Article Title: DNMT1 modulates interneuron morphology by regulating Pak6 expression through crosstalk with histone modifications
Figure Lengend Snippet: Enhanced Pak6 expression induced by EZH2 inhibition results in increased morphological complexity. (A-E) Representative microphotographs of dissociated E16 (+ 2 DIV) POA cells (A, B) and N2a cells (C, D) treated either with DMSO (A, C) or DZNep (B, D) and stained against TUBB3 (green channel) with a specific antibody, ACTB (red channel) with fluorescently labeled phalloidin and DAPI (blue channel). Quantification of cell morphology is shown in (E) as complexity index relative to DMSO controls. The complexity index is a product of primary neurite number, the branch point number of the longest process and the ratio between longest process length and the mean length of all other neurites. (E; n = 52 POA and 85 N2a cells for DMSO, n = 69 POA and 93 N2a cells for DZNep). (F-J) Representative microphotographs of dissociated E16 (+ 2 div) POA cells treated either with DMSO (F, H) or DZNep (G, I) in addition to control (F, G) or Pak6 siRNA (H, I), then stained against TUBB3 (green channel) with a specific antibody, ACTB (red channel) with fluorescently labeled phalloidin and DAPI (blue channel). The complexity index is quantified in (J) relative to DMSO + ctrl siRNA cell parameters (n = 39 POA cells for DMSO + ctrl siRNA, n = 43 POA cells for DZNep + ctrl siRNA, n = 51 POA cells for DMSO + Pak6 siRNA, n = 68 POA cells for DZNep + Pak6 siRNA). ‘n’ refers to the number of analyzed cells. **P
Article Snippet: Mouse Dnmt1 siRNA oligos (50 nM) or mouse Pak6 siRNA oligos (50 nM), containing a pool of at least three target-specific 19–25 nt siRNAs to knockdown gene expression ( Dnmt1 siRNA sc-35,203; Pak6 siRNA sc-44,879; Santa Cruz Biotechnology) or 50 nM control siRNA (BLOCK-iT Alexa Fluor red/green fluorescent oligo; Invitrogen) were applied for 5 h in antibiotic-free Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific) at 37°C, 5% CO2 , and 95% humidity.
Techniques: Expressing, Inhibition, Staining, Labeling