Journal: Breast Cancer Research and Treatment
Article Title: Heat shock factor 1 induces cancer stem cell phenotype in breast cancer cell lines
Figure Lengend Snippet: Mechanisms of CSC regulation by HSF1 a Western blots of three matched pairs of HSF1 high CSC-like BPLER vs. HSF1 low non-CSC-like HMLER cells (#2-4) reveal only minor differences in heat shock proteins HSP70 and HSP90. The cell lysates were probed with anti-HSP70 and anti-HSP90 antibodies. β -actin (Actin) was used as loading control. b Real-time PCR analysis of ZEB1 mRNA reveals no significant change with HSF1 over-expression ( left panel ) and knockdown ( right panel ) after 2 days DOX addition. The Zeb1 mRNA was quantified with real-time PCR after HSF1 over-expression in SUM159 and BT20 cells ( black bar, left panel ) or knockdown in BT474, T47D, and MCF7 cells ( black bar, right panel ) compared to control cells without DOX ( white bars ). GAPDH was used to normalize Ct value. The error bars represent standard deviation of the mean. c HSF1 over-expression or knockdown has no effect on heat shock protein (HSP) or EMT marker expression. Western blot analysis of HSF1, HSP 70, 90, E-cadherin(E-cad), and Vimentin(Vim) after HSF1 over-expression (HSF1) in SUM159 and BT20 cells (left panel) and knockdown (KD) in BT474, T47D, and MCF7 cells ( right panel ) cells compared to control cells (without DOX) revealed no significant changes. β -actin (Actin) was used as loading control. d HSF1 high CSC-like BPLER cells are more sensitive to protein translation inhibition compared to HSF1 low non-CSC-like HMLER cells. Three pairs of matched BPLER and HMLER cell lines (#2–4) were treated either with cycloheximide ( top panel ) or anisomycin ( bottom panel ) for 8 h, and cultured in drug-free medium for 4 days and cell viability was measured with celltiter-blue assay on day 4. The bar graphs show percent cell viability as compared to control cells (100 %, not shown here). HMLER: white bar ; BPLER: black bar
Article Snippet: Real-time RT-PCR Quantitative real-time RT-PCR was performed using SuperScript® III First-Strand Synthesis kit and SsoFast ™ EvaGreen® Supermix according to the manufacturers’ instructions and analyzed with BioRad CFX Manager Software.
Techniques: Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Standard Deviation, Marker, Expressing, Inhibition, Cell Culture, CtB Assay