Fig 1: MDM2 interacts with EGLN2 and promotes its degradation. (A) Predicted E3 ligases interact with EGLN2. Confidence score, MDM2: 0.765, SYVN1: 0.736, STUB1: 0.716. The capital letter codes indicate E3 hierarchical tree, R: Ring; U: UBOX. (B) Immunofluorescent staining of EGLN2 (red), MDM2 (green) and nuclear (blue) in primary rat neurons. Scale bar=15 µm. (C) Primary neurons were coinfected with lv-flag-MDM2 alone or in combination with lv-myc-EGLN2 expression particles. IP was conducted with anti-Myc or anti-Flag antibodies. The potential MDM2-EGLN2 complex was then detected by Western blot analysis with an anti-Flag or anti-Myc antibody. (D) QRT-PCR analysis (n = 3) of EGLN2 mRNA expression in neurons 48 h after lentiviral mediated MDM2 overexpression or inhibition. (E), (F) Western blot analysis of EGLN2 expression in neurons with enforced MDM2 overexpression (E) or knockdown (F). (G)–(I) CHX pulse-chase assay was performed in primary rat neurons with enforced MDM2 overexpression or knockdown. 48 h after lentiviral infection, cells were treated with 10 µM CHX for the indicated time, followed by Western blot analysis. Representative images were presented (G), and the relative EGLN2 protein levels were illustrated graphically (H)–(I) (n = 3)
Fig 2: LncRNA CERS6-AS1 facilitates HCC progression via modulating MDM2 expression.A–D MDM2 overexpression or miR-30b-3p inhibitors partly reversed the inhibition effect of CERS6-AS1 knockdown on migration and invasion of HepG2 and MHCC97H. Transwell assay, Scale bar: 100 µm; Wound healing assay, Scale bar: 250 µm. E–H MDM2 overexpression or miR-30b-3p inhibitors partly reversed the inhibition effect of CERS6-AS1 knockdown on aerobic glycolysis of HepG2 and MHCC97H. I MDM2 overexpression or miR-30b-3p inhibitors partly reversed the inhibition effect of CERS6-AS1 knockdown on proliferation of HepG2 and MHCC97H. J MDM2 overexpression or miR-30b-3p inhibitors partly reversed the inhibition effect of CERS6-AS1 knockdown on protein expression involved in EMT (N-cadherin, twist) and glycolysis (PKM2, GLUT1). *p < 0.05, **p < 0.01.
Fig 3: MIAT reduces MDM2 mediated K48-linked poly-ubiquitination of EGLN2. (A), (B) Primary rat neurons were subjected to infection of indicated lentiviruses/adenoviruses (Lv-myc-EGLN2, lv-HA-Ub, lv-Flag-MDM2 and AAV5-MIAT) for 48 h, followed by treatment with MG132 (10 µM, 6 h). Then, cell lysates were immunoprecipitated with an anti-Myc tag antibody. Ubiquitinated EGLN2 was detected by Western blot assay with an anti-HA antibody. For treatment in panel B, mutant HA-Ub (K48 and K63) was used as indicated. (C) Western blot analysis of K48-linked poly-ubiquitination of EGLN2 in MIAT silenced primary rat neurons after oxygen-glucose deprivation (OGD) treatment for 12 h. (D) Schematic image showing the structure of the wild-type full length (FL) and truncated constructs of Myc-tag labelled EGLN2. (E) The indicated Myc-tagged FL or truncated mutation constructs were co-transfected with Flag-MDM2 primary rat neurons. The MDM2/Myc-tag complexes were immunoprecipitated by anti-Flag antibodies. EGLN2 proteins were detected using anti-Myc antibodies. (F) Immunoprecipitation assay was used to detect the interaction between EGLN2 N88 and MDM2 in MIAT-knockdown primary rat neurons with 12 h reoxygenation after OGD treatment
Fig 4: LncRNA CERS6-AS1 accelerates HCC proliferation in vivo.A Morphologic characteristics of xenograft tumors from HepG2/shNC group and HepG2/shCERS6-AS1 group (n = 5, scale bar: 1 cm). B The volume of xenograft tumors. C The weight of xenograft tumors. D Representative images of H&E, Ki67, PCNA, p53 and MDM2 staining in the xenograft tumors and the relative expression levels in shNC and shCERS6-AS1 groups. Scale bar: 100 μm. E Representative images of MDM2, N-cadherin, E-cadherin, GLUT1, PKM2 and HK2 staining in the xenograft tumors and the relative expression levels in shNC and shCERS6-AS1 groups. Scale bar: 100 μm. F The statistical analysis of these protein expression levels in shNC and shCERS6-AS1 groups. *p < 0.05, **p < 0.01.
Fig 5: MIAT-EGLN2 axis modulates oxidized (GSSG) and reduced (GSH) glutathione ratio in rat neurons. (A)–(B) OCR was measured in an XF96 Seahorse analyser in primary rat cortical neurons 48 h after MIAT knockdown (A) or overexpression (B) (n = 3). (C) oxPPP flux in primary rat cortical neurons 48 h after MIAT knockdown (n = 3). (D) oxPPP flux in primary rat cortical neurons 48 h after MIAT overexpression alone or in combination with EGLN2 knockdown (n = 3). (E) Quantitation of GSSG levels as percentage of total glutathione (GSSG +GSH) levels in primary rat cortical neurons 6 h after OGD/R treatment (n = 3). Cell treatments were the same as in panel (D). All quantitative data are mean ±SD. (F) Schematic images showing I/R injury-induced MIAT upregulation enhances EGLN2 stability via reducing MDM2 mediated ubiquitin-proteasomal degradation
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