<i>as-</i>APF increases p53 expression by modulating USP2a and MDM2 in TRT-HU1, immortalized human normal bladder epithelial cells.

<p>(<b>A</b>) p53 levels were enhanced by <i>as-</i>APF treatment. TRT-HU1 cells were incubated in <i>as-</i>APF-containing serum free medium for 3 days before harvesting cells for western blot analysis with anti-p53 antibody. (<b>B</b>) Endogenous USP2a level is rapidly decreased in response to <i>as</i>-APF treatment in TRT-HU1 cells. (<b>C</b>) Association of USP2a and MDM2 in TRT-HU1 cells. IP was performed using whole cell lysates from TRT-HU1 cells in exponential growth with anti-USP2a antibody, followed by western blot analysis with antibodies against USP2a and MDM2. Normal mouse IgG was used as an IP control (Ctrl). (<b>D</b>) USP2a loss decreases MDM2 and cell proliferation. TRT-HU1 cells were transiently transfected with siRNA of USP2a (or siCtrl). One day after transfection, cells were treated with 1 µM <i>as-</i>APF (or mock peptide)-containing medium. Three days later, cells were harvested for western blot analysis and proliferation assay. *<i>p</i><0.05, NT, non-transfected cells. (<b>E</b>) Proliferation is enhanced by overexpression of USP2a^WT, but not by USP2a^MUT. Cells transfected with USP2a^WT, USP2a^MUT or vector control (Vec) constructs were treated with 1 µM <i>as-</i>APF or mock peptide for 3 days. Western blot and proliferation assay were performed as above. Data are represented as mean +/− SD. *<i>p</i><0.05.</p

Diagram proposing the points at which the USP2a-MDM2-p53 network mediates the effect of APF on urothelial cell proliferation.

<p>Diagram proposing the points at which the USP2a-MDM2-p53 network mediates the effect of APF on urothelial cell proliferation.</p

<i>as-</i>APF downregulates MDM2, leading to inhibition of cell proliferation.

<p>(<b>A</b>) Inverse expression pattern of p53 and MDM2 in bladder cell lines. Protein extracts were prepared from growing TRT-HU1, RT4 and T24 bladder cell lines for western blot analysis using antibodies against MDM2, p53 and β-actin. (<b>B</b>) MDM2, an E3 ubiquitin ligase, is decreased in response to <i>as-</i>APF. T24 cells were treated with APF (1 µM) for the indicated times (1, 2, 4, 6 or 8 h). Whole cell lysates were prepared for immnuoblot with anti-MDM2 antibody. Fold changes compared to time 0 were calculated based on band intensities after normalization to β-actin. (<b>C</b>) Silencing of MDM2 decreases cell proliferation with or without <i>as-</i>APF treatment. Transfected T24 cells with siCtrl or siMDM2 were incubated in 1 µM <i>as-</i>APF (or negative control peptide)-containing medium for 3 days. Cell proliferation was determined by crystal violet assay. *<i>p</i><0.05 (Student’s <i>t-</i>test).</p

p53 is required for the growth inhibitory effect induced by the chemically synthesized <i>as</i>-APF.

<p>(<b>A</b>) Dose and time-dependent growth inhibitory effect of synthetic <i>as-</i>APF. T24 cells were incubated with varying doses of <i>as-</i>APF as indicated. Cell proliferation was determined by crystal violet assay at 0, 1, and 3 days after treatment. Experiments were performed in triplicate. (<b>B</b>) <i>as-</i>APF increases p53 expression. T24 cells were incubated <i>as-</i>APF (0, 0.01, 0.1 or 1 µM) containing serum free medium for 3 days. Cell lysates were prepared for immunoblot analysis using antibodies against p53 or β-actin. Fold changes of p53/β-actin are shown in the graph. (<b>C</b>) Gene silencing of p53 recovers growth arrest by <i>as-</i>APF. T24 cells were transiently transfected using siRNAs for p53 (sip53) or control (siCtrl). 24 h after transfection, cells were serum starved for 16 h and incubated with <i>as-</i>APF containing medium (1 µM) for additional 3 days. Data are expressed as mean±SD. *<i>p</i><0.05 (Student’s t-test).</p

Regulation of USP2a and MDM2 in response to <i>as-</i>APF. (A

<p>) <i>as-</i>APF downregulates USP2a expression (immunoblot). T24 cells were incubated with 1 µM <i>as</i>-APF or control peptides for the indicated times (0, 0.5, 1, 1.5, or 2 h). Western blot with anti-USP2a antibody showed that the level of USP2a level was stably decreased in response to <i>as</i>-APF treatment, but this was only transiently reduced by control mock APF peptide. (<b>B</b>) Reduced USP2a by <i>as</i>-APF treatment (IF staining). (<i>i</i> and <i>ii</i>) After incubation with 1 µM <i>as-</i>APF for 2 h, cells were fixed and stained with anti-USP2a antibody and a Cy3-conjugated secondary antibody (red). Nuclei were counterstained with DAPI. Scale bar represents 10 µm. (<i>iii</i> and <i>iv</i>) USP2a expression in T24 cells transfected with a GFP-USP2a construct was diminished in response to APF treatment. Green, USP2a. (<b>C</b>) Association of MDM2 and USP2a. Exponentially growing T24 cells were used for co-IP and western blot analysis with antibodies against USP2a and MDM2. (<b>D</b>) Knockdown of USP2a results in reduced MDM2 expression. T24 cells were transiently transfected with USP2a siRNA (siUSP2a) or siCtrl, and whole cell lysates were used for western blot analysis. (<b>E</b>) Gene silencing of USP2a inhibits cell proliferation. 3 days after transient transfection with siRNA of USP2a, cell proliferation was measured by crystal violet.</p

USP2a^WT blocks the growth inhibitory effect of <i>as-</i>APF.

