Figure 4
Knockdown of pro-protein GDF-15 suppresses lidocaine-induced growth inhibition. (a) Knockdown of GDF-15 by siRNA. Twenty-four hours after transfection with control or GDF-15 siRNA, HeLa cells were treated with the indicated dose of lidocaine for 24 h. Cell lysates and culture medium were applied for Western blotting using GDF-15 and α-tubulin antibodies. Pro-protein GDF-15 expression in cell lysates and culture medium was suppressed by GDF-15 siRNA1 and 2. (b) Immunofluorescence assay of GDF-15 knockdown of HeLa cells. Twenty-four hours after transfection with control or GDF-15 siRNA, HeLa cells were treated with 3 mM lidocaine for 24 h. After lidocaine treatment of HeLa cells transfected with control siRNA, lidocaine-induced GDF-15 was detected in the perinuclear (arrow) and pericellular region (asterisk) as shown in Fig. 3d (Fig. 5, left panel). After lidocaine treatment, GDF-15 expression was suppressed in HeLa cells transfected with GDF-15 siRNA 1 and 2 (middle and right panels). Nuclei were stained with DAPI. The scale bar represents 10 μm. (c) Cell growth in GDF-15 knockdown of HeLa cells after lidocaine treatment. Twenty-four hours after transfection with control or GDF-15 siRNA, HeLa cells were treated with the indicated concentration of lidocaine for 24 h. Cell growth was evaluated using 0.5% crystal violet staining. Lidocaine-induced growth inhibition was suppressed in GDF-15 siRNA transfected HeLa cells. Each sample was assayed in triplicate. The results are presented relative to the growth of lidocaine-untreated HeLa cells after transfection with control siRNA and expressed as the mean ± SD. *p < 0.05.
