Effect of depletion and over expression of Hsc70 in TG cells on bacterial internalization. (A) Depletion of Hsc70. TG cells were treated for 48 h with siRNA targeting Hsc70 or without it (reagent only or no treatment), or β-actin or the control (QIAGEN AllStars Negative Control). Expression of the indicated proteins was monitored by immunoblotting. β-actin was used as an internal control. (B) Over expression of Hsc70. TG cells were transfected with or without (control) pcDNA4/TO-Hsc70 or vector only. (C) Bacterial internalization into Hsc70 depleted (siRNA) or over expressed (over exp.) TG cells was studied in a bacterial internalization assay. Lanes correspond to panels A and B. Data are the averages of triplicate samples from three identical experiments, and the error bars represent the standard deviations. Statistically significant differences between bacterial internalization into TG cells with (Hsc70) and without siRNA (control), and over expression and the control (vector) are indicated by asterisks (*, P < 0.01). (D) Distribution of Hsc70 in non-treated (control), Hsc70 depleted (siRNA), or over expressed (over expression) TG cells. Fluorescence microscopy of stained TG cells with the R2–25 antibody (upper panels) and phase contrast microscopy of the corresponding microscopic fields (lower panels) are shown.