Figure 8. RNAi knockdown of abscission machinery affects midbody-release and differentiation.

(a,b) NS5-cells were transfected with control (con, light grey) or Alix (dark grey) siRNAs, grown for 2 days, followed by immunoblotting of cells and P2 pellet of conditioned medium. (a) GAPDH and Alix immunoblots of cells (1/20 of total) and CRIK immunoblots of P2 (1/2 of total). (b) Quantification of immunoblots in (a), expressed as arbitrary units (A.U.). Mean of three transfections; bars, s.d. **P<0.01; ***P<0.001 (Student's t-test). (c–n) MKLP-1–GFP transgenic Neuro-2a cells on six-well (c–e), 384-well (f–j) or 24-well (k–n) plates were transfected with control (con, light grey) or Alix, Tsg101 or Cep55 (dark grey) siRNAs, grown for 3 days, and analysed directly (c–j) or after 2 days of differentiation using retinoic acid (k–n). (c) Alix and Tsg101 immunoblots of control and Alix or Tsg101 knockdown cells, and quantitative PCR for Cep55 of control and Cep55 knockdown cells (control set to 1). (d) CRIK and α-tubulin (Tub) immunoblots of control and knockdown cells (C, 1/20 of total) and P1/P2 pellets of 3-day-conditioned medium (1/2 of total). (e) Quantification of immunoblots as in (d), of CRIK in P1+P2 and α-tubulin (Tub) in cells (C), each expressed as fold change of control. Mean of three transfections with one siRNA each; bars, s.d. *P<0.05; ***P<0.001 (Student's t-test). (f,g) Nuclei (Hoechst) and cell-retained midbodies (GFP) of control (f) and knockdown (g) cells. Scale bars, 20 μm. (h) Automated image analysis of control images shown in (f); midbody positions (red) and cell outlines (yellow) obtained from GFP and Hoechst images, respectively. (i,j) Residual midbodies per cell (i) and cells per mm² (j) were calculated from 7–9 images per well using automated image analysis. Mean values of three independent experiments with two siRNAs per target; number of wells analysed per experiment: control, 22; target, 2. Bars, s.d. **P<0.01 (Welch two sample t-test). (k,l) β-III-Tubulin immunofluorescence (red) of control (k) and knockdown (l) cells; blue (Hoechst), nuclei; green (GFP), midbodies. Scale bars, 20 μm. (m,n) Average length (m) and number per cell (n) of neurites were determined from 10 to 30 randomly chosen cells per transfection. Mean of four transfections, using two siRNAs per target; bars, s.d. *P<0.05 (Student's t-test). In total, 70–124 cells were analysed for control and target siRNAs each. (o) Hypothetical relationship between midbody-retention versus -release and differentiation. DC, differentiated cell; mb, midbody; PC, proliferating cell.