Supplementary Figure 2: A cell line stably expressing Aire recapitulates Aire-dependent gene expression. | Nature Immunology

Supplementary Figure 2: A cell line stably expressing Aire recapitulates Aire-dependent gene expression.

From: The transcriptional regulator Aire coopts the repressive ATF7ip-MBD1 complex for the induction of immunotolerance

Supplementary Figure 2

(a) Immunofluorescence of HEK 293 cells stably expressing Flag tagged Aire. The left panel shows Aire staining in red, the center panel shows Flag (green), the right panel shows the overlay (yellow). Blue indicates nuclear DAPI staining. (b) Relative expression of the Aire-dependent genes S100A8, KRT14, and ALOX12 in the Aire-stable cell line compared to a cell line expressing vector only. (c) Western blot showing the efficiency of knockdown by plasmids expressing a scrambled shRNA (shNeg), an ATF7ip shRNA (shATF7ip), or an MBD1 shRNA (shMBD1). TATA-binding protein (TBP) is included as a loading control. (d) Aire and MBD1-VP16 do not induce the non Aire-regulated gene S100A10. Relative expression of S100A10 after transient transfection with plasmids expressing Flag only (Vector), Aire-Flag (Aire), Flag-MBD1-VP16 (MBD1-VP16), or Flag-MBD1-VP16-R22A (R22A). (e) Western blot showing the expression of Flag only (Vector), Aire-Flag (Aire), Flag-MBD1-VP16 (MBD1-VP16), or Flag-MBD1-VP16-R22A (R22A) in HEK 293 cells. TATA-binding protein (TBP) is included as a loading control. Data are representative of three experiments (mean and s.e.m of pooled data from three biological replicates in b,d). Data are representative of two experiments (a,c,e).

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