Fig 1: GPC6 increased the surface levels of NMDARs of hiPSC-neurons. (A) The expression of markers for intracellular proteins (β-actin) and membrane proteins (Na+-K+ ATPase) in respective fractions, and the cellular distribution of GluN1 (NMDARs). GPC6 induced an increase in the surface expression level of GluN1 and a corresponding decrease in intracellular expression, without little effects on the total level. (B) The cellular distribution of GluA1 (AMPARs). GPC6 also resulted in a slight increase in the surface expression level of GluA1, but the effect was weaker than that on GluN1. Total and intracellular GluA1 levels showed little change. The data were mean ± SE of three independent experiments.
Fig 2: GPC6 enhanced the NMDAR-mediated Ca2+ increase in hiPSC-neurons. Typical traces of the Ca2+ increases in control and GPC6-treated cells are shown above. In the control group, the average value of the L-Glu-induced Ca2+ increase was not significantly affected by AP5 (n = 53). AP5+DNQX almost completely suppressed the L-Glu-induced Ca2+ increase. In the GPC6-treated group, AP5 significantly suppressed the L-Glu-induced Ca2+ increase, and AP5+DNQX almost completely suppressed the L-Glu-induced Ca2+ increase (n = 68). ∗p < 0.05, ∗∗p < 0.01 vs. AP5(-) group. Tukey’s test following ANOVA.
Fig 3: Effects of GPC6 on the sensitivity of hiPSC-neurons to L-Glu-induced cytotoxicity. After a 5-days treatment with GPC6, cells were exposed to L-Glu (1, 10 mM, 24 h), and an MTT reduction assay was performed. L-Glu did not cause cell damage in the control group. In the GPC6-treated group (10 nM, 5 days), L-Glu caused a concentration-dependent decrease in MTT reduction. ∗∗p < 0.01 vs. the control in each group, Tukey’s test following two-way ANOVA (N = 6).
Fig 4: GPC6 increased the L-Glu-induced Ca2+ increase in hiPSC-neurons. (A) Left panel shows the typical control cells loaded with fura-2 AM. The 5-min area under the curve (AUC) of the ratio of the fluorescence intensities of 340 and 380 nm from the start of the response to L-Glu (100 μM, 2 min) (right panel) was quantified. (B) Typical Ca2+ traces from control and GPC6-treated cells are presented above. In the GPC6-treated group (n = 68), the average L-Glu-induced Ca2+ increase of all cells in the field of view was significantly higher than that of the control group (n = 53). ∗∗p < 0.01 vs. control group, Student’s t-test.
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