Fig. 2: LINC01554 protects G3BP2 from ubiquitin-proteasome degradation. | Oncogene

Fig. 2: LINC01554 protects G3BP2 from ubiquitin-proteasome degradation.

From: G3BP2 regulated by the lncRNA LINC01554 facilitates esophageal squamous cell carcinoma metastasis through stabilizing HDGF transcript

Fig. 2

A catRAPID (http://service.tartaglialab.com) suggested the interaction between G3BP2 and LINC01554. B RPI-seq platforms (http://pridb.gdcb.iastate.edu) strongly recommended the possibility of interaction between G3BP2 and LINC01554. C Colocalization of G3BP2 and LINC01554 was detected by FISH-IF. Scale bar, 20 μm. RIP (D) and RNA pull-down (E) assays showed the interaction of G3BP2 with LINC01554. IgG antibody served as a control for RIP assay. F Western blot analysis revealed that the protein expression of G3BP2 was upregulated by LINC01554 in LINC01554-transfected KYSE30 and KYSE150 cells. G Cycloheximide chase assay showed that the half-life of the G3BP2 protein in LINC01554-transfected KYSE30 cells was longer than that in empty vector cells (left). In vitro ubiquitination assay showed that the ubiquitination level of the G3BP2 protein was abolished in LINC01554-transfected KYSE30 cells (right). RIP (H) and RNA pull-down (I) assays showed that G3BP2 did not bind to LINC01554 after deletion of the RRM ___domain in G3BP2. IgG antibody served as a control for the RIP assay. FL-G3BP2, full-length G3BP2; ΔRRM-G3BP2, truncation of the RRM ___domain in G3BP2. J Western blot showed that the G3BP2 protein level was decreased in KYSE30 cells with truncation of the RRM ___domain. K Cycloheximide chase assay showed that the half-life of the G3BP2 protein was shorter in KYSE30 cells with truncation of the RRM ___domain than in cells with full-length G3BP2. L In vitro ubiquitination assay showed that the ubiquitination level of the G3BP2 protein was dramatically increased in KYSE30 cells with truncation of the RRM ___domain. M catRAPID platform (http://service.tartaglialab.com) was applied to predict the specific binding sequence of LINC01554 with G3BP2 protein. N Top, schematic structures showing a summary of in vitro transcribed, biotinylated full-length, truncated or antisense LINC01554 probes. Bottom, immunoblot analysis with anti-G3BP2 in KYSE30 cells followed by RNA pull-down assay with full-length, truncated or antisense LINC01554 probes. FL, full length (1–1931 bp); F1, fragment 1 (1–500 bp); F2, fragment 2 (400–900 bp); F3, fragment 3 (1400–1931 bp); AS, antisense. RIP (O) and RNA pull-down (P) assays showed that the interaction of G3BP2 and LINC01554 was weakened in KYSE30 cells transfected with truncated fragment 2 in LINC01554. IgG antibody served as a control for RIP assay. Q Western blot showed that the G3BP2 protein level declined in KYSE30 cells transfected with truncated fragment 2 within LINC01554. R Cycloheximide chase assay showed that the half-life of the G3BP2 protein with truncated fragment 2 in LINC01554-transfected KYSE30 cells was shorter than that in full-length transfected cells (left). In vitro ubiquitination assay showed that the ubiquitination level of the G3BP2 protein was elevated in KYSE30 cells transfected with LINC01554 with fragment 2 truncated (right). The data represent the mean ± SD from three biological replicates. **P < 0.01, ***P < 0.001.

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