Journal: Journal of experimental & clinical cancer research : CR

Article Title: Energy stress-induced circZFR enhances oxidative phosphorylation in lung adenocarcinoma via regulating alternative splicing.

doi: 10.1186/s13046-023-02723-z

Figure Lengend Snippet: 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 proteinhigh 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

Article Snippet: Western blotting was performed as described previously, [16] using the following primary antibodies: HNRNPLL (26769-1-AP, Proteintech, USA), MYO1B (sc-393,053, Santa Cruz Biotechnology, USA), FLAG (14,793 S, Cell Signaling Technology, USA), phospho-AKT (4060, Cell Signaling Technology), phospho-mTOR (5536T, Cell Signaling Technology), and β-actin (ab6276, Abcam, UK).

Techniques: Positive Control, Luciferase, Activity Assay, Transfection, Silver Staining, Western Blot, Negative Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Fluorescence, Staining, Plasmid Preparation, Software, Ubiquitin Proteomics, Immunoprecipitation, Two Tailed Test

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Energy stress-induced circZFR enhances oxidative phosphorylation in lung adenocarcinoma via regulating alternative splicing.

doi: 10.1186/s13046-023-02723-z

Figure Lengend Snippet: Fig. 4 The effects of circZFR and HNRNPLL on alternative splicing of MYO1B. a GSEA results of the differential genes affected by HNRNPLL knockdown. b, c Distribution of alternative splicing events affected by circZFR (b) and HNRNPLL (c). SE skipped exon, RI retained intron, A5SS alternative 5’ splice site, A3SS alternative 3’ splice site, MXE mutually exclusive exons. d Venn diagram illustrating the overlap of altered exon skipping events upon circZFR overexpres sion or HNRNPLL depleting. P value was calculated by χ2 test. e HNRNPLL binding site analyses on alternatively spliced exons. f Pie chart showing the screening of potential functional target of both circZFR and HNRNPLL.g Kaplan-Meier analysis of the DFI of the LUAD patients. h Top 6 hallmark pathways from GSEA enriched in patients with high MYO1B PSI scores from TCGA project. i Left: Diagram of the splicing variants of MYO1B mRNA and the primers for RT-PCR detection of exon 23 (primer 1) and exons 23 and 24 (primer 2) inclusion/exclusion. Right: RT-PCR validation of MYO1B exons upon knockdown or overexpression of circZFR in A549 cells. in%, percentage of inclusion. Red, exon 23, blue, exon 24, purple, exon 23/24. j The fluorescence images of circZFR (green) and MYO1B exon23 (red). Nuclei was stained with DAPI (blue). Scale bars, 50 μm. k Splicing pattern of MYO1B in LUAD cell lines (upper panel) and tissues (lower panel) as detected by RT-PCR. T, tumor tissue, N, nontumorous tissue. l Correlation analysis on circZFR and MYO1B PSI in LUAD tissues. m The effects of HNRNPLL on MYO1B exons in A549 cells. n Interaction of MYO1B exons and HNRNPLL examined by RT-PCR with RIP assays in A549 cells. Data are shown as mean ± SD (n = 3). Similar results were obtained in three independent experiments or typical photographs of one representative experiment. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t test

Article Snippet: Western blotting was performed as described previously, [16] using the following primary antibodies: HNRNPLL (26769-1-AP, Proteintech, USA), MYO1B (sc-393,053, Santa Cruz Biotechnology, USA), FLAG (14,793 S, Cell Signaling Technology, USA), phospho-AKT (4060, Cell Signaling Technology), phospho-mTOR (5536T, Cell Signaling Technology), and β-actin (ab6276, Abcam, UK).

