Figure 6: Recombinant PK2 (rPK2) activates Akt and ERK (p44/42) signalling pathways to protect N27 cells from MPP⁺ toxicity.

(a,b) N27 cells were untreated or treated with 25 nM rPK2 in the absence or presence of 2 μM PKRA7 for 3 h. Cells were collected and subjected to western blot analysis of ERK (a) and Akt (b) activation. (c) N27 cells were untreated or treated with 300 μM MPP⁺ in the absence or presence of 25 nM rPK2, 100 nM Akt inhibitor API1 or 50 μM ERK inhibitor PD98059 for 24 h. Cell viability was then measured by MTS assay. Data are expressed as the mean±s.e.m. of three independent experiments using one-way ANOVA with Bonferroni post-test comparison with n=7–22. (d,e) CRISPR/Cas9-based knockdown of PK2 in N27 cells infected with CRISPR/Cas9 PK2 KO lentivirus or CRISPR/Cas9 control non-target lentivirus. PK2 expression was analysed by western blot (d) and real-time RT–PCR (e) (Student’s t-test with n=6). (f,g) CRISPR/Cas9 PK2 KO or CRISPR/Cas9 control lentivirus-infected N27 cells were treated with or without 300 μM MPP⁺ in the presence or absence of 25 nM rPK2. Cell viability was analysed by MTS assay (f) (one-way ANOVA with Bonferroni post-test comparison and n=8; NS, non-significant, P=0.9065) and Akt activation was measured by western blot (g) 24 h after MPP⁺ treatment. Asterisks denote statistical significance (*P<0.05, ^**P<0.01 and ^***P<0.001. NS, non-significant).