Fig. 4.

Knockdown of SULF2 downregulates p-Akt, cyclin D1 and Bcl-2 and upregulates BAD in Huh7 cells. (A) Huh7 cells were plated onto cover slips in six-well plates and transiently transfected with either GFP-expressing plasmids expressing a scrambled shRNA sequence or plasmids expressing an shRNA targeting SULF2 mRNA. After 24 h, cells were immunostained for phospho-Akt ser473 and examined by confocal microscopy. Nuclei were counterstained with DAPI. (B) Whole cell lysates of Huh7 vector, Huh7 SULF2 shRNA-3 and Huh7 SULF2 shRNA-4 cells were used for Western immunoblotting using antibody against phospho-Akt, cyclin D1, Bcl-2 and Bcl-XL. Total Akt and actin were used as loading controls. (C) Huh7 cells were either transiently or stably transfected with plasmids expressing a scrambled shRNA sequence or a sequence targeting SULF2. For the transient transfection, the transfection efficiency was monitored by transfection of a GFP plasmid and was 70–80%. Whole cell lysates were prepared and Western immunoblotting performed using antibody to BAD at 1:250 dilution; actin was used as a loading control. The BAD level was increased in Huh7 cells with both transient and stable knockdown of SULF2.