Additional file 3 of circPARD3 drives malignant progression and chemoresistance of laryngeal squamous cell carcinoma by inhibiting autophagy through the PRKCI-Akt-mTOR pathway

Additional file 3: Figure S1. The flow chart for screening and verifying autophagy suppressive circPARD3 in LSCC. Figure S2. FD-LSC-1 and Tu 177 cells were transfected with Cy3 labeled si-NC (si-NC-Cy3), NC mimics (NC mimics-Cy3), or NC inhibitor (NC inhibitor-Cy3) for 48 h. Nuclei were stained with DAPI (blue). Transfection efficiency was evaluated by imaging with confocal microscopy. Red dot represents siRNA, miRNA mimics, or miRNA inhibitor. Scale bar, 50 μm. Figure S3. Verification of the structure features of circPARD3. a Expression of circPARD3 in FD-LSC-1 and Tu 177 cells was verified by RT-PCR. Agarose gel electrophoresis showed that divergent primers amplified circPARD3 in cDNA but not genomic DNA (gDNA). GAPDH served as a negative control. b Stability of circPARD3 and linear PARD3 mRNA was assessed by RNase R treatment and RT-PCR analysis. Figure S4. FD-LSC-1 and Tu 177 cells were infected with circPARD3 overexpression lentiviruses (circPARD3-OE) or transfected with si-circPARD3 (si-circ-1, si-circ-2) for 48 h. Expression level of linear PARD3 mRNA was determined by qPCR analysis. Error bars represent SD of three independent experiments. N.S., no significant. Figure S5. Expression levels of potential circPARD3 target miRNAs in FD-LSC-1 and Tu 177 cells with overexpression (a) or knockdown (b) of circPARD3 were determined by qPCR analysis. Error bars represent SD of three independent experiments. * P < 0.05, **P < 0.01. Figure S6. The effects of miR-145-5p on LSCC cell autophagy. a and b FD-LSC-1 and Tu 177 cells were transfected with miR-145-5p mimics (a) or inhibitor (b) for 48 h. Expression levels of p62 and LC3B were detected by western blotting. c FD-LSC-1 and Tu 177 cells were transfected with miR-145-5p mimics or inhibitor for 48 h. Autophagic flux was analyzed by confocal microscopy. Representative images (Top) and statistical data (Bottom) were shown. Scale bar, 25 μm. Error bars represent SD of three independent experiments. * P < 0.05, **P < 0.01. Figure S7. mCherry-EGFP-LC3B labeled FD-LSC-1 and Tu 177 cells were transfected with miR-145-5p mimics alone or infected with circPARD3 overexpression lentiviruses simultaneously for 48 h. Autophagic flux was analyzed by confocal microscopy. Representative images (Top) and statistical data (Bottom) were shown. Scale bar, 25 μm. Error bars represent SD of three independent experiments. * P < 0.05, **P < 0.01. Figure S8. FD-LSC-1 and Tu 177 cells were transfected with miR-145-5p mimics or NC mimics for 48 h, then expression levels of PAK2, SLC38A2, and TBL1XR1 were determined by qPCR analysis. Error bars represent SD of three independent experiments. N.S., no significant. Figure S9. The effects of PRKCI overexpression on LSCC cell proliferation, migration, invasion, and chemoresistance. a Cell proliferation of FD-LSC-1 and Tu 177 cells overexpressing PRKCI was determined by colony formation assays. b and c The migration (b) and invasion (c) abilities of FD-LSC-1 and Tu 177 cells overexpressing PRKCI were evaluated by Transwell assays. Scale bar, 200 μm. d FD-LSC-1 and Tu 177 cells overexpressing PRKCI were treated with various concentrations of Cisplatin for 24 h. Cell viability was determined by CCK8 assays. Error bars represent SD of three independent experiments. **P < 0.01. Figure S10. FD-LSC-1 and Tu 177 cells were transfected with si-circPARD3 for 24 h, then treated with Akt activator SC79 (5 μg/ml) or mTOR activator MHY1485 (10 μM) for 24 h. Autophagic flux was analyzed by confocal microscopy. Representative images (Left) and statistical data (Right) were shown. Scale bar, 25 μm. Error bars represent SD of three independent experiments. **P < 0.01.