UbB siRNA could down-regulate the SP and spheroids formation.

<p><b>A</b>, HeLa cells were transfected with 10 nM UbB-targeting siRNA, or stealth RNAi control, for 72 hours. Real-time PCR analysis of relative UbB mRNA expression was proceeded. GAPDH was used as a reference control. <b>B</b>, The quantitative analysis of the UbB protein level by western blotting. Cells were treated as described in A. Beta-actin was used as a reference control. <b>C</b>, HeLa and HeLa/TSA mammosphere cells were infected with LV-UbBsi, which was cloned with UbBsi2, for 72 hours and subjected to analysis by western blotting for UbB protein level. <b>D</b>, LV-UbBsi-infected mammosphere cells were analyzed for the UbB, UbC, UbA52 and UbA80 mRNA levels. GAPDH was used as a reference control. The columns represent the average of three separate experiments; error bars, SD. <b>E</b>, LV-UbBsi-infected HeLa/TSA mammosphere cells were analyzed for the Sox2, Oct4 and Nanog mRNA levels. GAPDH was used as a reference control. The columns represent the average of three separate experiments; error bars, SD. <b>F</b>. LV-UbBsi-infected HeLa/TSA mammosphere cells were analyzed for the Sox2, Oct4 and Nanog protein levels. Beta-actin was used as a reference control. <b>G</b>, The analysis of the side-population cell fraction in LV-UbBsi-infected HeLa and HeLa/TSA cell cultures. <b>H</b>, The spheroid formation assays were performed on LV-UbBsi-infected cells.