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. 2019 Apr 8;234(11):19895–19910. doi: 10.1002/jcp.28588

Figure 2.

Figure 2

CircFNDC3B did not influence FNDC3B expression. (a) The pcDNA3.1‐circFNDC3B‐mini vector was constructed without a restriction enzyme site by Gibson Assembly DNA clone technology. The light green arrows show the inverted repeat supporting circFNDC3B circularization. Pink triangle primers or blue triangle primers were designed to amplify vector or circFNDC3B exons 5 and 6. (b) The circFNDC3B expression vector successfully induced an increase in circFNDC3B expression in MGC‐803 cells (EV: empty vector; OVER: overexpressing circFNDC3B; WT: wild‐type). (c) The three siRNAs targeting the circFNDC3B junction sequence were designed and only exhibited the sense strand. (d) BGC‐823 cells were transfected with three siRNAs specifically targeting circFNDC3B (S1, S2, S3) or a negative control siRNA for 48 hr, and the level of silencing circFNDC3B was detected by qRT‐PCR (WT: wild‐type; NC: negative control; siRNA: S1, S2, S3). (e,g) MGC‐803 cells were transfected with the circFNDC3B vector or an empty vector. Cells were cultured for 48 hr, and then we examined FNDC3B mRNA and protein expression by qRT‐PCR and western blot. The results showed that overexpression of circFNDC3B did not significantly increase the mRNA or protein level of FNDC3B (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B). (f,h) BGC‐823 cells were transfected with siRNA (S1, S2, S3) or a negative control siRNA. Cells were cultured for 48 hr, and then we examined circFNDC3B and FNDC3B mRNA and protein expression by qRT‐PCR and western blot, respectively. The silencing of circFNDC3B with S1 and S2 did not statistically affect FNDC3B mRNA or protein levels (WT: wild‐type; NC: negative control; siRNA: S1, S2, S3). (i,j) MGC‐803 cells were transfected with the pEGFP‐FNDC3B vector or an empty vector. After 48 hr, FNDC3B levels and circFNDC3B levels were detected by western blot and qRT‐PCR. The results suggest that FNDC3B mRNA increased circFNDC3B (WT: wild‐type; EV: empty vector against EGFP‐FNDC3B; FNDC3B mRNA: overexpressing FNDC3B mRNA). Data were expressed as the mean ± SEM and were analyzed by independent samples t test, n = 3. *p < 0.05, ** p < 0.01, *** p < 0.0001. FNDC3B: fibronectin type III domain‐containing protein 3B; qRT‐PCR: quantitative reverse transcription‐PCR; SEM: standard error of the mean; siRNA: small interfering RNA [Color figure can be viewed at wileyonlinelibrary.com]