FBP negatively affects p21 and BCCIP expression in Huh7 cells. (A) Stably FBP-knockdown (FBP-kd) Huh7 cells by expressing FBP1 targeting shRNA: Huh7 cells were transfected with lentivirus vector encoding FBP1 targeting shRNAs or with empty vector alone. Stable clones (NP-4, NP-5) were selected after several passages via puromycin selection and were confirmed for stable knockdown of FBP1 expression by Western blot analysis as compared to cells transformed with the vector alone (NP-2, NP-3). (B). Expression of p53 and p21 in FBP-kd cells. Huh7 cells stably transduced with FBP1 shRNA (NP-4) or empty vector alone (NP-2) were grown for 48 h. Cell lysates were normalized with respect to protein and Western blotted for the expression of FBP1, p53, p21, and actin. Lane 1, control Huh7 cells; lane 2, FBP-kd Huh7 cells; lane 3, Huh7 cells transduced with a lentivirus vector only. (C) Construction of FBP1 expression clone resistant to FBP1-shRNA. The FBP1-shRNA targeted sequences spanning codons 248 to 254 and from 560 to 567 were subjected to point mutation in FBP1 expression clone (pCIA-CMV-FBP) without changing the amino acid sequence. (D) Transient expression of FBP1 in FBP-kd cells suppressed the expression of p21. FBP-kd cells were transfected with shRNA resistant FBP1 expression clone ((pCIA-CMV-FBPSHR) or empty pCIA-CMV vector; 48-h later cells lysates were Western blotted for FBP1, p53 and p21 and Actin. Lane 1, control Huh7 cells, lane 2, Huh7 cells transduced with lentiviral vector alone; lane 3, FBP-kd cells; lane 4, FBP-kd cells transfected with empty vector; lane 5, FBP-kd cells transfected with shRNA resistant FBP1 expression clone. (E) Expression level of p21 in control and p53-kd, BCCIP-kd, and FBP-kd Huh7 cells. Lane1, control Huh7 cells; lane 2, lentivirus vector control; lane 3, p53-kd; lane 4, BCCIP-kd and lane 5, FBP-kd Huh7 cells. A colon cancer cell line, HCT116 without p53 (-/-) (lane 6) or with wild-type p53 (+/+) (lane 7) was used as controls.