Sp1 and Sp3 are critically involved in PDPN promoter activation. A) Cotransfection of the core promoter construct (-857/+171) without (Wild-Type) and with Sp-binding site mutations (Mutant) using increasing amounts of pCMV-Sp1 and pMCS-Sp3 or a combination of both expression plasmids along with pRL-TK into MG63 and Saos-2 cells. The total amount of expression plasmids was filled to 0.17 μg with empty vector. Luciferase values were normalized to the internal control Renilla luciferase and fold-induction versus mock-transfected cells was evaluated. B) Real-time PCR analysis of podoplanin transcription in MG63 and Saos-2 cells upon transfection with Sp1 and Sp3 expression vectors. Transcript levels are relative to GAPDH mRNA content. The average ± S.D. from two independent experiments performed in triplicates is shown. C) Drosophila SL2 cells were transiently transfected with Wild-Type promoter, Sp.4-1mut construct and pGL3-Basic. Cells were cotransfected with Drosophila expression vectors pPac-empty, pPac-Sp1, pPac-Sp3, or both. Fold promoter activation versus pPac-empty transfections was evaluated. The average ± S.D. from three independent experiments performed in triplicates is shown. D) The role of Sp.1 for PDPN promoter activity. Cotransfection of the shortened promoter constructs (-76/+171) and (-38/+171) with pCMV-Sp1 or pMCS-Sp3 along with pRL-TK into MG63 and Saos-2 cells. The statistical difference of the promoter activity in all panels is shown against mock-transfected cells. *, p < 0.05, **, p < 0.01, ***, p < 0.001.