Techniques: Alternative Splicing, Knockdown, Binding Assay, Functional Assay, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Over Expression, Fluorescence, Staining, Two Tailed Test

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Energy stress-induced circZFR enhances oxidative phosphorylation in lung adenocarcinoma via regulating alternative splicing.

doi: 10.1186/s13046-023-02723-z

Figure Lengend Snippet: Fig. 5 MYO1B-fl partially recapitulates the biological function of circZFR via AKT-mTOR signaling. a Subcellular distribution of different MYO1B isoforms. Nuclei was stained with DAPI (blue). Scale bars: 50 μm. b, c The changes of OCR levels (b) and cell vitality (c) in A549 cells after transfecting with control or MYO1B-fl siRNAs. d Western blot of the indicated proteins in the extracts of A549 cells. e The protein levels of MYO1B-fl, phospho-AKT and phospho-mTOR induced by circZFR in A549 cells. f-jMYO1B exons (f), OCR levels (g), cellular ATP (h), cell proliferation (i) and protein expression (j) in A549 cells transfected with HNRNPLL vector alone or co-transfected with MYO1B siRNA. k-oMYO1B exons (k), OCR levels (l), cellular ATP (m), cell proliferation (n) and protein expression (o) in A549 cells transfected with circZFR vector alone or co-transfected with MYO1B siRNA. Data are shown as mean ± SD (n = 3). Similar results were obtained in three independent experiments or typical photographs of one representative experiment. *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA followed by Tukey’s test

Article Snippet: Western blotting was performed as described previously, [16] using the following primary antibodies: HNRNPLL (26769-1-AP, Proteintech, USA), MYO1B (sc-393,053, Santa Cruz Biotechnology, USA), FLAG (14,793 S, Cell Signaling Technology, USA), phospho-AKT (4060, Cell Signaling Technology), phospho-mTOR (5536T, Cell Signaling Technology), and β-actin (ab6276, Abcam, UK).

Techniques: Staining, Control, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Energy stress-induced circZFR enhances oxidative phosphorylation in lung adenocarcinoma via regulating alternative splicing.

doi: 10.1186/s13046-023-02723-z

Figure Lengend Snippet: Fig. 6 CircZFR promotes tumor growth in vivo. a Survival was analyzed and compared between patients with high and low levels of circZFR. b Multi variable analysis of circZFR in LUAD TMA. c The expression of circZFR analyzed by CISH in TMA was correlated with T stage. Scale bar indicates 100 μm. d, e Representative data of xenograft tumors isolated from PDTX models (d) and tumors in nude mice-bearing HCC827 cells (e). f-h The inclusion levels of MYO1B exon23 (f), volumes (g) and weights (h) in xenograft tumors isolated from PDTX models. (n = 7 mice per group). i Multi-label IHC staining of H&E, HNRNPLL, phospho-AKT and phospho-mTOR. Scale bar indicates 100 μm. j-l The inclusion levels of MYO1B exon23 (j), volumes (k) and weights (l) of subcutaneous xenograft tumors isolated from nude mice (n = 7 mice per group). Data are shown as mean ± SD (n = 7). *p < 0.05, **p < 0.01, two-tailed Student’s t test

Article Snippet: Western blotting was performed as described previously, [16] using the following primary antibodies: HNRNPLL (26769-1-AP, Proteintech, USA), MYO1B (sc-393,053, Santa Cruz Biotechnology, USA), FLAG (14,793 S, Cell Signaling Technology, USA), phospho-AKT (4060, Cell Signaling Technology), phospho-mTOR (5536T, Cell Signaling Technology), and β-actin (ab6276, Abcam, UK).

Techniques: In Vivo, Expressing, Isolation, Immunohistochemistry, Two Tailed Test

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Energy stress-induced circZFR enhances oxidative phosphorylation in lung adenocarcinoma via regulating alternative splicing.

doi: 10.1186/s13046-023-02723-z

Figure Lengend Snippet: Fig. 7 Graphical illustration of mechanism in LUAD progress. The elevated circZFR levels upon glucose stress protect HNRNPLL protein from degradation by ubiquitination, which promotes the inclusion of MYO1B exon 23 to enhance OXPHOS via AKT-mTOR signaling to promote tumor growth

Article Snippet: Western blotting was performed as described previously, [16] using the following primary antibodies: HNRNPLL (26769-1-AP, Proteintech, USA), MYO1B (sc-393,053, Santa Cruz Biotechnology, USA), FLAG (14,793 S, Cell Signaling Technology, USA), phospho-AKT (4060, Cell Signaling Technology), phospho-mTOR (5536T, Cell Signaling Technology), and β-actin (ab6276, Abcam, UK).

Techniques: Ubiquitin Proteomics