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. 2022 Apr 9;107(2):458–473. doi: 10.1093/biolre/ioac070

Figure 9.

Figure 9

SMAD4 is required for activin A-induced upregulation of HAS2 and versican expression, and SMAD4 binds to human HAS2 and VERSICAN promoters in primary hGL cells. (A and B) Primary hGL cells (n = 3) were transfected with 50 nM siCtrl or 50 nM siSMAD4 for 48 h and then the cells were treated with Ctrl or 25 ng/mL activin A for an additional 12 h. The mRNA levels of HAS2 (A) and versican (B) were examined using RT-qPCR. (C and E) The SMAD4 binding sites in the human HAS2 and VERSICAN promoter are highlighted by a box. The primers (forward and reverse) for ChIP assay are listed underlined. (D and F) Primary hGL cells were treated with 25 ng/ml of activin A for 6 h before being subjected to a ChIP assay. Cell extracts were precipitated with anti-SMAD4 or control IgG antibodies. Subsequent RT-qPCR was performed using specific primers complementary to the SMAD4 binding site at HAS2 and VERSICAN promoter regions. Percent input method was used to analyze the ChIP-qPCR data. The results are expressed as the mean ± SEM from at least three independent experiments (* indicates P < 0.05). Ctrl, control.