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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: A novel lncRNA MDHDH suppresses glioblastoma multiforme by acting as a scaffold for MDH2 and PSMA1 to regulate NAD+ metabolism and autophagy

Fig. 4

MDHDH binds MDH2 and PSMA1, and promotes their colocalization and MDH2 degradation by promoting the binding of ubiquitinated MDH2 and PSMA1. (A and B), Schematic diagram of the truncated biotin-labeled MDHDH RNA probe (upper). Western blot analysis showing the interaction between truncated MDHDH and MDH2 or PSMA1 (bottom). MDH2 mainly interacted with Δ1, PSMA1 mainly interacted with Δ2, and both interacted with the RNA main stem–loop structure (Δ5). (C), Secondary structure of RNA truncation probes (Δ1, Δ2, Δ5). The red frame section shows the key stem–loop structure of each truncated probe. Construction of truncated probes with deletion mutations (Δ-del). RNA pulldown experiments showed that the binding of three RNA probes (Δ1-del, Δ2-del, Δ5-del) to MDH2 and/or PSMA1 was diminished after deletion of the stem–loop structure. (D), Fluorescence assessment of MDH2 (green) and PSMA1 (red) enhanced colocalization (yellow) by MDHDH in U87 cells (scale bar = 10 μm, correlation scatter plot shown in the same panel). (E), Western blot of MDH2/PSMA1 using cytoplasmic and nuclear lysates isolated from control and MDHDH-overexpressing modified U87 cells to examine the effect of MDHDH on the subcellular location of MDH2 and PSMA1. (F), Western blot to detect MDH2 after 0, 4, 8 and 12 hours of cycloheximide (CHX, 100 mg/mL) treatment in the control and MDHDH-overexpressing U87 cells. Decay curve of MDH2 protein in the control and MDHDH-overexpressing U87 cells based on semiquantitative analysis of bands in the left panel. (G), U87 and U251 cells expressing either MDHDH or the control vector were cultured in the presence or absence of MG132 (20 μM) for 6 h. The cell lysates were analyzed by Western blotting with an anti-ubiquitin antibody. (H), Lysates from U87 cells transfected for MDHDH overexpression or with the control vector were subjected to immunoprecipitation with anti-MDH2 antibody or mouse IgG followed by immunoblotting analysis with anti-ubiquitin antibody. The MDH2 expression level of the overexpression group was approximately 50% of that of the control group, and the double eluate of the overexpression group was added to reflect the ubiquitination level of MDH2 in the overexpression group. (I and J), Western blot of MDH2/PSMA1 Co-IPs using cell lysates isolated from the control and MDHDH-overexpressing modified U87 and U251 cells to examine the effect of MDHDH on the interaction of MDH2 and PSMA1. (K and L), Co-IP and RNA pulldown experiments in a cell-free system. The cell-free system utilized recombinant proteins instead of cell lysate, and the binding of recombinant MDH2 and recombinant PSMA1 was enhanced by MDHDH

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