DEV020180 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. NICD fail to maintain progenitor properties when co-expressed with Sox21. (A-C) Misexpression of NICD together with Sox21 caused cells to downregulate Sox3 (A), exit the cell cycle (B) and upregulate the expression of NeuN (C), even in the presence of NICD misexpression. (D-F) Black and white representation of A-C. Scale bar: 40 µm.

• Supplemental Figure S2 -

  Fig. S2. High levels of NICD counterbalance the effects of dnCSL overexpression. (A-C) Misexpression of NICD for 42 hours could counterbalance the activity of transfected dnCSL and several of the electroporated cells remained Sox3 positive (A) but Tuj1 (B) and E47 (C) negative. Scale bar: 50 µm.

• Supplemental Figure S3 -

  Fig. S3. Notch-mediated block of neurogenesis depends on intact SoxB1 function. Black and white representation of images presented in Fig. 2. (A-D) Misexpression of NICD (A) prevented the generation of neurons expressing NeuN (D) but retained expression of the progenitor protein Sox3 (A) and the incorporation of BrdU (C). (E-H) Expression of a dominant-negative version of CSL (dnCSL) (A), unable to bind DNA, induced cells to downregulate Sox3 (F), exit the cell cycle (G) and upregulate the expression of NeuN (H). (I-L) Overexpression of Sox3EnR (I) caused cells to downregulate Sox1 (J), exit the cell cycle (K) and upregulate the expression of NeuN (L), even in the presence of NICD misexpression.). (M-P) Combined expression of Sox3 and dnCSL (M) efficiently blocked the generation of NeuN⁺ cells (P) and maintained cells in a self-renewing (O) and Sox1-expressing state (N). Scale bar: 50 µm.

• Supplemental Figure S4 -

  Fig. S4. NICD, but not Sox3, attenuates the expression of Ngn2. Black and white representation of images presented in Fig. 3. (A, B) Expression of NICD (A) attenuated Ngn2 expression (B). (C,D) Transfection of dnCSL (C) increased Ngn2 expression (D). (E,F) Misexpression of Sox3 (E) did not alter the level of Ngn2 expression (F). Scale bar: 40 µm.

• Supplemental Figure S5 -

  Fig. S5. Expression of Ngn2 or E47 induces neurogenesis. (A-F) Expression of Ngn2 or E47 promoted the downregulation of progenitor markers (A,D), cell cycle exit (B, E) and the upregulation of markers for terminal neuronal differentiation (C,F). Scale bars: 40 µm in D; 10 µm in F.

• Supplemental Figure S6 -

  Fig. S6. Hes1, or the combination of Hes1 and Hes5, repress proneural protein, but not E-protein expression. (A-C) Transfection of a Hes1 in combination with an eGFP expression vector (A) efficiently attenuated Ngn2 expression (B) but had no effect on E47 expression (C). (D-F) Similar results were achieved by overexpressing a combination of Hes1 and Hes5 (D-F). (G-H) Cells co-expressing Hes1 and E47 maintain progenitor properties (G) and fail to upregulate Tuj1 (H). Scale bar: 50 µm in H.

• Supplemental Figure S7 -

  Fig. S7. Ngn2, but not E47, can rescue Hes5-dependent maintenance of progenitor properties. Black and white representation of images presented in Fig. 5. (A-C) Misexpression of Hes5 (A) suppressed Ngn2 (B) and Tuj1 expression (C). (D-I) Co-transfection of Ngn2 and Hes5 (D) promoted electroporated cells to differentiate into post-mitotic neurons (E-F), whereas misexpression of Hes5 in combination with E47 blocked neuronal differentiation (G-I). Scale bar: 50 µm.