Skip to main content
. 2019 Feb 12;10(2):135. doi: 10.1038/s41419-019-1354-2

Fig. 5. DJ-1 regulates mRNA stability of ATF4 through binding with ATF4 mRNA but not transcription level of ATF4.

Fig. 5

cDNA was prepared using oligo dT primers. Synthesized cDNAs were used as templates. mRNA levels of ATF4 and β-actin were determined by real-time PCR in MEFs (a, b, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA with Tukey post-test, n = 4) and mouse cortical neurons (d, e, *p < 0.05, **p < 0.01, two-way ANOVA with Tukey post-test, n = 3). Data are averages from three or four independent experiments in MEFs or mouse cortical neurons. c Luciferase assay was performed by using DJ-1 WT and KO MEFs after transfection with pGL3 (empty vector) and pGL3 fused with ATF4 promoter vector after treatment with Tun for 6 h. Data are averages from six independent experiments. f Heterozygous DJ-1 and DJ-1 KO mouse cortical neuron were collected after treatment with DMSO (0 h) as a vehicle control or actinomycin D for 15, 60, or 120 min. Data are averages from four independent experiments (*p < 0.05, ****p < 0.0001, two-way ANOVA with Tukey post-test, n = 4). RIP assay was performed by using MEFs (g), mouse cortical neuron (h, j) treated with or without Tun for 3 h or DMSO (0 h) as vehicle control, and SH-SY5Y+ (i) cells. DJ-1 protein was isolated by immunoprecipitation (IP) using anti-goat-DJ-1 antibody and protein G beads. DJ-1-associated RNA was isolated with Trizol and amplified using oligo dT primers for reverse transcription and ATF4 or β-Actin primers for PCR. An input was detected by immunoblotting with the DJ-1 or β-Actin antibody. k Recombinant GST or GST-DJ-1 immobilized on glutathione-Separose beads pull-down ATF4 mRNA. Pulldowns were immunoblotted with anti-GST antibody or detected by RT-PCR for ATF4 mRNA. l RIP assay is conducted as in h using DJ-1 KO mouse cortical neurons infected with adenovirus-expressing DJ-1 WT, L166P, or pcDNA3 (empty vector) as control for 30 h. An input was detected by immunoblotting with the DJ-1 or β-Actin antibody. m Analysis of ATF4 mRNA stability (*p < 0.05, ***p < 0.001, two-way ANOVA with Tukey post-test, n = 5) was conducted as in f using DJ-1 KO mouse cortical neurons infected with adenovirus-expressing DJ-1 WT, L166P, or pcDNA3 (empty vector) as control