Fig 1.
Kinetic analysis of [3H]TdR-incorporation (A) and cell cycle distribution (B) during TPA-induced differentiation of U937 cells. For the proliferation assay, U937 cells (5 × 104 cells/well) were treated with 32 nM TPA in 96-well plates and pulsed for 4 h with 1 μCi of [3H]TdR at the indicated times. Each value is expressed as the mean ± SD (n = 3 with three replicates per independent experiment). *p < 0.05 compared to the control. After equivalent cultures (2.0 × 105 cells/mL) were incubated, cells were harvested at the indicated times and stained with PI for flow cytometry.
Fig 2.
Kinetic analysis of the expression of LAPTM5 mRNA (A) and LAPTM5 protein (B) during TPA-induced differentiation of U937 cells. U937 cells were treated with 32 nM TPA for the indicated time and then total RNA and cell lysates were extracted. For northern blot analysis, total RNA (10 μg) were electrophoresed, transferred, and probed with 32P-labeled human LAPTM5, integrin α6 subunit, and 18S rRNA cDNA. After individual cell lysates were prepared, western blot analyses using anti-human LAPTM5, anti-COX-1, anti-COX-2, anti-APOC2, anti-NF-κB, anti-IκBα, anti-p47 phox, anti-cyclin A, and anti-GAPDH antibodies were performed as described in the Materials and Methods. A representative study is shown; two additional experiments yielded similar results.
Fig 3.
Northern blot analyses of LAPTM5-specific mRNA in various human normal tissues (A) and in various human malignant cell lines (B). Human multiple-tissue northern membranes and human immune system multiple-tissue northern membrane, each containing 2 μg of poly(A)+ RNA per lane, were sequentially hybridized using 32P-labeled human LAPTM5 and β-actin cDNA. The northern blot contained 10 μg of total RNA in each lane, which was isolated from each cell line, was hybridized with 32P-labeled human LAPTM5 and 18S rRNA cDNA.
Fig 4.
Kinetic analysis of [3H]TdR-incorporation (A) and western blot analysis of LAPTM5 protein (B) during TPA-induced differentiation of HL-60 cells into monocytes/macrophages or DMSO-induced differentiation of HL-60 cells into granulocytes. For the proliferation assay, HL-60 cells (1 × 105 cells/well) were treated with 32 nM TPA or 1.25% DMSO in 96-well plates and pulsed for 4 h with 1 μCi of [3H]TdR at the times indicated. Equivalent cultures were incubated and the cells were harvested at the indicated times for preparation of cell lysate. Western blot analysis was performed as described in the Materials and Methods. Representative results are presented; two additional experiments yielded similar results.
Fig 5.
Localization of GFP (A) and GFP-LAPTM5 (B) in HeLa cells, and immunofluorescence staining by anti-LAPTM5 of HeLa cells transfected with pCAGGS (C) or pCAGGS-LAPTM5 (D). To observe localization of GFP and GFP-LAPTM5, HeLa cells transfected with either a GFP vector or a GFP-LAPTM5 vector were fixed with 4% paraformaldehyde for 30 min. For immunostaining of ectopically expressed LAPTM5 in HeLa cells, cells transfected with pCAGGS or pCAGGS-LAPTM5 were fixed with cold methanol and stained with rabbit polyclonal anti-human LAPTM5 antibody and DAPI as described in the Materials and Methods. Images were visualized at ×200 using the LSM 700 confocal laser scanning microscope. Representative results are presented; two additional experiments yielded similar results.
Fig 6.
Effect of ectopic LAPTM5 expression on cell viability (A), cell cycle distribution (B), and apoptotic cell death measured by FITC-Annexin V and PI staining (C) and forward scatter dot plot (D) in HeLa cells. Untreated HeLa cells or those transfected with pCAGGS or pCAGGS-LAPTM5 were harvested 48 h after transfection. Cell cycle distribution and apoptotic cell death were determined by flow cytometric analyses with PI staining and FITC-Annexin V/PI double staining, respectively. The forward scatter properties of individual unstained live, early apoptotic, and late apoptotic cells were measured to analyze changes in cell size during the induced apoptosis. For the cell viability assay, the individual cells harvested 12 h after transfection were added to a 96-well plate (1 × 104 cells/well) and incubated for an additional 36 h, with MTT added for the final 4 h. The cells were sequentially processed to assess the colored formazan crystal produced from MTT as an index of cell viability. Each value is expressed as the mean ± SD (n = 3 with three replicates per independent experiment). *p < 0.05 compared to the control. Representative results are presented; two additional experiments yielded similar results.
Fig 7.
Flow cytometric analyses of Δψm loss (A), Bak activation (B), and western blot analyses of LAPTM5, Bcl-2, Bcl-xL, Mcl-1, Bak, Bax, Bid, Bim, and GAPDH (C), and activation of caspase-9, -8, and -3, PARP cleavage, and GAPDH (D) in HeLa cells ectopically expressing LAPTM5. HeLa cells untreated and transfected with pCAGGS or pCAGGS-LAPTM5 were harvested 48 h after transfection. Flow cytometric analyses of Bak activation and Δψm loss, and western blot analyses were performed as described in the Materials and Methods. Representative results are presented; two additional experiments yielded similar results.
Fig 8.
Effect of Mcl-1 overexpression on the ectopic LAPTM5-induced apoptotic sub-G1 peak (A), and changes in the level of pro-apoptotic and anti-apoptotic regulatory proteins (B) in HeLa cells. After HeLa cells were transfected with pCAGGS/pcDNA3.1, pCAGGS-LAPTM5/pcDNA3.1, pCAGGS-LAPTM5/pcDNA3.1-Mcl-1, or pCAGGS/pcDNA3.1-Mcl-1 for 48 h, cells were harvested and subjected to cell cycle analysis and western blot analysis as described in the Materials and Methods. Representative results are presented; two additional experiments yielded similar results.
Fig 9.
Effect of the pan-caspase inhibitor (z-VAD-fmk), the pan-cathepsin inhibitor (Cathepsin inhibitor I), and the cathepsin D inhibitor (Pepstatin A) on the ectopic LAPTM5 overexpression-induced apoptotic sub-G1 peak (A) and Δψm loss (B) in HeLa cells. After HeLa cells were transfected with pCAGGS or pCAGGS-LAPTM5 for 12 h, 30 μM z-VAD-fmk, 2.5 μM Cathepsin inhibitor I, or 1.0 μM Pepstatin A were added to the individual cells and incubated for an additional 36 h. The apoptotic sub-G1 peak and Δψm loss of the cells were analyzed using flow cytometry as described in the Materials and Methods. Representative results are presented; two additional experiments yielded similar results.