Extended Data Figure 4. Stressed neurons robustly produces cis p-tau, cis p-tau is released from stressed neurons and neurotoxic, but are effectively blocked by cis, but trans, mAb.

a-c, Quantification results of Fig. 4a, b and f, respectively. The results are expressed as means ± s.d. and p values determined using the two-way ANOVA test (a) and Student's t test (c). d, Hypoxia induces cis p-tau, which is blocked by cis mAb. SY5Y neurons expressing a control vector were cultured in the hypoxia chamber in the absence or presence of cis or trans mAb for the times indicated, followed by IB for cis p-tau. e, Hypoxia induces cis p-tau before tau aggregation. SY5Y neurons were subjected to hypoxia for the times indicated, followed by sarkosyl extraction before IB with Tau5 mAb and quantification. f, Hypoxia induces cell death, which are blocked by cis, but trans, mAb. SY5Y neurons were cultured in the hypoxia chamber in the absence or presence of cis or trans mAb for the times indicated, followed by live and dead cell assay using the LIVE/DEAD® Viability/Cytotoxicity Kit. g, Stressed neuron lysates are neurotoxic, which is neutralized by cis, but not trans, mAb. Cell lysates were prepared from stressed SY5Y neurons and then added to growing SY5Y neurons directly (Control) or after immunodepletion with cis or trans mAb to remove cis or trans p-tau, respectively for 3 days, followed by live and dead cell assay. h, cis p-tau is released from stressed neurons. SY5Y neurons were cultured in the absence of serum for the times indicated and culture media were collected and centrifuged, followed by analyzing the supernatants for cis and trans p-tau with actin as a indicator of cell lysis.