of pFOXO3a. (e) The difference in mRNA levels of CHUK, IKBKB, IKBKE, and IKBKG between 4T1ApoA1 and 4T1WT cells. CHUK: component of inhibitor of nuclear factor Kappa B kinase complex; IKBKB: inhibitor of nuclear factor Kappa B kinase B (IKB) subunit beta; IKBKE: inhibitor of IKB subunit epsilon; IKBKG: inhibitor of IKB subunit gamma. (f) The expression of pAkt, p-S315-FOXO3a (S315 site phosphorylated by Akt), and phosphorylated IκB (p-IκB) in 4T1 and HCC1937 cells transfected with pApoA1 was detected by western blotting. (g) Tumor samples were collected from mice orthotopically inoculated with 4T1WT or 4T1ApoA1 cells, and phosphorylated p65 was measured with IHC. (h) The level of nFOXO3a and KRT14 in 4T1WT and 4T1ApoA1 cells transfected with a series of plasmids encoding flag-conjugated wild-type FOXO3a (WT) or mutant FOXO3a, which mutate the sites phosphorylated by Akt, ERK, and IKK (Akt-mut-O3a, ERK-mut-O3a, and IKK-mut-O3a). (i) The expression of KRT14 in 4T1ApoA1 cells transfected with IKBKB, IKBKE, or IKBKG encoding plasmids. **p < 0.01, ***p < 0.001