Fig. 4.

Bit1 regulates Erk activation through Erk phosphatase activity, and Erk down-regulation partially restores the anoikis resistance of Bit1 KO cells. (A) HeLa cells were transfected with control siRNAs or with three different specific Bit1 siRNAs. (Left) Three days after transfection, cells were lysed, and the total lysate was subjected to immunoblotting (Left) to detect the phosphorylated Erk (pErk), total Erk (tErk), active Mek (pMek), total Mek, active Akt (pAkt), total Akt (tAkt), Bit1, and β-actin. (Right) The relative p-Erk/t-Erk ratio with the level in the control siRNA was taken as 100%. *, P < 0.05, compared with control siRNA (Student's t test). The values represent the average of four independent experimental results. (B) Three days after siRNA transfection, control siRNA- and Bit1 siRNA-treated HeLa cells were transfected with the empty vector or C-terminally GFP-tagged Bit1. (Left) After 48 h, cells were harvested, and p-Erk, t-Erk, GFP-tagged Bit1, and β-actin were detected by immunoblotting (Left). The normalized pErk/tErk intensity from three independent experiments. *, P < 0.01 (Student's t test) (Right). (C and D) Stable Bit1 knockdown and control clones were established in the HeLa cancer cell line as described in Materials and Methods. Exponentially growing, stable Bit1 knockdown cells (clones nos. 8, 21, and 44), control clones (nos. 1 and 16), and the parental cell line were harvested and subjected to immunoblotting (C) and anoikis assay; *, P < 0.001, compared with control clones (Student's t test) (D). (E) Total cell lysates from HeLa cells transfected with control siRNA or Bit1 siRNA-1, as well as lysates from stable control16 and Bit1 RNAi44 clones, were subjected to an Erk phosphatase assay as described in Materials and Methods. A representative immunoblot of isolated His-6-tagged Erk2 is shown to reveal pErk2 or total Erk2 levels. The relative intensity of pErk2/tErk2 was determined, and the values represent the average of at least three independent experiments. *, P < 0.01, compared with respective control (Student's t test). (F) Stable control 16 and Bit1 RNAi44 clones were transfected with control- or Erk2-specific siRNAs; 72 h after transfection, cells were harvested and subjected to immunoblotting with antibodies against total Erk2 and phosphorylated Erk1/2 (pErks), and to an anoikis assay. Results are representative of three independent experiments. *, P < 0.001 (Student's t test).