Additional file 1 of CircVAPA promotes small cell lung cancer progression by modulating the miR-377-3p and miR-494-3p/IGF1R/AKT axis
Creators: name: Hua, Jinghan
name: Wang, Xiaolin
name: Ma, Liying
name: Li, Jingxin
name: Cao, Guozhen
name: Zhang, Shaobo
name: Lin, Wenchu
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Virology

description: Additional file 1: Figure S1. (a) The relative expression of circVAPA, CircHIPK3 and 18S rRNA in SCLC cells were detected by RT-qPCR after RNase R treatment. CircHIPK3 was a positive control, while 18S rRNA was a negative control. (b-c) Quantification of circVAPA copy numbers in DMS273 and H82 cell lines. The red and blue dots indicate the Ct value and the amount of RNA in DMS273 and H82 SCLC cells, respectively. More experimental details are provided in the Methods section. (d) Prediction of the potential translation ability of circVAPA. An interaction model shows that circVAPA harbors potential internal ribosome entry site (IRES), but not open reading frame (ORF), which is analyzed by the circRNADb and ORFfinder databases. (All data are presented as the mean ± SD; ns, no significance; ***P &lt; 0.001 by two-tailed Student’s t-test). Figure S2. (a) Schematic diagram of knocking down circVAPA using two independent siRNAs target circVAPA junction. (b-c) The apoptosis rate was analyzed by flow cytometry after downregulation (b) or overexpression (c) of circVAPA in SCLC cells. (All data are presented as the mean ± SD; ***P &lt; 0.001 by two-tailed Student’s t-test). Three independent assays were performed in the above assays. Figure S3. (a) The effect of circVAPA on miR-377-3p and miR-494-3p expression levels in SCLC cells were detected by RT-qPCR. (b and c) RT-qPCR analysis of miR-377-3p (b) and miR-494-3p (c) expression in SCLC cells transiently transfected with the corresponding mimic, respectively. (d) After RNA pull-down by biotin-labeled circVAPA probe or negative probe, the miR-101 enrichment level in DMS273 cells was detected by RT-qPCR. (e) RT-qPCR analysis of miR-101 expression in SCLC cells transiently transfected with the corresponding mimic. (f) Western blot analysis of the effect of miR-101 on IGF1R, AKT, and its downstream protein expression in DMS273 (left) and H82 (right) SCLC cells. β-actin was used as an internal reference. (g-j) Cell viability analysis of SCLC cells transiently transfected with siRNAs, plasmids, miRNA inhibitors or mimics as indicated. SCR, siRNA with scrambled sequences; si-circVAPA, the co-transfection of two independent siRNAs target circVAPA; EV, the empty vector; circVAPA, the circVAPA overexpression plasmid; miR-377 mimic/miR-494 mimic, transiently overexpressing miR-377-3p/miR-494-3p, respectively; miR-377 inhibitor/miR-494 inhibitor, transiently suppressing miR-377-3p/miR-494-3p, respectively. (All data are presented as the mean ± SD; N.S., not significant; *P &lt; 0.05; **P &lt; 0.01; ***P &lt;0.001 by two-tailed Student’s t-test). Three independent assays were performed in the above assays. Figure S4. (a-d) RT-qPCR analysis of common candidate targets of miR-377-3p &amp; miR-494-3p in SCLC cells transiently transfected with the corresponding mimics or inhibitors. The common targets of miR-377-3p and miR-494-3p were predicted by miRWalk and ENCORI, and they were further in comparison with upregulated genes in the GEO dataset GSE149507. Overlapped genes matching the condition where |fold change| &gt; 1 and p-value &lt; 0.001 were selected. NC, negative control cells for overexpressing or suppressing miR-377-3p/miR-494-3p; miR-377 mimic/miR-494 mimic, transiently overexpressing miR-377-3p/miR-494-3p, respectively; miR-377 inhibitor/miR-494 inhibitor, transiently suppressing miR-377-3p/miR-494-3p, respectively. (All data are presented as the mean ± SD; **P &lt; 0.01; ***P &lt;0.001 by two-tailed Student’s t-test). Three independent assays were performed in the above assays. Figure S5. (a) RT-qPCR analysis of miR-377-3p/miR-494-3p expression in DMS273 cells transiently transfecting the indicated mimics. (b) RT-qPCR analysis of the effects of miR-377-3p/miR-494-3p on IGF1R in SCLC cells. (c-f) Quantification of miR-377-3p, miR-494-3p, and IGF1R copy numbers in DMS273 and H82 cell lines. The red and blue dots indicate the Ct value and the amount of RNA in DMS273 and H82 SCLC cells, respectively. More experimental details are provided in the Methods section. NC, negative control cells for overexpressing or suppressing miR-377-3p/miR-494-3p; miR-377 mimic/miR-494 mimic, transiently overexpressing miR-377-3p/miR-494-3p, respectively; miR-377 inhibitor/miR-494 inhibitor, transiently suppressing miR-377-3p/miR-494-3p, respectively. (All data are presented as the mean ± SD; ***P &lt; 0.001 by two-tailed Student’s t-test). Three independent assays were performed in the above assays. Figure S6. (a) Cell viability analysis of the effect of circVAPA overexpression or IGF1R inhibitor (drug BMS-536924) on SCLC cells. (b) Western blot analysis of the effects of circVAPA depletion or IGF1R inhibitor (drug BMS-536924) addition on S6RP and p-S6RP protein expression in H82 cells. (c and d) Cell viability (c) and colony formation (d) analysis of the H82 cells with circVAPA knockdown or the control with or without IGF1R inhibitor. (e) RT-qPCR analysis of circVAPA and VAPA mRNA expression in stable DMS273 cells transfected with the lentivirus-shRNA. Vector was the negative control for knocking down circVAPA. (f) Immunohistochemistry analysis of ki67 in tumors. Scale bar, 50 μm. si-circVAPA, the co-transfection of two independent siRNAs target circVAPA; IGF1Ri, the addition of IGF1R inhibitor (drug BMS-536924). (All data are presented as the mean ± SD; ns, no significance; ***P &lt; 0.001 by two-tailed Student’s t-test). Three independent assays were performed in the above assays.