Fig. 5: HNRNPH1-stabilized LINC00662 blocks ubiquitin-proteasome-dependent GRP78 degradation.

A Immunoblot analysis of the protein levels of HNRNPH1 and GRP78 after LINC00662 activation or knockout. β-Actin was used as the internal control. B Relative LINC00662 levels when silencing HNRNPH1 with specific siRNAs in A2780 and OVCA433 cells. C Half-life of LINC00662 in A2780 and OVCAR-3 cells with HNRNPH1 knockdown after treatment with 2.5 μM actinomycin D for the indicated times. D, E A2780 cells transfected with LINC00662 siRNAs were treated with CHX (50 mg/ml) for the indicated times. D Immunoblot analysis of GRP78 levels in whole-cell extracts. E Densitometric analysis of the GRP78 protein bands, whereby the relative fold change in the level is with respect to the level at 0 h. F A2780 cells transfected with LINC00662 siRNAs were treated with MG132 (25 mM) for 12 h; immunoblot analysis of GRP78 levels in the indicated cells. G Lysates from A2780 cells with LINC00662 knockdown or OVCAR-3 cells with LINC00662 overexpression were immunoprecipitated (IP) with either control IgG or an anti-GRP78 antibody and then immunoblotted to assess ubiquitin and GRP78 levels. H, I Immunoprecipitation to detect the association between GRP78 and AMFR after LINC00662 knockdown. In brief, lysates of A2780 cells transfected with LINC00662 siRNA or scrambled control siRNA and treated with 25 mM MG132 for 4 h were subjected to coimmunoprecipitation with antibodies specific for GRP78 and AMFR followed by western blot analysis using the indicated antibodies. J Heatmap of DEGs identified by RNA-seq. K GO enrichment analysis of DEGs. L MAPK signaling pathway enrichment in A2780 cells transfected with LINC00662 siRNA or scrambled control siRNA, as shown by GSEA. M Immunoblot analysis of c-Src (Tyr419/Tyr424) and p38 (Tyr182) phosphorylation in A2780 and OVCA433 cells transfected with LINC00662 siRNAs and in OVCAR-3 and SK-OV-3 cells with transient overexpression of LINC00662. Data are shown as means ± SEMs. A two-tailed Student’s t-test was used for statistical analysis. **P < 0.01; ***P < 0.001. P < 0.05 was considered significant.