Western blot analyses of protein expression in A549 cells after DOX and LMB treatment.

<p>A, Effects of pre-DOX+LMB treatment on the protein expression of p53, phospho-p53 (Ser15), phospho-p53 (Thr55), p21, and survivin in A549. Cells were treated with 0.5 µM DOX 24 h before treatment with LMB (1 nM or 5 nM). After 48 h LMB treatment, cells were harvested for Western blot analysis to determine protein levels. Blots were also probed for α-tubulin to confirm equal protein loading. B, The relative protein intensities of p53, phospho-p53 (Ser15), phospho-p53 (Thr55), p21, and survivin as compared with the intensity of α-tubulin. The intensity of each band was quantified using Quantity One software. Data are means ± SD, n = 3. The experiments were conducted in triplicate. LMB1: 1 nM LMB; LMB5: 5 nM LMB; *, <i>P</i><0.05 compared to control; **, <i>P</i><0.01 compared to control; #, <i>P</i><0.05, compared to LMB5; ##, <i>P</i><0.01, compared to LMB5.</p

Effects of DOX and LMB on cell cycle and apoptosis of A549 cells.

*<p><i>P</i><0.05 in comparison to control;</p>**<p><i>P</i><0.01 in comparison to control.</p>#<p><i>P</i><0.05 in comparison to LMB alone;</p>##<p><i>P</i><0.01 in comparison to LMB alone.</p

Nuclear proteome profiling in A549 cells after DOX and/or LMB treatment.

<p>A, Western blot of nuclear and cytoplasmic protein extractions from A549; α-tubulin served as an internal control for cytoplasmic proteins, and histone 3 served as a control for nuclear proteins. B, 2D-DIGE analyses of nuclear proteins in A549 cells and 3D views of SQSTM1 in A549 cell with vehicle control or LMB treatment. Nuclear proteins treated with LMB or vehicle control were labeled with Cy3 (green channel) and Cy5 (red channel), respectively. Nuclear proteins were separated based on isoelectric point (PI, horizontal axis) and molecular weight (MW, vertical axis). Approximately 1,000 protein spots were detected in nuclear extractions of A549 cells. Spots labeled with red color indicate decreased expression after LMB treatment, while spots labeled with green color indicate increased expression after LMB treatment (left panel). Magnification of 5 protein spots (right upper panel) and 3D view of vehicle control and LMB treated (right bottom panel) (identified by LC/MS/MS as sequestosome 1 (SQSTM1/p62)). C, Protein sequence and tandem mass spectrometry identification of SQSTM1. The MS/MS fragmentation spectrum (obtained after trypsin digestion) of AYLLGKEDAAR for SQSTM1 is shown. The resultant MS/MS data were processed using Mascot. D, Western blot analysis of SQSTM1 in cytoplasm and nucleus of A549 cells after LMB treatment. E, Effects of LMB alone or pre-DOX+LMB treatment on protein expression of SQSTM1 in A549 cells. The relative protein intensity of SQSTM1 was compared with the intensity of corresponding α-tubulin. The intensity of each band was quantified using Quantity One software. Data are means ± SD. Experiments were conducted in triplicate. LMB1: 1 nM LMB; LMB5: 5 nM LMB; **, <i>P</i><0.001 compared to control.</p

Cytotoxic effects of DOX and LMB on A549 cells.

<p>A, Cytotoxic effects of DOX alone and DOX+LMB on cell viability of A549 cells as determined by the MTT assay. Data are expressed as the percentage by comparing to vehicle control for DOX and LMB (0.5 nM) for DOX+LMB. Values are represented as means ± SD, n = 6. B, Cytotoxic effects of LMB alone and LMB+DOX on cell viability of A549 cells as determined by the MTT assay. Data are expressed as the percentage by comparing to vehicle control for LMB and DOX (0.5 µM) for LMB+DOX. Values are means ± SD, n = 6. C, Cytotoxic effects of DOX alone and pre-LMB+DOX on cell viability of A549 cells at 48 h as determined by the MTT assay. Data are expressed as the percentage by comparing to vehicle control for DOX and pre-LMB for pre-LMB+DOX. Values are means ± SD, n = 6. D, Cytotoxic effects of LMB alone and pre-DOX+LMB on cell viability of A549 cells at 48 h as determined by the MTT assay. Data are expressed as the percentage by comparing to vehicle control for LMB and pre-DOX for pre-DOX+LMB. Values are means ± SD, n = 6. Experiments performed in triplicate yielded similar results.</p

Pathology analysis of the mice injected with Ba/F3 cells, control p185 cells and p185 cells transduced with Abi1 shRNA

() Spleen weight of mice injected with Ba/F3 cells (control) and the p185 cells expressing with (p185 + Abi1 shRNA) or without (p185) Abi1 shRNA. () Histology of spleens and livers from the mice receiving Ba/F3 cells (control) and the p185 cells expressing with (p185 + Abi1 shRNA) or without (p185 ctrl) Abi1 shRNA. Arrowheads indicate the massively expanded p185 cells.<p><b>Copyright information:</b></p><p>Taken from "Abi1 gene silencing by short hairpin RNA impairs Bcr-Abl-induced cell adhesion and migration and leukemogenesis "</p><p></p><p>Carcinogenesis 2008;29(9):1717-1724.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2527646.</p><p></p

