Fig. 7

METTL3 knockdown induced dysregulation of cell cycle genes and apoptotic changes in primary neuronal cultures. (A, B) Representative immunofluorescence image (A) and quantification analysis of m⁶A immunoreactivity (B) in primary cortical neurons after AAV-GFP-shRNA infection revealed significantly decreased neuronal m⁶A modification by METTL3 depletion. (C) Quantitative PCR analysis of cell cycle genes in primary neurons after Mettl3 knockdown mediated by AAV-shRNA infection. GAPDH was used as a reference gene for quantitative gene expression analysis. (D) Total pure RNA from adult rat cortical tissue was immunoprecipitated with a specific m⁶A antibody (Novus) or a mouse IgG control. CCND2 mRNAs were pulled down and detected by quantitative PCR. (E-J) Primary rat cortical neurons from E16–18 embryos were infected with AAV-GFP-shMettl3 or AAV-GFP-shCtrl on DIV 7. (E-F) Six days after AAV injection, neurons were treated with actinomycin D (5 μg/ml). Representative mRNA profiles of GAPDH (E) and CCND2 (F) at 0-, 3-, and 6-h time points after treatment were shown. 18S rRNA was used as reference for quantitative analysis. (G-J) Eight to nine days after AAV infection, neuron extracts were used for immunoblot analysis (G, I) and quantification of protein changes were shown (H, J) (Data are means±SEM from at least 3 independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001; B-F, H, J unpaired student’s t-test)