Differential gene expression in human hepatocellular carcinoma Hep3B cells induced by apoptosis-related gene BNIPL-2

Figure 1 Agarose gel (10 g/L) electrophoresis analysis of re-combinant pTRE2hyg-BNIPL-2 digested with BamHI and ClaI. Lane 1: Lambda DNA/Hind III markers, Lane 2: SD 006 markers, Lane 3: pTRE2hyg-BNIPL-2 digested with BamHI and ClaI.

Figure 2 Western blot analysis of BNIPL-2 expression after Dox induction. A: Different cell protein was gotten from 37 clones after hygromycin selection. The clone Tet-on-BNIPL-2/13 showed a low background and high induction fold of BNIPL-2 regulated by Dox. The intensity of each band was quantified by densitometry. Lane 1: lysate of Tet-on-BNIPL-2/13, Lane 2: lysate of Tet-on-BNIPL-2/13+Dox, Lane 3: lysate of Tet-on-BNIPL-2/17, Lane 4: lysate of Tet-on-BNIPL-2/17+Dox, Lane 5: lysate of Tet-on-BNIPL-2/21, Lane 6: lysate of Tet-on-BNIPL-2/21+Dox, Lane 7: lysate of Tet-on-BNIPL-2/28, Lane 8: lysate of Tet-on-BNIPL-2/28+Dox. B: Hep3B-Tet-on-BNIPL-2/13 cells were treated with Dox at different concentrations (0, 500, 1 000, 2 000 μg/L) for 24 h. C: Hep3B-Tet-on-BNIPL-2/13 cells were treated with Dox (2 000 μg/L) for different time as indicated (0, 12, 24, 48 h). Equal amount of total cell protein was analyzed using BNIPL-2 antibody. β-actin was used as an internal control.

Figure 3 Parallel analysis of gene expression profiles in Hep3B-Tet-on-BNIPL-2/13 cells before or after BNIPL-2 overexpression. Total RNA samples were purified from 1×10⁷ cells using TRIzol reagent before (A) or after (B) BNIPL-2 overexpression. They were reverse-transcribed into cDNA, labeled with [α-³²P] dATP, and hybridized to Atlas human cDNA expression array (#7740-1) according to the manufacturer’s protocol. Notes: The genes in the row G are housekeeping genes. Arrows indicate the three examples of differentially expressed genes (A6b, p120 antigen; C5n, Rad; D6f, p21).

Figure 4 Semi-quantitative RT-PCR assays of Rad, CLK-1, p21, and p38 MAPK before or after BNIPL-2 overexpression, β–actin was also amplified as an internal control. The figure was con-sisted with the hybridization results. Lane 1: Before BNIPL-2 inducible expression, Lane 2: After BNIPL-2 inducible expression.