Fig. 1. Human Lats2 recombinant adenovirus construction and dosage-dependent expression. (A) The schematic drawing represents a replication-defective Lats2 recombinant adenovirus, Ad-Lats2, and the control virus, Ad-EGFP. (B) Dosage-dependent expression of human LATS2 protein in human lung cancer cell lines. A549 and H1299 were infected by Ad-Lats2 at indicated MOI and harvested 2 days after virus infection for Western blot using a monoclonal anti-LATS2 antibody (9B4). Arrows indicate the degraded LATS2 proteins. β-ACTIN was used as the loading control. ITR: inverted terminal repeat; Pcmv: early promoter from CMV virus, IRES: internal ribosome enter site.

Fig. 2. Expression of exogenous human Lats2 induces apoptosis. (A) Genomic DNA fragmentation in Lats2-transduced human lung cancer cells. Genomic DNA was extracted from Ad-Lats2 or control virus-transduced cells 4 days (for A549) or 5 days (for H1299) after virus infection, and separated on a 1.5% agarose gel as described in Materials and methods. (B) Lats2-induced cleavage of PARP nuclear protein. Ad-Lats2 or control virus-transduced A549 and H1299 were respectively harvested for Western blot with anti-PARP antibodies 2 or 3 days after virus infection. Full-length PARP (116 kDa) and its cleavage product (85 kDa) were indicated by molecular weight. Arrow indicates a specific band recognized by anti-PARP antibodies. Its identity is not clear (see Discussion). The MOI used in this experiment was 20 pfu/cell for A549 and 30 pfu/cell for H1299. (C) Flow cytometric analysis of Annexin V uptake in adherent Ad-Lats2 or control virus-transduced A549 or H1299 cells, 2 days (for A549) or 3 days (for H1299) after virus infection.

Fig. 3. Human Lats2 induces apoptosis by activating a caspase cascade. (A) Expression of Lats2 results in the cleavage of procaspase-3, 7, and 9. A549 cells were infected with Ad-Lats2 and control virus Ad-EGFP (MOI of 20 pfu/cell) and harvested 2.5 days after infection for Western blot analyses. Similar experiments were performed using H1299 cells with a higher MOI (30 pfu/cell) and the cells were harvested at 3.5 days after infection. Arrow indicates a specific band recognized by anti-caspase-7 antibody and its identity is not clear (see Discussion). (B) Kinase-inactive Lats2 (Lats2-kd) does not mediate the cleaving procaspases. 293 cells were transiently transfected with either Lats2, Lats2-kd, or vector alone as indicated resulting in approximately 50% cell transfection. Thirty-six hours after transfection, the cells were harvested for Western blot with anti-caspase-7 and caspase-9 antibodies separately. (C) Caspase-9 activity assay. A549 and H1299 cells were infected with Ad-Lats2 or control virus Ad-EGFP (MOI of 15 pfu/cell) and were harvested 2 days (for A549) or 3 days (for H1299) after virus infection. Two hundreds μg of cell lysate protein were used for caspase-9 activity assay. The results are the average of two the independent experiments. (D) Caspase inhibitors delay Lats2-mediated apoptosis. The A549 cells were treated with either z-DEVD-fmk (30 μM), z-DQMD-fmk (30 μM), or z-LEHD-fmk (30 μM) for 1 h before virus infection (MOI of 10 pfu/cell). Following infection, Ad-Lats2-infected cells were cultured in media containing caspase inhibitors, and the percentage of dead cells were determined at the indicated time points by a trypan blue exclusion assay. A549 cells treated with DMSO (solvent) alone were also included as a control. The experiments were each repeated three times.

Fig. 4. Lats2 downregulates the level of anti-apoptotic proteins of the Bcl-2 family. The levels of BCL-2 and BCL-x_L were downregulated in A549 and H1299 cells transduced by Ad-Lats2 (A), as well as in 293 cells transfected with Lats2, but not with Lats2-kd (B). A549 cells were infected with Ad-Lats2 or control virus at MOI of 20 pfu/cell and harvested 2.5 days after infection for Western blot analysis. Similar experiments were performed with H1299 cells using a higher MOI (30 pfu/cell) and the cells were harvested at 3.5 days after infection. Transient transfection of 293 cells was described in Fig. 3B. The amount of cell lysate protein used for each Western blot was 10 μg for BAX and 30 μg for BCL-2 and BCL-x_L. β-ACTIN was used as the loading control.

Fig. 5. Expression of exogenous Bcl-2 and Bcl-x_L blocks Lats2-mediated apoptosis. (A) Expression of exogenous Bcl-2 and Bcl-x_L in A549 and H1299 cells. Top panel: Western blot of A549 and its derivative cell lines showing Bcl-2 expression. Bottom panel: Western blot of H1299 and its derivative cell lines showing Bcl-x_L expression. Although endogenous BCL-x_L protein is easily detectable in H1299 and H1299/pCDNA3 cell lines, its expression is much weaker compared with the two Bcl-x_L expressing stable cell lines. β-Actin was used as the loading control. (B) Expression of exogenous Bcl-2 blocked the cleavage of procaspases-3, 7, and 9. Bcl-2 expressing stable cell lines and control cell lines (A549 and A549/pCDNA3) were infected by Ad-Lats2 (MOI of 15 pfu/cell). Two days after infection, 150 μg of cell lysate protein was processed for Western blot with anti-caspase-3, anti-caspase-7, and caspase-9 antibodies, respectively. (C and D) Trypan blue dye exclusion assays show that expression of exogenous Bcl-2 or Bcl-x_L blocks Lats2-mediated apoptosis. A549/Bcl-2 stable cell lines and controls were infected with Ad-Lats2 (MOI of 10 pfu/cell) and viable and dead cells were counted at indicated time points. The values indicated on the y axis are the mean ± SD of the percentage of viable cells from three independent experiments that were each performed in duplicate (C). Similar experiments were performed using H1299/Bcl-x_L stable cell lines and corresponding controls (D).

Fig. 6. Initiator caspase-9, not caspase-8, mediates Lats2-induced apoptosis. (A) Both initiator caspases-8 and 9 were processed in Ad-Lats2-transduced cells. A549 and H1299 cells were infected with Ad-Lats2 or control virus Ad-EGFP (MOI of 15 pfu/cell). They were harvested for examination of caspase cleavage by Western blot at the indicated times after virus infection. (B) Cleavages of both caspase-8 and 9 are blocked in Bcl-2 and Bcl-x_L expressing stable cell lines. The experiments were performed as described in Fig. 5B with the exception that lysates were blotted with anti-caspase-8 and anti-caspase-9 antibodies, respectively.