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. 2023 Jul 10;63(2):98. doi: 10.3892/ijo.2023.5546

Figure 3.

Figure 3

circRPS19 upregulates HK2 protein expression via USP7. (A) mRNA and (B) protein levels of PKM2, HK2 and PFK1 were assessed by RT-qPCR and WB in AGS cells following circRPS19 ablation. (C) IP showed HK2 ubiquitination in AGS cells following circRPS19 depletion. (D) RIP assay was used to examine the interaction between circRPS19 and HK2 protein in AGS cells. (E) Co-IP followed by mass spectrometry analysis was used in AGS cells to determine enzymes affecting ubiquitination of HK2 protein. (F) mRNA and (G) protein levels of USP7 were assessed by RT-qPCR and WB in AGS cells following circRPS19 ablation. (H) FISH assay was used to determine co-localization of circRPS19 and USP7 mRNA in AGS cells. (I) RIP assay was used to detect abundance of circRPS19 and USP7 mRNA in RISC. **P<0.01, ***P<0.001, ****P<0.0001 compared with control group. circRPS19, circRNA ribosomal protein S19; HK2, hexokinase 2; USP7, ubiquitin-specific processing protease 7; PKM2, pyruvate kinase M2; PFK1, 6-phosphofructokinase subunit alpha; RT-q, reverse transcription-quantitative; IP, immunoprecipitation; FISH, Fluorescence in situ hybridization; RISC, RNA-induced silencing complex; n.s., not significant; si, small interfering; NC, negative control; WB, western blot; IB, immunoblot; Ub, ubiquitin.