Article Figures & Data
Figures
- Figure 1.
Ral regulates CD24. A, representative blots of Ral knockdown in UM-UC-3 and HeLa cells using control GL2, RalA, RalB, and the RalA/B duplex. Protein was quantitated using the bicinchoninic acid assay and equal amounts loaded. Top, blots probed for RalA; bottom, blots probed for RalB. B, oligonucleotide microarray analysis of CD24 mRNA expression in UM-UC-3 cells 72 hours after transfection with indicated siRNAs. Columns, % control CD24 expression in indicated Ral siRNA-treated sample; bars, SD. C, quantitative RT-PCR analysis of CD24 expression in UM-UC-3 and HeLa cells 72 hours after treatment with RalA/B or GL2 siRNA. Columns, % control CD24 expression; bars, SD. D, immunoblotting for CD24 in UM-UC-3 and HeLa cells 72 hours after treatment with RalA/B or GL2 siRNA. Blots were stripped and reprobed for tubulin as a loading control. RalA/B knockdown results in down-regulation of CD24. *, P ≤ 0.05.
- Figure 2.
Growth curves and soft agar assays for CD24-depleted cells. After 4 days of growth, CD24 knockdown results in significantly less cells compared with GL2 nontargeting control in (A) UM-UC-3 (∼53% less), (B) DU145 (∼66% less), (C) HeLa (∼57% less), (D) MCF-7 (∼68% less), and (E) SAOS-2 (∼26% less). Points, representative of multiple experiments using duplicate wells serially assayed using Alamar Blue, with fluorescence reported for each day; bars, SD. Inset, immunoblotting for CD24 72 hours after treatment with CD24 or GL2 siRNA in indicated cell lines. Top, anti-tubulin loading control; bottom, anti-CD24. F, in UM-UC-3 (15 days) and DU145 (25 days) cells, CD24 knockdown results in significantly reduced clonogenicity on soft agar compared with GL2 control. Columns, representative averages of counts of colonies in 1-cm diameter areas of triplicate wells in assays done at least twice; bars, SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
- Figure 3.
Microscopy and cell cycle analysis of CD24-depleted cells. A, phase-contrast microscopy of GL2 siRNA-treated UM-UC-3 cells reaching confluence at 72 hours after transfection. B, CD24 siRNA-treated UM-UC-3 cells at 48 hours are flattened and produce phase dark cytoplasmic protrusions. C, CD24 siRNA-treated cells at 72 hours rounded up and lost adhesion, and floating cells were apparent in culture. D, GL2 siRNA-treated UM-UC-3 cells at 72 hours exhibit latitudinal and longitudinal stress fibers on phalloidin staining, as previously reported. E, CD24 siRNA-treated cells at 48 hours seem to lose stress fiber organization of the actin cytoskeleton. F, CD24 siRNA-treated cells at 72 hours show loss of stress fibers, rounding, and an apoptotic figure visible on Hoechst 33342 nuclear staining (arrow). G, cell cycle analysis by propidium iodide staining for UM-UC-3 bladder cancer cells, 4 days after transfection with CD24 or GL2 siRNA, as indicated. Propidium iodide stains the DNA complement in cells, and DNA fragmentation, a hallmark of apoptosis, is represented by a shift in DNA content to the left of the main G0-G1 peak at 200, the hypodiploid range subtended by the M1 markers indicated. H, similar analysis to that in (G) on DU145 prostate cancer cells.
- Figure 4.
CD24 in human bladder cancer. A, Affymetrix analysis expression analysis of CD24 mRNA shows fold overexpression in bladder cancer compared with normal bladder mucosa. Columns, fold change; bars, SE. B, tissue microarray staining for CD24. Representative scoring of 1+, 2+, and 3+ levels. C, Kaplan-Meier estimation of disease-free survival as a function of CD24 immunohistochemical expression staining.
Volume 66, Issue 4