The knock down of Abi1 expression attenuated Bcr-Abl-induced activation of Lyn kinases

Ba/F3 cells (lanes 1 and 4), control p185 cells (lanes 2 and 5) and p185 Abi1 shRNA cells (lanes 3 and 6) were starved in RPMI 1640 plus 0.1% bovine serum albumin for 6 h. The cells were then treated without (lanes 1–3) or with (lanes 4–6) recombinant rat IL3 (20 ng/ml) for 10 min. The lysates (2 × 10 cells) from indicated cell lines were immunoprecipitated (IP) with anti-Lyn antibodies, separated on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to western blot (WB) analysis using anti-Src p416 antibody, which recognizes activated Lyn (upper panel). The membrane then was stripped and reprobed with anti-Lyn antibodies (lower panel). Two Lyn isoforms, p53Lyn and p56Lyn, were indicated.<p><b>Copyright information:</b></p><p>Taken from "Abi1 gene silencing by short hairpin RNA impairs Bcr-Abl-induced cell adhesion and migration and leukemogenesis "</p><p></p><p>Carcinogenesis 2008;29(9):1717-1724.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2527646.</p><p></p

Abi1 knockdown inhibited Bcr-Abl-stimulated actin cytoskeleton remodeling, MT1-MMP clustering, as well as cell adhesion and migration on fibronectin-coated surfaces

() Inhibition of Bcr-Abl-induced abnormal actin remodeling by Abi1 knockdown. Ba/F3 cells and the Ba/F3 cells expressing p185, p185 and p185 plus Abi1 shRNA were fixed and stained with TRITC-conjugated phalloidin. The cells with F-actin-rich structures (invadopodia-like structures) were visualized by fluorescence microscopy as shown by arrowheads (upper panel) and were counted (lower panel, represented as mean ± SD percentage of three randomly picked areas). () The p185 cells expressing GFP–MT1-MMP were transduced with either control retrovirus (control) or the retrovirus expressing Abi1 shRNA. The knock down of Abi1 expression was confirmed by western blotting (data not shown). The distribution of GFP–MT1-MMP was visualized by fluorescence microscopy. A similar result has been described previously (). () Effects of Abi1 knockdown on Bcr-Abl-stimulated cell adhesion (lower panel) and migration (upper panel) on fibronectin-coated surfaces. Ba/F3 cells and the p185 cells transduced with either non-silencing shRNA or Abi1 shRNAs were grown in fibronectin-coated six-well plate (2.5 × 10 per well) for 16 h. The total cells and the cells that were adherent to fibronectin-coated surfaces were counted and the percentage of adherent cells calculated. The vertical axis shows the percentage of the adherent cells and is expressed as the mean ± SD of triplicate wells. For cell migration on fibronectin-coated membrane, 1 × 10 cells were tested in Transwell migration assay. The vertical axis shows the percentage of the migrated cells and is expressed as the mean ± SD of triplicate wells; * < 0.01.<p><b>Copyright information:</b></p><p>Taken from "Abi1 gene silencing by short hairpin RNA impairs Bcr-Abl-induced cell adhesion and migration and leukemogenesis "</p><p></p><p>Carcinogenesis 2008;29(9):1717-1724.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2527646.</p><p></p

Abi1 knockdown did not affect Bcr-Abl-induced protein tyrosine phosphorylation and IL3-independent growth

() Profiles of protein tyrosine phosphorylation in Ba/F3 cells and p185 cells transduced with either non-silencing shRNA (p185) or Abi1 shRNAs (Abi1R). Indicated cell lines were starved in RPMI 1640 plus 0.1% bovine serum albumin for 6 h prior to harvest. Total lysates from 1 × 10 cells were separated on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to western blot analysis with anti-phosphotyrosine antibody. () IL3-independent growth of Ba/F3 cells as well as the p185 cells transduced with either non-silencing shRNA (p185) or Abi1 shRNA (Abi1R).<p><b>Copyright information:</b></p><p>Taken from "Abi1 gene silencing by short hairpin RNA impairs Bcr-Abl-induced cell adhesion and migration and leukemogenesis "</p><p></p><p>Carcinogenesis 2008;29(9):1717-1724.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2527646.</p><p></p

Systolic blood velocity.

<p>High resolution ultrasound (HRUS) was used to measure blood flow velocities in the luminal center of the abdominal region of the aorta in 8–10 animals from each group. Note that both CMVpr-hIL10- and LOX1pr-hIL10-HCD-treated animals had significantly lower blood velocity than the AAV/Neo-HCD-treated animals, similar to ND controls (<i>p</i><0.05). However, CMVpr-hIL10-HCD- and LOX1pr-hIL10-HCD-treated animals were statistically similar to each other (<i>p</i> = 0.377).</p

Analysis of the aortic lumen by high resolution ultrasound (HRUS).

<p>HRUS was used to measure the cross sectional area of the thoracic region of the aortas in 8–10 animals from each animal group. Shown is a quantification of the cross-sectional area for the abdominal/thoracic region of the aorta. Note that both CMVpr-hIL10- and LOX1pr-hIL10-HCD-treated animals had a significantly larger cross sectional area than the AAV/Neo-HCD-treated animals, indicating significant efficacy (<i>p</i><0.05). However, CMVpr-hIL10-HCD- and LOX1pr-hIL10-HCD-treated animals were statistically similar to each other (<i>p</i> = 0.235).</p