FIG. 8.

Lipid rafts and response to radiation and EGFR inhibition. (A) Imaging of lipid rafts in SCC-61 and rSCC-61 cells. SCC-61 and rSCC-61 cells were either untreated or irradiated with 2 Gy followed by staining for confocal microscopy with cholera toxin subunit B (CT-B) conjugated to AlexaFluor594 (red) and EGFR antibody conjugated to AlexaFluor488 (green). The scale bars represent a distance of 20 μm. (B) The role of ROS in lipid raft formation in SCC-61. The SCC-61 cells were treated with 2 and 20 U of catalase and for 24 h followed by staining with AlexaFluor594 conjugated CT-B. A gradual decrease in the structural integrity of lipid rafts is observed. The scale bars represent a distance of 20 μm. (C) Inhibition of lipid rafts by MβCD and Lovastatin in SCC-61. The SCC-61 cells were treated for 24 h with 1 mM MβCD, 2 μM Lovastatin or 1 mM MβCD plus 2 μM Lovastatin and imaged using AlexaFluor594 conjugated CT-B. A decrease in the lipid rafts formation is observed under all treatment conditions. The scale bars represent a distance of 20 μm. (D) Inhibition of lipid rafts results in improved response to Erlotinib in SCC-61. After 24 h pretreatment of SCC-61 with MβCD, Lovastatin, or MβCD plus Lovastatin, the cells were treated with 2 μM Erlotinib for 48 h, The cell viability was determined using the MTT assay. An increased response to Erlotinib was observed under all treatment conditions and achieved statistical significance under combined MβCD and Lovastatin treatment (*p=0.0189, n=4). (E) Inhibition of lipid rafts results in decreased response to radiation in SCC-61. After 24 h pretreatment of SCC-61 with MβCD plus Lovastatin, the cells were treated with 0.5 and 2 Gy radiation. After 48 h, the cell viability was determined using the MTT assay. A statistically significant increase in resistance to radiation was noted at both radiation doses in the MβCD and Lovastatin-treated cells relative to the untreated control (0.5 Gy: ***p<0.001; 2 Gy, n=4: ***p<0.001, n=4).