Figure 1.
Anoikis insensitivity of A549 cells is associated with lack of caspase activation.
A. Cells were cultured being attached (A) or detached from the ECM for 24 hours (D24) and 48 hours (D48) and were harvested for apoptosis analysis using the Cell Death Elisa ELISA. In parallel, cells were also treated with 1 µM staurosporine (STS) for 24 hours and subjected to Cell Death Elisa ELISA. B. Cytosolic fractions were obtained from cells in A and analysed for the presence of cytochrome c by western blot. C. The presence of caspase-3-like activity in the cell lysates from cells treated in A was determined by the cleavage of a fluorescent substrate z-DEVD-AFC (DEVD). D. and E. Cells in A were subjected to western blotting to detect the processing of pro-caspase 3 (D) and PARP (E). In A and C, three independent experiments were performed in triplicates, * indicates p<0.05 as compared to attached conditions (Student's t test).
Figure 2.
Mitochondrial Bit1 induces anoikis in A549 cells.
A. Cells were transfected with the C-terminally myc-tagged mitochondrial localized Bit1 (Bit1 mito) or the empty vector construct. 24 h post-transfection, cells were cultured being attached or detached from the ECM. Following 48 h in culture, cells were harvested and analyzed for apoptosis by measuring the amount of DNA histone fragments (Cell Death Elisa). B and C. Cells were transfected with the N-terminally myc tagged cytoplasmic localized Bit1 (Bit1 cyto) (B), Bit1 cell death domain (CDD) (C), or vector construct and 48 h later, cells were subjected to Cell Death ELISA. D. Vector or Bit1 mito transfected cells were cultured being attached (A) or detached (D) from the ECM for the indicated times. Cells were then harvested and subjected to western blotting against specific antibodies to caspase-3, PARP, Bcl-2, Bcl-xl, Bax, Bad, myc, and B-actin. E. Bit1 mito transfected cells were cultured in attached or detached conditions in the presence of z-Vad-fmk (50 uM) or DMSO for 48 h. Cells were then harvested and subjected to Cell Death ELISA. In A, B, and C, three independent experiments were performed in triplicates, * indicates p<0.05 by Student's t test.
Figure 3.
Knockdown of Bit1 expression protects A549 cells from anoikis.
A. and B. Cells were cultured in attached (A) or detached (D) conditions for the indicated times and then subjected to subjected to Cell Death ELISA (A) or western blotting (B) with antibodies against caspase-3, PARP, and B-actin. In A, three independent experiments were performed in triplicates; *, p<0.05 as compared to attached cells (Student's t test). C. and D. Cells were transfected with control- or Bit1 specific siRNAs, and 48 h later, cells were subjected to immunoblotting (C) with antibodies against Bit1 and B-actin to confirm the knockdown of Bit1 expression. In parallel, control- and Bit1- siRNA treated cells were cultured in attached (A) or detached (D) conditions for the indicated times and subjected to Cell Death Elisa (D). E and F. Stable A549 control shRNA and Bit1 shRNA knockdown clones and pools were generated (as described in materials and methods) and subjected to western blotting (E) using specific antibodies to Bit1 and B-actin. In F, the control shRNA and Bit1 shRNA pools were cultured in attached (A) or detached (D) conditions for the indicated times and subsequently analysed for apoptosis by Cell Death Elisa. In A, D, and F, three independent experiments were performed in triplicates, * indicates p<0.05 as compared to control cells (Student's t test).
Figure 4.
Bit1 attenuates the anchorage-independent growth of A549 cells.
A., B. and C. Cells transfected with vector or Bit1 mito construct were subjected to soft agar assay as described in materials and methods. The representative colonies are shown in A. The extent of colony formation was quantified by counting the number of visible colonies with a diameter greater than 30 uM (B) and by alamar blue staining and fluorescent assay (C). D, E and F. The stable A549 control shRNA and Bit1shRNA knockdown pools were also subjected to soft agar assay. The representative colonies are visualized in A and quantified by counting the number of visible colonies (E) and alamar blue assay (F). In B, C, E and F, three independent experiments were performed in triplicates, * indicates p<0.05 as compared to corresponding control cells (Student's t test).
