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. 2016 Sep 14;10:423. doi: 10.3389/fnins.2016.00423

Figure 4.

Figure 4

The effect of Sig-1R gene silencing on the DMT-mediated cell survival and HIF-1α expression of human iPSC-derived cortical neurons, moMACs and moDCs in hypoxia. Gene knockdown of Sig-1R was performed as in Section Materials and Methods. NT, non-treated control; ctrl siRNA, non-targeting negative control siRNA; Sig-1R siRNA, Sig-1R-specific siRNA. (A) Western blot validation of Sig-1R silencing. A typical experiment out of three (neuron) or four (moMAC/moDC) is demonstrated. (B) Effect of Sig-1R knockdown on cell viability in hypoxia. Cellular survival was monitored as in Figure 2. Cells were treated with 50 μM DMT before hypoxia treatment (hypoxia+DMT) or left untreated (hypoxia ctrl). Cultures with prior exposure to targeting Sig-1R siRNA (hypoxia+DMT+Sig-1R siRNA) or scrambled oligo (hypoxia+DMT+siRNA control) were also tested within the same experimental setup. (C) Densitometry data of HIF-1α Western blots in hypoxia following Sig-1R gene silencing. Black bars: HIF-1α protein expression after 6 h of hypoxia treatment (6 h) as compared to baseline (0 h); green bars: baseline (0 h) vs. 6 h hypoxia + 50 μM DMT treatment; blue bars: baseline (0 h) vs. hypoxia + 50 μM DMT administration in control siRNA-treated cultures; red bars: baseline (0 h) vs. 6 h hypoxia + 50 μM DMT treatment in targeting Sig-1R siRNA-treated cultures. (B,C) Results of three independent experiments are shown as Mean ± SEM. Asterisk indicates significance as compared to control siRNA (p < 0.05).