Journal: International Journal of Molecular Medicine
Article Title: circOMA1 delivered by exosomes regulates DRD2-mediated prolactinoma resistance
doi: 10.3892/ijmm.2026.5812
Figure Lengend Snippet: circOMA1 promotes ubiquitin-mediated degradation of DRD2 and induces CAB resistance in MMQ cells by upregulating KBTBD7. (A) Western blot detection of DRD2 protein in circNC and circOM cells (top) with quantification (bottom). (B) DRD2 protein levels in circNC and circOM cells were measured by western blotting after treatment with 100 μ g/ml CHX for varying lengths of time. (C) Western blot analysis of DRD2 in circOM cells at the indicated time points after CHX treatment with or without treatment with 20 μ M MG132. (D) Co-IP analysis of DRD2 ubiquitination in circNC and circOM cells (green arrow indicates DRD2). (E) Western blot and densitometric analysis of KBTBD7 expression in circNC and circOM cells. (F) Representative immunofluorescence images showing distribution and co-localisation of DRD2 and KBTBD7 in MMQ cells; the line profile at right indicates co-localisation. Scale bar, 10 μ m. (G) Co-IP demonstrating the interaction between DRD2 and KBTBD7 in MMQ cells. (H) Immunoblot analysis of KBTBD7, DRD2 and PRL after circOMA1 knockdown for 72 h in circOM cells. (I) Immunoblot detection of DRD2 in circOM cells after KBTBD7 knockout by CRISPR/Cas9. (J) CCK-8 determination of circOM cell sensitivity to CAB after KBTBD7 knockdown (R 2 =0.9240). (K) Western blot detection of KBTBD7 and DRD2 in PRL-PitNET-S (n=4) and PRL-PitNET-R (n=6) tissues. (L) Representative immunofluorescence images showing distribution and co-localisation of KBTBD7 and DRD2 in PRL-PitNET tissues. Scale bar, 10 μ m. Data are presented as the mean ± SEM of at least three repeats. Differences between groups were compared using a unpaired Student's t-test. * P<0.05 and ** P<0.01. CAB, cabergoline; NC, negative control; circ, circular RNA; PRL-PitNET, prolactin-secreting pituitary neuroendocrine tumour; PRL-PitNET-S, PRL-PitNET-sensitive; PRL-PitNET-R, PRL-PitNET-resistant; CHX, cycloheximide; MG132, proteasome inhibitor; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; Co-IP, co-immunoprecipitation; circ, circular RNA; DRD2, dopamine D2 receptor; KBTBD7, Kelch-repeat and BTB domain-containing protein 7.
Article Snippet: CHX (a protein synthesis inhibitor) and MG132 (a proteasome inhibitor) were purchased from MedChemExpress. circOM, circNC or MMQ cells were treated with 100 μ g/ml CHX alone or combined with 20 μ M MG132 for 0, 4, 8, or 12 h, after which, western blotting was performed.
Techniques: Ubiquitin Proteomics, Western Blot, Co-Immunoprecipitation Assay, Expressing, Immunofluorescence, Knockdown, Knock-Out, CRISPR, CCK-8 Assay, Negative Control, Immunoprecipitation
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