Fig. 4: DENR regulation of the translation of JAK2 is dependent on three uORFs.
From: DENR controls JAK2 translation to induce PD-L1 expression for tumor immune evasion
a uORFs of human and mouse JAK2 5′ UTR features: 1 amino acid uORFs (red), 2 amino acid uORF (orange). b EGFP-HA-SV40 Poly(A) reporters of murine WT Jak2 5ʹ UTR with three uORFs or mutated 5ʹ UTR with 1 uORF or 0 uORF. c WT B16/F10 cells (1 × 107) were divided into six-well plates; 24 h later, 650 ng of plasmid with individual reporters with 1 µl Lip3000 was added, and the cells incubated for 14–16 h. The EGFP was analyzed by fluorescence microscope. Scale bars, 100 μm. The experiment was repeated independently three times with similar results. d The protein levels of HA-tag as performed in c were analyzed by western blotting. The experiment was repeated independently three times with similar results. e, f Control or DENR KO B16/F10 cells were incubated with reports as described, and the percentage of EGFP positive cells were analyzed by FACS (n = 3 independent samples). Results are representative of three biological replicates. Three technical replicates are shown. Data are presented as mean values ± SD, unpaired two-tailed Student’s t-test: ** p < 0.01, *** p < 0.001, ns: not significant. p values from left to right: < 0.001, 0.0061, 0.2399. g The corresponding mRNA levels of EGFP reports in e were measured (n = 3 independent samples). Results are representative of three biological replicates. Three technical replicates are shown. Data are presented as mean values ± SD, unpaired two-tailed Student’s t-test: ns, not significant. p values from left to right: 0.8567, 0.4271, and 0.3341. h Venn diagram shows the overlap of more than 1 aa (amino acid) uORF genes with the TE (translation efficiency) impaired genes in DENR KO NIH3T3 cells. Source data are provided as a Source Data file.
