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. 2015 Oct 22;7(2):1367–1379. doi: 10.18632/oncotarget.6011

Figure 3. Down-regulation of p55PIK or blocking NF-κB signaling led to decreased KIT and re-sensitized GIST882IR cells to Imatinib.

Figure 3

A. Decreased p55PIK expression in GIST882IR cells infected with lentivirus expressing shRNA against p55PIK. GIST882IR cells were infected with Lenti-shRNAp55PIK or control Lenti-sc (MOI = 10) overnight. Cell lysates were prepared and protein level of p55PIK was determined by Western blotting. B. Restoration of Imatinib sensitivity in GIST882IR cells infected with Lenti-shRNAp55PIK. GIST882IR cells were infected with Lenti-shRNAp55PIK or Lenti-sc overnight and treated with various concentration of Imatinib for 72 h. Cell viability was measured. *p < 0.05; **p < 0.01. C. Down-regulation of p55PIK decreased expression of KIT in GIST882IR cells. Cultured GIST882IR cells were infected with Lenti-shRNAp55PIK or Lenti-sc, then transfected with KIT promoter reporter plasmids. Cellular lysates were prepared 48 h after transfection, the protein level of KIT and the luciferase activity was determined. D. BrdU incorporation assay showed that down-regulation of p55PIK re-sensitized GIST005 cells to Imatinib. Cultured GIST005 cells were infected with Lenti-shRNAp55PIK or Lenti-sc, after transfection for 72 h, Imatinib (10 μmol) was added in cultured cells, BrdU incorporation was checked at 72 h after Imatinib was added. E. Down-regulation of p55PIK or blocking NF-κB signaling led to decreased KIT in GIST005 cells. Cultured GIST005 cells were infected with Lenti-shRNAp55PIK, Lenti-sc or NF-κB signaling inhibitor Bay11–7082 (BAY) (final concentration: 5 μM). Cellular lysates were prepared 48 h after transfection or BAY treated, The expression of KIT, p55PIK and the phosphorylation of NF-κB p65 (pp65(Ser536)) were examined by Western blotting. F. Restoration of Imatinib sensitivity in GIST005 cells infected with Lenti-shRNAp55PIK or using NF-κB signaling inhibitor Bay11–7082 (BAY). GIST005 cells were infected with Lenti-shRNAp55PIK or Lenti-sc overnight and were incubated with various concentrations of Imatinb in the presence of BAY (final concentration: 5 μM) or solvent (DMSO) for 72 h. Cell viability was determined. *p < 0.05; **p < 0.01.