DEV009951 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Par3 shRNA efficiently reduces Par3 protein expression in vitro. E12 cortical cell transduced with either control (A-C) or Par3b shRNA (D-F) -containing lentivirus, double labelled for GFP (green) and Par3 (red) and stained with DAPI (blue). Note the higher immunoreactivity of control-transduced cells for Par3 (arrows in A-C) in comparison to shRNA-transduced cells (arrows in D-F). Arrowhead (B,C) points to a Par3-positive mitotic cell.

• Supplemental Figure S2 -

  Fig. S2. Identification of Par3b shRNA-transduced cells followed in time-lapse experiments. See Movie 2. After final live image (A), cells were fixed and immunolabelled for GFP (B, green) and MAP2 (C, red). Subsequently, the tissue culture plate was placed back in the microscope and a single picture taken, enabling the identification of MAP2-positive neurons (arrow).

• Supplemental Figure S3 -

  Fig. S3. Par6 overexpression does not affect the cell cycle kinetics of cortical progenitors. Quantification of the cell cycle length of E13 cortical progenitors transduced with control (diamonds) or Par6 (squares) and progressing from the first to the second round of cell division, second to third, and third to fourth. Note that the cell cycle length is not significantly affected after Par6 overexpression and that both control and Par6-transduced cells display a progressive lengthening of the cell cycle at later divisions.

• Supplemental Figure S4 -

  Fig. S4. Analysis of GFP+ cell-pairs after Par6 overexpression in vitro. Primary cell cultures of E13 cortex infected with control (A-C) or Par6 (D-F) retrovirus and double labelled for GFP (green in A,C,D,F) and Ki67 (red in B,C,E,F) after 2div. DAPI staining reveals cellular nuclei (blue in C and F). White arrows point to GFP+ Ki67+ cells and the black arrow points to a GFP+ Ki67− cell. (G) Quantification of GFP+ cell-pairs containing proliferating (Ki67+) and differentiating (Ki67−) cells after 2div. Note the increased proportion of pairs in which both cells are Ki67+ after Par6 overexpression (P<0.05; unpaired t-test).

• Movie 1 -

  Movie 1. E12 cortical progenitors transduced with control lentivirus. Note the presence of a GFP+ postmitotic cell (white circle) and GFP+ progenitors undergoing several rounds of cell division. Numbers in the upper left corner indicate the time when images were taken (day- hour: minutes: seconds).

• Movie 2 -

  Movie 2. E12 cortical progenitors transduced with Par3b shRNA lentivirus. GFP+ cells do not divide during the 5 days of live imaging.

• Movie 3 -

  Movie 3. E13 cortical progenitors transduced with Par6 retroviruses. GFP+ progenitor cell undergo several rounds of cell division, giving rise to the clone shown in Fig. 6G (white circles).

• Movie 4 -

  Movie 4. Phase-contrast images of the clone shown in Movie 3. Clonally related cells are easily distinguished from neighboring cells. Tracking of daughter cells in these images enabled the classification of cell divisions as well as the measurement of cell cycle length (see Fig. 6 and Fig. S3).