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. 2019 Sep 11;39(37):7408–7427. doi: 10.1523/JNEUROSCI.2994-18.2019

Figure 2.

Figure 2.

Activation of the ApoE signaling pathway (DLK-MKK7-ERK/MAP-kinase) is reproduced by multiple experimenters in a highly independent manner. Data are from human neurons cultured on MEFs in the absence of serum; recombinant ApoE (10 μg/ml, produced in HEK293 cells, see Figure 2-1 and Figure 7-2 or control solutions were added at day 10 (D10), and neurons were analyzed at D12 as indicated. The human neuronal cultures, ApoE and control solutions were prepared by two experimenters in parallel (“A ApoE” and “B ApoE”), anonymized by an independent third individual, and used by the same experimenters (“dataset A” and “dataset B”) in a blinded manner. A, Dataset A showing that ApoE induces an increase in ERK phosphorylation and in the levels of DLK, APP, and synapsin-1 (Syn1) proteins with an ApoE4>ApoE3>ApoE2 potency rank order (top, representative immunoblots; bottom, summary graphs). Protein levels measured using fluorescent secondary antibodies were normalized for the Tuj1 signal examined on the same blots as an internal standard and additionally for the levels observed in control neurons. Because ApoE solutions were anonymized, samples on the immunoblot are not in a logical order. B, Dataset B showing that ApoE induces an increase in ERK phosphorylation and in the levels of DLK and APP proteins with an ApoE4>ApoE3>ApoE2 potency rank order (synapsin-1 was not analyzed). Only summary graphs are shown; for representative immunoblots, see Figure 2-2. C, ApoE2, ApoE3, and ApoE4 increase neuronal APP and PSD95 but not MAP2 gene expression as assessed by mRNA measurements in human neurons treated with the two independently produced ApoE preparations. APP and PSD95 mRNA levels were normalized for those of MAP2 as an internal standard and for the levels observed in the absence of ApoE (1.0). Data in bar graphs are means ± SEM (n ≥ 3 independent experiments); statistical significance was evaluated by two-way ANOVA with Tukey's multiple-comparisons test (*p < 0.05, **p < 0.01; ***p < 0.001) as detailed in the boxes. Note that in all analyses in which ApoE has an effect on a measured parameter, ApoE3 is always significantly more potent than ApoE2 and less potent than ApoE4.