Additional file 3 of CircIL4R activates the PI3K/AKT signaling pathway via the miR-761/TRIM29/PHLPP1 axis and promotes proliferation and metastasis in colorectal cancer

Additional file 3: Figure S1. a. qRT-PCR validation of novel circRNAs expressed in different CRC cell lines and FHC cells; the circRNAs are clustered in a heatmap. b. qRT-PCR validation of circIL4R and IL4R mRNA expression in DLD1 cells transfected with siRNAs into DLD1 cells. c. The viability of DLD1 cells with circIL4R knockdown was detected by CCK-8 assays at the indicated time points. d. A colony formation assay was conducted to determine the proliferation of DLD1 cells stably transfected with sh-Ctrl or sh-circIL4R. e. An EdU assay was performed to assess the proliferation of DLD1 cells transfected with the indicated siRNAs. f and g. Representative images and quantification of Transwell and wound healing assays of DLD1 cells transfected with the indicated siRNAs. The data are presented as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Figure S2. a-c. qRT-PCR validation of miR-761 expression in CRC cells transfected with miR-761 mimics or inhibitor into CRC cells. d-i. The CCK-8 and Transwell assays showed that the inhibitory effect of circIL4R knockdown on the proliferation, migration and invasion of HCT116 and DLD1 cells was reversed by miR-761 inhibitor, whereas the stimulatory effect of circIL4R overexpression on the proliferation, migration and invasion of LoVo cells was reversed by miR-761 mimics. j and k. Western blots showed that the reduction in p-AKT levels in HCT116 cells transfected with circIL4R siRNAs was reversed by miR-761 inhibitor, whereas the increase in p-AKT levels in LoVo cells transfected with circIL4R was reversed by miR-761 mimics. *P < 0.05, **P < 0.01, ***P < 0.001. Figure S3. a. TRIM29 expression was upregulated in CRC samples based on the TCGA COAD and READ databases. b and c. The transfection efficiency of the indicated TRIM29 siRNAs or overexpression plasmids was verified at the protein and mRNA level by western blot and qRT-PCR, respectively. d and e. qRT-PCR validation of circIL4R expression in HCT116 and DLD1 cells stably transfected with circIL4R knockdown (sh-circIL4R) or negative control (sh-Ctrl) lentivirus. f and g. Analysis of the metastatic nodules from dissected lungs. h. Relative expression of circIL4R in a normal colorectal epithelium cell line (FHC) and CRC cell lines (HCT116, DLD1, LoVo, SW620, HT29 and SW480). GAPDH served as internal reference. i and j. CRC cells were cotransfected with miR-541-3p mimics and either WT or Mut circIL4R luciferase reporter vectors, and the activity of the luciferase reporter was determined. k. Schematic illustration of the experimental pipeline by which circIL4R activates the PI3K/AKT signaling pathway via the miR-761/TRIM29/PHLPP1 axis and promotes proliferation and metastasis in CRC. *P < 0.05, **P < 0.01, ***P < 0.001. Figure S4. a. Representative images showing the expression of circIL4R in TMAs detected by ISH. b. Staining intensities of circIL4R in CRC tissues compared with paired ANTs. N, paired adjacent non-cancerous tissues. C, colorectal cancer tissues (n = 165, P < 0.001). c. The ISH score of circIL4R in CRC tissues was significantly higher than that in paired ANTs. d. The ISH score of circIL4R in CRC tissues with stage III/IV was significantly higher than those with stage I/II. e. The Kaplan–Meier survival curves showed that CRC patients with high circIL4R expression in the TMAs exerted poor OS. f. Representative images showing the expression of TFAP2C in TMAs detected by IHC. g. The IHC score of TFAP2C in CRC tissues was significantly higher than that in paired ANTs. h. Representative images showing the expression of TRIM29 in TMAs detected by IHC. i. The IHC score of TRIM29 in CRC tissues was significantly higher than that in paired ANTs. j. Pearson’s correlation analysis of the correlation between circIL4R and TFAP2C expression based on TMAs specimens. k. Pearson’s correlation analysis of the correlation between circIL4R and TRIM29 expression based on TMAs specimens. *P < 0.05, **P < 0.01, ***P < 0.001.