PMC

Memantine induced autophagic ultrastructural features in T-98 G cells according to TEM. A: The control cells showed the normal cytoplasm including mitochondria, ribosomes, and endoplasmic reticulum. B: After incubation with 400 μM memantine for 48 h, the cells developed autophagic vacuoles with lamellar structure in the cytoplasm. TEM: transmission electron microscopy.

Expression levels of NMDAR1 and NMDAR2B in U-251 MG and T-98 G cells. The cell lines were analyzed using western blotting (A) and RT-PCR (B). NMDAR1 was present only in T-98 G cells, but NMDAR2B was detectable in both glioma cell lines. β-actin and GAPDH were used as internal controls. NMDAR: N-methyl-D-aspartate receptor, RT-PCR: reverse transcription polymerase chain reaction, GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Memantine-induced cytotoxicity in glioma cell lines. After incubation with memantine (0–600 μM), malignant glioma cells were grown for 48 h starting at a density of 10⁴/well. The number of viable cells was counted and expressed as a percentage of the untreated control. The results are representative of 3 independent experiments. *p<0.05, Student’s t-test.

Memantine upregulated LC3-II and beclin-1 in T-98 G cells. A: Protein levels of LC3-I and LC3-II were analyzed in T-98 G cells treated with 0 and 400 μM memantine for 48 h. The graph shows the results of densitometric quantitation of western blots, presented as the LC3-II/LC3-I ratio relative to the control (0 μM of memantine). LC3-II was significantly increased by 400 μM memantine. B: Protein expression levels of AMPKα, beclin-1, APG5L, and ULK1 were evaluated using western blotting after incubation with memantine (0 and 400 μM) for 48 hr in T-98 G cells. The results of densitometric quantitation of immunoblots are presented as the ratio relative to the control (0 μM of memantine). Beclin-1 was significantly upregulated after the treatment with 400 μM of memantine. *p<0.05, Student’s t-test. AMPK: AMP-activated protein kinase.

A knockdown of NMDAR1 using siRNA transfection in T-98 G cells. T-98 cells incubated in EMEM was used as the control value. A: After the transfection with anti-NMDAR1 siRNA (10 nM) for 48 hr, the expression of NMDAR1 was evaluated using western bloting. B: Cell viability of control and siRNA-transfected T-98 G cells was analyzed after incubation with various concentration of memantine (0–600 μM). Significant differences in cell viability was observed at 400, and 600 μM of memantine. C: Protein level of LC3-I and LC3-II was evaluated in control and NMDAR1 knockdown cells using western blotting after incubation with 400 μM memantine. The results of densitometric quantitation of immunoblots were represented as the LC3-II/LC3-I ratio relative to control cells. *p<0.05, Student’s t-test. NMDAR: N-methyl-D-aspartate receptor, siRNA: small interfering RNA, EMEM: Eagle’s minimum essential medium.