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Fig. 5 | Cancer Cell International

Fig. 5

From: RNF8 is responsible for ATRA resistance in variant acute promyelocytic leukemia with GTF2I/RARA fusion, and inhibition of the ubiquitin–proteasome pathway contributes to the reversion of ATRA resistance

Fig. 5

RNF8 triggers RARA Lys48-linkage polyubiquitination and inhibits ATRA-induced differentiation. a During western blot analysis, HL60 cells were separately transfected with RNF8 expressing plasmid or empty vector and treated with 0 μM, 5 μM, and 10 μM MG132 for 4 h, with results indicating MG132 could reverse RNF8-mediated RARA-expressing inhibition. b Linkage-specific ubiquitination on RARA in response to overexpression of RNF8 or GTF2I-RARA. Coimmunoprecipitation following co-overexpression of RNF8, RARA-Ha, Ub-His/K63-Ub-His/K48-Ub-His, and GTF2I-RARA in 293T. RARA and its post-translational derivatives were purified by an HA antibody, and linkage-specific ubiquitinated RARA was detected by immunoblot using a His antibody. c 293T cells were cotransfected with the RARE reporter genes and plasmid encoding RNF8 or empty vector. Relative luciferase activity of each group in response to various concentrations of ATRA is shown in the histogram. d 293T cells were cotransfected with the RARE reporter genes and siRNF8. Relative luciferase activity in response to various concentrations of ATRA is shown in the histogram. e, f HL60 cells were transfected with a plasmid encoding RNF8. The CD11b differentiation marker was assessed by flow cytometry under exposure to various concentrations of ATRA for 3 days. For morphological analysis, each sample was stained with Wright-Giemsa and observed under an optical microscope (×1000) after 1 μM of ATRA treatment for 3 days. g, h GTF2I-RARA-HL60 cells were transfected with siRNF8. CD11b differentiation marker assay and Wright-Giemsa staining procedures were completed in the same manner as described above. Error bars represent the mean of experiments in triplicate. *p < 0.05, **p < 0.01. WCE: whole cell extracts

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