Figure 8.

Blocking ERK1/2 and PKC activities using PD98059 and chelerythrine resulted in a larger decrease in sAPPα levels in A549 and H1299 cell media. Cells (0.2 × 10⁵) were grown in 10% FBS-supplemented media for 24 h. The following day, the cell monolayers were incubated in serum-free media for 24 h, then treated for 72 h with the ERK1/2 inhibitor (PD98059, 50 μM) PKC inhibitor (chelerythrine, 7.5 μM), neostigmine (Neo, 50 μM), ACh (100 nM), mBDNF (5 nM), or in combination. Levels of sAPPα released into the culture media of A549 (A) and H1299 (B) cells during the 3-day incubation period were measured as described in the Methods section on the same amount of protein (3 µL of 600 µg/mL total protein) of each sample. ERK1/2 activity was measured in A549 (C) and H1299 (D) cells as described in the Methods section. Data from five independent assays, each carried out in triplicate, were averaged, normalized, and expressed as fold change relative to untreated cells (control) using the GraphPad 9.4.1 software. The graphs summarize the results expressed as means ± SD (n = 5). Asterisks indicate a statistically significant difference from the corresponding control of each cell line while absence of asterisks indicates no significance, Mann–Whitney test. Statistical differences between different groups were analyzed by an ordinary one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc multiple comparison test. * p < 0.05, ** p < 0.01.