Fig. 4.

Autophagy modulates PU.1 protein stability in T_H9 cells. a Naive CD4⁺ T cells were isolated from Atg5^fl/+*CD4-Cre and Atg5^{fl/fl*CD4-Cre}mice and differentiated into T_H9 cells for 24 h. Representative western blot of T_H9 cell-related transcription factors in Atg5-deficient T_H9 cells compared to controls. Data are representative of two experiments. b Naive CD4⁺ T cells were isolated and differentiated into T_H9 cells for 24 h in the presence of metformin (1 mM) or chloroquine (25 µM). PU.1, GATA-3 and β-actin expression was analysed by western blot and its quantification is shown in c. d Same as in b and Sfpi1 expression was determined by quantitative RT-PCR at 24 h from three experiments. e Naive CD4⁺ T cells were isolated and differentiated into T_H9 cells for 16 h in the presence or absence of chloroquine (25 μM). Then, cells were treated with 25 μg ml⁻¹ of DRB, an inhibitor of transcription, for 8 h. Sfpi1 mRNA expression was analysed by qRT-PCR at 0 and 8 h of DRB treatment and f PU.1 expression was analysed by western blot at 8 h of DRB treatment. Experiments performed twice. All qRT-PCR results were normalized to the expression of Actb (mean+s.d.), NS, not significant, analysis of variance (ANOVA) test. g Naive CD4⁺ T cells were isolated from Atg5^fl/+*CD4-Cre and Atg5^{fl/fl*CD4-Cre} mice and differentiated into T_H9 cells. PU.1 protein expression was assessed by western blot after being treated with 25 μg ml⁻¹ cycloheximide to inhibit protein synthesis for 3, 6 and 12 h. PU.1 protein expression is plotted in h as a percentage of total PU.1 protein at time zero, and reflects the values obtained from western blot shown. The western blot shown is representative of two independent experiments. Mean (+s.d.), *P<0.05 two-way ANOVA test