Fig. 3.

Induction of p21 protein expression by CQ and more prominently by AQ. (A–B) TCR-triggered CD4+ T cells were incubated with different amounts of CQ or AQ for 24 h. Whole cell extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibody against p21, p27, PCNA, p53, LC3B, ATG5, or actin (A). The intensity of the p21 protein band was quantitatively analyzed by a densitometry scan (B). (C) CQ- or AQ-treated T cells were fixed in 4% paraformaldehyde and stained with antibody against p21 and LAMP1, followed by subsequent incubation with Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibody. The nuclei were blue counterstained with DAPI. (D) Activated CD4+ T cells were treated with cycloheximide (+CHX) in the presence of either CQ or AQ and harvested for immunoblotting analysis. (E) Total RNA was obtained from CQ- or AQ-treated T cells and subjected to reverse transcription and real time-PCR analysis. The relative level of p21 mRNA was calculated after normalization to the actin level. *, p < 0.05; **, p < 0.005; ***, p < 0.0005. Three independent experiments were performed and data are given as the mean ± SD. (F) Highly transfectable 293T cells were transfected with the p21 promoter reporter gene with or without p53 expression vector and additionally treated with CQ (50 μM) or AQ (50 μM) for 24 h. Reporter activity was measured using luciferase assay kit and the relative luciferase unit was calculated after normalization. The activity under vehicle-treated conditions was set to 1. *, p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)