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Fig. 5 | Cell Communication and Signaling

Fig. 5

From: Crystal structure of a cytocidal protein from lamprey and its mechanism of action in the selective killing of cancer cells

Fig. 5

The cytocidal activity of LIP against tumor cells disappeared upon PI-PLC or SMase treatment. (a) MCF-7 cells were incubated with (+) or without (−) PI-PLC (5 U/mL) for 2 h and then incubated with LIP and stained with PI for flow cytometric analysis. Histogram showing statistics of the above results (right pane). Means ± SDs are shown (n = 3 per group). (b) MCF-7, HepG2, H293T and MCF-10A cells were pretreated with PI-PLC and then stained with Alexa555-cholera toxin subunit B (CT-B) prior to staining with Alexa488-tagged LIP. The cells were observed and photographed by 3D-SIM superresolution microscopy. (c) MCF-7 cells were incubated with (+) or without (−) PI-PLC prior to incubation with LIP. After the cells were washed to remove free LIP, the proteins were separated by SDS-PAGE and detected by immunoblotting with anti-LIP antibodies (left panel). Immunoblotting of proteins in H293T cells incubated with LIP (right panel). (d) MCF-7 cells were pretreated with SMase and then stained with Alexa555-cholera toxin subunit B (CT-B) prior to staining with Alexa488-tagged LIP. (e) The mean immunofluorescence intensity, which was measured as the average gray level, and the area ratio of the Alexa488-tagged  LIP area were examined using Image Pro Plus 6.0. (f) After the preincubation of MCF-7 cells in the presence (+) or absence (−) of SMase, the cells were treated with LIP. Cell death rates were analyzed by the LDH method. Each histogram represents the average value of triplicate experiments (**P < 0.01). Means ± SDs are shown

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