Journal: OncoTargets and therapy
Article Title: Targeted TPX2 increases chromosome missegregation and suppresses tumor cell growth in human prostate cancer
Figure Lengend Snippet: TPX2 depletion changes the cell fitness in LNCap prostate cancer cell line. Notes: Scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) was transfected into LNCap human prostate cancer cells for 96 h. ( A ) RT-PCR was used to detect the mRNA levels of TPX2 in LNCap cells. siRNA-transfected cells were harvested and lysed for immunoblotting to determine the protein levels of TPX2. β-actin was used as a loading control. Protein expression was quantified by densitometric analysis. ( B ) Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay, and the luminescence units indicating cell growth were measured and plotted as the growth curve. ( * The cell growth was inhibition in si-TPX2 treated cells compared with si-CTL treated cells). ( C ) Representative data and ( D ) quantitative results of siRNA-transfected cells that were seeded in ultralow attachment 96-well microplates for spheroid formation assays by using ImageJ software. ( E ) LNCap spheroids presented irregular morphology; hence, viable cells were measured using the CellTiter-Glo Luminescent Cell Viability Assay, and the luminescence units indicating cell growth were measured and plotted as the bar plot. ( F ) Scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) was transfected into LNCap cells for 72 h, followed by cell harvesting. The knockdown cells were fixed and stained with DAPI to examine the proportions of the cell cycle by using an image-flow cytometry assay. ( G ) Representative data for cell cycle proportions were analyzed and quantified using Nucleoview NC-3000 software. ( H ) Western blot analysis of the levels of TPX2, CDK1, cyclin B1, CDK2, cyclin A, and cyclin E proteins in TPX2-targeted cells. β-actin was used as a loading control. ( I ) Representative images of TPX2 and Phalloidin (a high-affinity F-actin probe conjugated to the red-orange fluorescent dye, tetramethylrhodamine) immunofluorescence in TPX2-depleted LNCap cells. TPX2 expression was decreased in si-TPX2 treated cells, and morphologic examination of the cell nucleus (DAPI stain) revealed that transfection with the TPX2 RNAi oligo pool resulted in increased multinucleated cells. Scale bar =25 mm; magnification 400×. ( J ) Histograms of multinucleation in LNCap cells. All representative graphs are from two independent experiments. Values are presented as mean ± SD (Student’s t -test, P
Article Snippet: Small interfering RNA (siRNA) and knockdown of gene expression The TPX2 siRNA oligos pool (1: 5′-GGACGAACCGGUAGUGAU-3′; 2: 5′-GCAUAAGGCAAAUCCAAUA-3′; 3: 5′-GUACCAUUGUUAAGCCUUU-3′; 4: 5′-GAAAUU CUACCCUCUAAGA-3′) was synthesized by Sigma-Aldrich Co. All transient transfections of the TPX2 siRNA oligos pool at a final concentration of 20 nM were accomplished with LipofectAMINE RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s protocols.
Techniques: CTL Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Viability Assay, Inhibition, Software, Cell Harvesting, Staining, Flow Cytometry, Cytometry, Western Blot, Immunofluorescence