Figure 5.
TLE1 inhibits Bit1 anoikis function.
A. and B. A549 cells were transfected with Bit1 mito and/or with GFP-tagged TLE1 construct. 24 h post transfection, the expression of exogenous Bit1 and TLE1 in transfected cells was confirmed by western blotting using specific antibodies to myc and GFP tags. The amount of plasmid transfected into cells was normalized with the vector construct. In parallel, transfected cells were grown in suspension for 48 h and then subjected to Cell Death ELISA assay (B). C. A549 can HOP-92 cells were transfected with Bit1 cyto or GFP-TLE1 construct. 48 h post-transfection, cells were harvested and analyzed for apoptosis by Cell Death Elisa. D. A549 cells were transfected with control- or TLE1 specific siRNAs, and 48 h later, cells were subjected to immunoblotting with antibodies against TLE1 and B-actin to confirm the knockdown of TLE1 expression. E. A549 cells were transfected with vector or Bit1 mito construct, and 24 hr later, the mitochondrial Bit1 treated cells were transfected with control- or TLE1-siRNAs as indicated. Cells were then cultured in suspension for 48 hr and subjected to Cell Death Elisa. In B, C, and E, three independent experiments were performed in triplicates, * indicates p<0.05 by Student's t test.
Figure 6.
TLE1 blocks Bit1-AES complex formation by sequestering AES in the nucleus.
A. and B. A549 cells were transfected with GFP-TLE1 together with N-terminally myc tagged Bit1 construct. The amount of DNA was normalized with the empty vector in each transfection. 24 h post-transfection, cells were harvested and cell extracts were prepared, immunoprecipitated with agarose-immobilized anti-myc, and immunoblotted with anti-AES and anti-myc antibodies (A). In parallel, transfected cells were subjected to nuclear isolation as indicated in the materials and methods. The resulting nuclear fraction was run on SDS-PAGE and western blotted with an anti-AES antibody. The Histone H2B was used as a nuclear marker (B). C. A549 cells were transfected with vector or GFP-TLE1 construct, and 24 h post-transfection, cells were subjected to immunoprecipitation with agarose-immobilized anti-GFP and western blotting with anti-AES and anti-GFP antibodies.
Figure 7.
Suppression of Bit1 enhances tumorigenecity of A549 cells in vivo.
A. A549 derived control shRNA and Bit1 shRNA cells (1.0×106) were injected subcutaneously in 8 week old BALB/c nude mice. Tumors were measured periodically with a calliper on the days after injection as indicated. B. and C. Mice were anesthetized and sacrificed on the day 35 after injection. Subcutaneous tumors were surgically excised, and the tumors were then photographed (the representative tumors are shown in (B) and weighed (C). In D and E, tissue sections of control shRNA and Bit1 shRNA xenografts were subjected to TUNEL assay to detect cellular apoptosis. Representative images of TUNEL stained tumor sections are shown in (C). Arrows indicate apoptotic cells characterized by a brown staining. D. TUNEL positive tumor cells in serial sections of control shRNA and Bit1 shRNA tumors were quantified. In C and E, * indicates p<0.05 as compared to control tumors (Student's t test).
Figure 8.
Bit1 is downregulated in NSCLC tissues.
A. Lung tumor tissue array slides were stained with the affinity purified rabbit anti-Bit1 antibody. Images are representative of each respective case type: normal lung (i, ii 10×), squamous cell carcinoma (iii, iv, viii 10×), adenocarcinoma (v, vi 10×) and large cell carcinoma (vii). B. The average Bit1 immunostaining intensity in normal bronchial and alveolar epithelial tissues and in various types of NSCLC cancer was determined as described in materials and methods. The normal bronchial and alveolar epithelial tissues was statistically significant from the NSCLC tumors using the ANOVA and subsequent Tukey post-hoc analysis (*, P<0.05).