Fig. 2

Identification of the autophagy suppressive circRNA circPARD3 in LSCC. a-c RNA sequencing was performed on 107 LSCC tissues and paired ANM tissues (Cohort 2) to screen differentially expressed circRNAs, mRNAs, and miRNAs. GSEA analysis of differentially expressed mRNAs (a). NES, normalized enrichment score. KEGG pathway analysis of differentially expressed mRNAs (b). Top 15 pathways were plotted according to enriched gene ratio and p value. c Hierarchical clustering of circRNA differential expression profiles between 107 LSCC tissues and 107 paired ANM tissues. The heatmap was generated from the top 15 differentially expressed circRNAs. d FD-LSC-1 and Tu 177 cells expressing LC3B-GFP were transfected with siRNAs or si-NC for 24 h, then cells were seeded into 48-well plates and incubated for additional 24 h. LC3 dots were analyzed on high-content screening system. Representative images of si-circ_00043 transfected cells were shown. Scale bar, 20 μm. e Schematic diagram of the genomic location and splicing pattern of circPARD3 is shown (Left). Validation of the head-to-tail splicing of circPARD3 by RT-PCR and Sanger sequencing (Right). f The relative RNA levels of circPARD3 and linear PARD3 mRNA in FD-LSC-1 and Tu 177 cells treated with actinomycin D at the indicated time points were determined by qPCR. g circPARD3 levels in cytoplasm and nucleus of FD-LSC-1 and Tu 177 cells were determined by qPCR analysis. 18S and U6 RNA were used as positive controls of in the cytoplasm and nucleus, respectively. h RNA fluorescence in situ hybridization for circPARD3 was performed in FD-LSC-1 and Tu 177 cells. Nuclei were stained with DAPI (blue) and circPARD3 probes were labeled with Cy3 (red). Scale bar, 20 μm. Error bars (f and g) represent SD of three independent experiments. **P < 0.01