<p>(<b>A–B</b>) USP2a^WT abrogated <i>as-</i>APF’s effect on MDM2 and p53 levels (IF imaging). MDM2 or p53 were labeled with specific primary antibodies and Cy3.5 conjugated secondary antibody (red). Nuclei were stained with DAPI (Blue). (<b>C–D</b>) Enforced expression of USP2a^WT alleviates the <i>as-</i>APF-induced growth inhibition. T24 cells were transiently transfected with vector, USP2a^WT or USP2a^MUT constructs. One day after transfection, cells were incubated in the 1 µM <i>as-</i>APF containing serum free medium for an additional 3 days. Proliferation was measured by crystal violet assay and representative cell images are shown in C.</p

Catalytically active USP2a, USP2a^WT, alters MDM2 and cyclin D1 expression.

<p>(<b>A</b>) Enforced expression of wild type USP2a (USP2a^WT) enhances MDM2 level and decreases p53 expression (western blot). T24 cells were transfected with varying doses of USP2a^WT plasmid. 2 days later, whole cell lysates were prepared and subjected to western blot with MDM2 or p53 antibodies. Experiments were performed 3 times and blot images are representative. Fold change was calculated based on band intensities and normalized to β-actin (right graph). (<b>B</b>) USP2a^WT, but not catalytically inactive USP2a^MUT, enhances MDM2 expression (Western blot). T24 cells were transfected with Vector, USP2a^WT or USP2a^MUT constructs, and prepared for western blot. Data are representative of at least 3 experiments. (<b>C</b>) Proliferation is enhanced by overexpression of USP2a^WT (BrdU staining). 3 days after transfection with USP2a^WT construct (or Vector control), T24 cells were stained with BrdU staining reagent for visualization of proliferating cells. Green indicates proliferating cells. Nuclei were stained with DAPI (Blue). (<b>D</b>) Cyclin D1 transcription is induced by USP2a^WT (promoter assay). Cyclin D1 luciferase promoter construct, MDM2 and varying doses of USP2a^WT (0, 0.1, 1 or 2 µg) were transfected into T24 cells. Three days after transfection, cyclin D1 promoter activity was measured by luciferase assay. *<i>p</i><0.05. **<i>p</i><0.005. (<b>E–F</b>) Cyclin D1 protein expression and nuclear localization are increased by USP2a^WT overexpression (IF imaging). Immunofluorescence staining for cyclin D1 in T24 cells was performed 2 days after overexpression of USP2a^WT. Cells were fixed and stained with specific antibody to cyclin D1 (red) and mounted with DAPI (blue) solution. Scale bar represents 10 µm. (<b>F</b>) Localization of cyclin D1 was quantified. N, only nuclear resident cyclin D1; C+N, localized in nucleus as well as cytoplasmic compartments.</p

Additional file 1: of AKT1low quiescent cancer cells persist after neoadjuvant chemotherapy in triple negative breast cancer

S2 Antibody target specificity is unaffected by sequence of primary antibody application. Merged (right) and single color (left) confocal microscopy images at × 60 of an untreated primary TNBC tumor stained in an alternate sequence: pan-AKT ➔ H3K9me2 ➔ HES1 (c.f. standard sequence of H3K9me2 ➔ pan-AKT ➔ HES1) demonstrating consistent cytoplasmic pan-AKT (green) and HES1 (red) staining and nuclear H3K9me2 (yellow) staining in an example QCC (white arrows). (PDF 3624 kb

Additional file 2: of AKT1low quiescent cancer cells persist after neoadjuvant chemotherapy in triple negative breast cancer

S1 Determination of fluorescence intensity thresholds and staining reproducibility. For each marker (HES1, H3K9me2, pan-AKT) the proportion of cells at a specific fluorescence intensity level was different between sequential sections from control tumor 4, stained simultaneously (S1A and S1B, respectively). QCC percentage (red bars) and QCC density (box and whisker plots) in control tumors 1–4 increased proportionally at 25%, 33%, and 50% thresholds (S1C, S1D, and S1E, respectively). (PDF 2666 kb

Literature Lab: a method of automated literature interrogation to infer biology from microarray analysis-4

<p><b>Copyright information:</b></p><p>Taken from "Literature Lab: a method of automated literature interrogation to infer biology from microarray analysis"</p><p>http://www.biomedcentral.com/1471-2164/8/461</p><p>BMC Genomics 2007;8():461-461.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2244637.</p><p></p> Heat map of the top 100 genes with increased expression in HL60 cells treated with ATRA compared to untreated HL60 cells (Red – high normalized expression, Blue – low normalized expression). C) Percentage of cells positive for nitro blue tetrazolium (NBT) reduction (Mean +/- St dev). D) Literature Lab ranking, association scores, and confidence calls for cell physiology, metabolism, and pathway terms (Association score is the log of the product of frequency (logPF))