Figure 2.

HIC1 Is Induced by RA, and It Contributes to the Suppressive Activity of iTreg Cells

(A) Cells were stimulated in Th0 or iTreg conditions for the indicated time points (hours), followed by HIC1 and FOXP3 measurement by WB. A representative blot from two biological replicates is shown.

(B) Cells were activated in presence of indicated concentrations of RA for 24 hr followed by measurement of HIC1 expression by WB. A representative image as well as mean of three biological replicates are shown. Error bars represent SE.

(C) Cells were nucleofected with three siRNAs against HIC1 (si1–3) and one non-targeting (NT), followed by a rest for 24 hr. Cells were then cultured for 72 hr in iTreg culture conditions, and HIC1 expression was measured by WB. A representative blot is shown at the bottom, and the bar chart shows the mean ± SE of four biological replicates. Significance was measured by two-tailed paired t test. ^∗p < 0.05, ^∗∗p < 0.01.

(D) FOXP3 expression was measured in HIC1-sufficient (red) and HIC1-deficient (blue) cells after 72 hr of culturing in iTreg conditions. Gray color shows isotype control. The bar charts show percentage FOXP3-positive cells (left) and median fluorescence intensity (MFI) (right) of three biological replicates (mean ± SE).

(E) Histogram plots showing the proliferation of responder cells at a responder/suppressor ratio of 1:0.5 after 72 hr of activation in absence (responder only) or presence of HIC1-sufficient (NT) or HIC1-deficient (siHIC1) iTreg cells. The number on the histogram plot shows the percentage of proliferating cells. The line plot shows the percentage of proliferating responder cells at different responder/suppressor ratios. The shaded area shows the minimum and maximum value of the three replicates. Significance was measured using two-tailed paired t test. ^∗p < 0.05.

(F) Same as (E), except that the responder cells were cultured in the presence of activated T cells (Th0) instead of iTreg cells.