Fig. 4. Effect of WT or mask mutant TMPRSS6 expression on Hamp promoter responses.

HepG2 cells were transfected with a full-length Hamp promoter reporter construct in all experiments shown in A-D, and with a normalizing renilla luciferase construct. Tmprss6, full length construct encoding the native protein. Tmprss6^msk, the truncated mutant sequence expressed by mask mice. Tmprss6^S762A, protease-dead Tmprss6 mutant. delN, GFP cytoplasmic domain, TMPRSS6 transmembrane and ectodomain. delC, GFP ectodomain and TMPRSS6 transmembrane and cytoplasmic domains. A. The effect of Tmprss6 and Tmprss6^msk on Hamp promoter activity in cells cotransfected with a hemojuvelin-encoding vector or stimulated with BMP2, BMP4, BMP9, IL-6, or IL-1α. B. Comparison of the Hamp promoter-suppressing effects of Tmprss6, Tmprss6^msk, and Tmprss6^S762A in cells subjected to the stimuli indicated. C. Localization of delN and delC constructs in HepG2 cells by confocal fluorescence microscopy. D. Comparison of the Hamp promoter-suppressing effects of Tmprss6, Tmprss6^msk, delN and delC constructs in cells subjected to the stimuli indicated. E. Response of Hamp promoter deletion constructs to induction by IL-6 (length indicated with respect to the cap site), and the relative suppression of responses by co-transfection with Tmprss6 or Tmprss6^msk constructs. In all experiments (A-E), duplicate transfections were performed. Incubations with cytokines were performed for 16 hours before luminescence was read to estimate hepcidin promoter activity. Error bars indicate 1 SEM.