Fig. 3

CircZFR stabilizes HNRNPLL to promote OXPHOS. a The enrichment of circRNAs was detected by RIP in A549 cells. circPRKCI was used as positive control. b Luciferase activity of circZFR transfected with candidate miRNA mimics. c Silver staining (upper panel) and western blotting (lower panel) of proteins retrieved by circZFR probe. Scramble probe was used as a negative control. d RT-PCR analysis of circZFR enriched by Flag-HNRNPLL proteins. e The HNRNPLL RNA levels in LUAD and normal lung tissues. f The HNRNPLL protein levels in LUAD and adjacent tissues from CPTAC cohort. g The HNRNPLL protein expression in lung tissues (upper panel) and the changes affected by circZFR (lower panel). h The fluorescence images of circZFR (green) and HNRNPLL (red) in A549 cells. Nuclei was stained with DAPI (blue). Scale bars, 25 μm. i Correlation analysis on circZFR RNA and HNRNPLL protein levels in LUAD tissues. ΔCT values were normalized according to ACTB. HNRNPLL protein levels was determined by western blotting and normalized according to β-actin. j Heatmap of differentially expressed genes (left) and top 5 biological processes from GO enriched (right) in the HNRNPLL protein^high patient group. k-n HNRNPLL protein expression (k), cell proliferation (l), OCR levels (m) and ATP levels (n) in A549 cells transfected with circZFR vector alone or co-transfected with HNRNPLL siRNA. o The expression kinetics of HNRNPLL in A549 cells treated with CHX and circZFR expression vector. p The predicted 3D model of circZFR and HNRNPLL by HDOCK software. q Ubiquitination site prediction and docking capability determined by distance on circZFR. r HNRNPLL ubiquitination treated with circZFR expression vector was assayed by immunoprecipitation. Data are shown as mean ± SD (n = 3) or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t test