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. 2025 Sep 26;16:1618677. doi: 10.3389/fimmu.2025.1618677

Figure 2.

Graphs comparing Foxp3 mRNA expression under different conditions. (A) and (B) show the percentage of remaining Foxp3 mRNA over time with actinomycin-D treatment in splenic Tregs, comparing KH-3B vs. KH-3 and WT control vs. KO, showing significant differences in half-lives. (C) Bar graph depicts Foxp3 mRNA fold change in T cell subsets, noting significant differences between WT control and KO groups. (D) Bar graph shows RIP mRNA fold enrichment in spleen Tregs, comparing IgG1, Foxp3, and β-actin, with notable enrichment for Foxp3. Statistical significance is indicated.

HuR directly interacts with Foxp3 mRNA and controls its steady-state levels and mRNA stability. (A) Foxp3 mRNA stability was evaluated in splenic Tregs (CD4+CD25+) isolated by magnetic column separation from WT mice treated with KH-3 or sham control (KH3-B) for 2 hours, followed by actinomycin (D) HuR inhibition reduced Foxp3 mRNA half-life (1.9 hours with KH-3) compared to control (4.3 hours). Data represents seven WT mice. (B) Foxp3 mRNA stability was assessed in column-isolated splenic Tregs from WT and Foxp3 YFP/Cre HuR fl/fl (HuR-KO Tregs) mice. HuR-KO Tregs showed a shorter Foxp3 mRNA half-life (1.9 hours) compared to WT (4.2 hours). Data represents seven WT and four HuR-KO mice. (C) Steady-state Foxp3 mRNA levels were measured in splenic and thymic Tregs. Foxp3 mRNA was significantly lower in HuR-KO Tregs compared to WT control, while CD4+CD25− cells showed minimal expression with no significant differences. Data represent three HuR-KO and three WT mice. (D) RNA immunoprecipitation (RIP) from WT splenic Tregs showed Foxp3 mRNA enrichment in HuR pull-downs compared to IgG1 control, with β-actin as a positive control. Each point represents pooled cells from four WT mice. A total of 12 mice were used across three independent experiments. **p<0.01, ****p<0.0001.