Research Paper Volume 14, Issue 20 pp 8221—8242

Figure 4. Degradation of APP and Tau by AR12 and neratinib requires LAP and macroautophagy in HCN2 neuronal cells and in BV2 microglia. (A, B) HCN2 cells were transfected with a scrambled siRNA or with siRNA molecules to knock down expression of Rubicon, Beclin1 or ATG5. In parallel, cells were transfected with an empty vector plasmid in (B), or transfected with plasmids to express APP or Tau in (A). After 24h, cells were treated with vehicle control, AR12 (2 μM), neratinib (50 nM) or the drugs in combination for 6h. Cells were fixed in place and immunostaining performed to determine the expression of Tau, APP and ERK2. (n = 3 +/-SD) Endogenous expression of APP was 5% of the value for APP expressed from a plasmid. Endogenous expression of Tau was 6% of the value for Tau expressed from a plasmid. * p < 0.05 less than vehicle control; ** p < 0.05 less than corresponding AR12 value; ▼p < 0.05 greater degradation than corresponding value in cells transfected to express APP or Tau. (C) BV2 microglial cells were transfected with a scrambled siRNA or with siRNA molecules to knock down expression of Rubicon, Beclin1 or ATG5. In parallel, cells were transfected with plasmids to express APP or Tau. After 24h, cells were treated with vehicle control, AR12 (2 μM), neratinib (50 nM) or the drugs in combination for 6h. Cells were fixed in place and immunostaining performed to determine the expression of Tau, APP and ERK2. (n = 3 +/-SD). * p < 0.05 less than vehicle control. (D) RAW macrophages (+/+ and -/- for Rubicon) were transfected to express Tau or APP. After 24h, cells were treated with vehicle control, AR12 (2 μM), neratinib (50 nM) or the drugs in combination for 6h. Cells were fixed in place and immunostaining performed to determine the expression of Tau, APP and ERK2. * p < 0.05 less than vehicle control; ** p < 0.05 less than corresponding neratinib value.