Fig. 5

PSMA1 was required for MDHDH regulation of GBM cells and the AMPK/mTOR pathway. A, Graphic representation of the relative PSMA1 expression level (TPM) in different tissues (GBM vs. GTEx, LGG vs. GTEx). B U87 and U251 cells were transfected with PSMA1 siRNA and compared with the basal PSMA1 expression level of the control groups. The knockdown efficiency was analyzed with qRT–PCR, and β-actin served as the internal control. C-E NAD+ and NAD+/NADH ratio in MDHDH-, MDHDH+siPSMA1- or negative control vector-transfected U87 and U251 cells (Student’s t test; column means of triplicate assays). F OCR and ECAR in MDHDH-, MDHDH+siPSMA1- or negative control vector-transfected U87 and U251 cells. The OCR and ECAR were measured using a Seahorse analyzer. G The mitochondrial transmembrane potential was tested using JC-1 fluorescent probes of U87 cells transfected with MDHDH, MDHDH+siPSMA1 or negative control vector. CCCP-treated U87 cells were used as a positive control (scale bar = 50 μm; column means of triplicate assays). H PSMA1 was required for the MDHDH-regulated AMPK/mTOR pathway and promoted glioma autophagy. The lysates of GBM cells cultured with glucose-free DMEM for 2 h were used as the positive control group. I Representative images of U87 cells transfected with Ad-mCherry-GFP-LC3B adenovirus after transfection with the MDHDH overexpression vector (OE MDHDH group), MDHDH overexpression vector + PSMA1 siRNA (OE MDHDH+siPSMA1 group) or control vector (NC group). U87 cells cultured in EBSS medium for 24 hours were used as a positive control (scale bar = 10 μm). (J), Western blot of LC3B-I/LC3B-II in MDHDH-, MDHDH+siPSMA1- and control modified U87 and U251 cells