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. 2010 Jul;177(1):404–414. doi: 10.2353/ajpath.2010.091079

Figure 3.

Figure 3

In vitro up-regulation of Shh inhibits sTOP/sFOP-FLASH in HEK293 cells. A: HEK293 cells were transiently transfected with the super(8×)TOP-FLASH and the super(8×)FOP-FLASH reporter plasmid, respectively, and stimulated with 20 mmol/L LiCl. After transfection with increasing amounts of pCMV-Sport6.1-Shh plasmid, activation of the sTOP-FLASH reporter was measured after 24 hours. B: To check the mShh biological activity in HEK293 cells transfected with the 8 × 3′Gli-BSδ51LucII or the 8 × 3′GliM3-BSδ51LucII luciferase reporter, after stimulation of HEK293 cells either by mGli1 transfection as a positive control or by Shh-CM, luciferase activity was measured after 24 hours (**P < 0.0081; *P < 0.0157). Validation of mShh protein expression in HEK293 cells transfected with the pCMV-Sport6.1-Shh or vector control (VC) by Western blot analysis of cell lysate and CM on a 12% SDS-polyacrylamide gel electrophoresis. C and D: Shh effect on sTOP/sFOP-FLASH reporter in HEK293 cells. Stimulation of canonical Wnt signaling either by 20 mmol/L LiCl or LEFΔN-βCTA transfection. C: 500 ng pCMV-Sport6.1-Shh-plasmid (*P < 0.0147; **P < 0.0015) or (D) Shh-CM were used (**P < 0.002; *P < 0.0241). Assays were repeated in three individual experiments; SD is shown as SEM.