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Fig. 4 | Journal of Hematology & Oncology

Fig. 4

From: CircRNA-CREIT inhibits stress granule assembly and overcomes doxorubicin resistance in TNBC by destabilizing PKR

Fig. 4

CircRNA-CREIT promoted PKR degradation via the ubiquitin–proteasome system. A Western blotting for PKR protein levels after circRNA-CREIT overexpression or knockdown. B, C TNBC cells with circRNA-CREIT overexpression or knockdown were treated with cycloheximide (CHX) for the indicated times. Western blotting analysis (upper) and statistical analysis (lower) of PKR levels upon CHX treatment are presented, with the level at 0 h as a control. D Western blotting analysis of PKR protein levels regulated by circRNA-CREIT with or without MG132 treatment. E Effects of circRNA-CREIT overexpression on the ubiquitination of PKR proteins. MDA-MB-231 cells were cotransfected with circRNA-CREIT overexpression plasmids and HA-tagged ubiquitin expression plasmids or the corresponding empty vectors. The cell lysates were incubated with anti-PKR or anti-IgG antibodies and protein A/G magnetic beads. The proteins precipitated in the co-IP assay were analyzed by Western blotting. IB immunoblot. F Western blotting assay showing the upregulation of K48-linked ubiquitination in circRNA-CREIT-overexpressing MDA-MB-231 cells. IB: immunoblot. HA-Ub-K48only: the cells were transfected with plasmids expressing HA-tagged ubiquitin with all lysines mutated except K48. HA-Ub-K63only: the cells were transfected with plasmids expressing HA-tagged ubiquitin with all lysines mutated except K63. G Identification of the candidate E3 ligases of PKR by bioinformatics prediction and MS analysis for RNA pull-down products, illustrated by a Venn diagram. H The interaction between PKR and HACE1 proteins was predicted by the ZDOCK server. The predicted structure was visualized by Discovery Studio software. I, J The interaction between circRNA-CREIT and HACE1 was verified by RNA pull-down assays (I) and RIP assay (J). Three independent experiments were conducted for each result. ns no significance; **p < 0.01 compared with the controls

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