Analysis of the cystatin C promoter. (A) Diagram of the self-inactivating retrovirus reporter (SIRV-GFP) carrying the cystatin C promoter. Both LTRs are inactivated and have no promoter activity. Ψ⁺, the extended viral packaging signal; MCS, multiple cloning site; H₄P-Puro^r; the puromycin resistant gene driven by the histone H₄ promoter. Sequences and mutations in element-1 to element-3 are shown. The numbers indicate the nucleotide positions relative to the start codon. (B) Cystatin C promoter activity in live cells, as monitored by flow cytometry of GFP signals. Tot2 cells were transduced with SIRV-GFP carrying wild-type (WT) or mutant (Mut-1 to Mut-3) promoters, and then with MSCV-ER or MSCV–IRF-8/ER. Cells were either left untreated or treated with 1 μM β-estradiol (Est) for 13 hours. (C) Quantification of the promoter activity measured in panel B. The activity is shown as mean fluorescent intensity (MFI) of GFP signals.