Knockdown of NRP2 expression causes increased mRNA expression of NRP1, while simultaneous knockdown of both NRPs has no effect on the total area of SC clusters.

<p>SCs were transfected with the siNRP1 pool, the siNRP2 pool or a combination of both. (A) After 5 days the measured NRP1 mRNA expression levels were 32, 178, and 47% respectively when compared to the siGlo condition. This indicates NRP1 mRNA upregulation after knockdown of NRP2. mRNA expression levels of NRP2 after knockdown with the siNRP1 pool, the siNRP2 pool or a combination of both were 88, 36 and 42% of endogenous levels respectively. The presented values are averages of 4 independent SC transfections (p<0.05). (B) A simultaneous transfection with the siNRP1 and siNRP2 pool has no effect on the total area of SC clusters. There is a small but significant decrease in the number of clusters (78%; p<0.001) and a moderate increase in cluster size (130%; p<0.05). The presented values are averages of 3 independent experiments (P<0.05). Error bars indicate the SEM.</p

Schwann cells form clusters when confronted with meningeal cells and these cells express neuropilins and class-3 semaphorins.

<p>(A) SCs labeled with S100 (red) form a clear border and form cluster-like aggregates when confronted with MCs (blue nuclei without cellular staining). (B) NRP1 and NRP2 are expressed in SCs and in MCs. SCs express 8 fold more NRP2 than NRP1. MCs express ∼2 fold more NRP2 than NRP1. MCs express 10 fold more NRP1 and almost 3 fold more NRP2 than SCs. Scalebar = 250 µm (C) endogenous Sema3A–F expression of SCs and MCs. SCs express predominantly Sema3B, C and E. MC express predominantly Sema3C and at 10 fold lower levels Sema3A, E and F. These results represent the average mRNA levels of 3 independent SC and MC cultures. Error bars indicate the standard error of the mean (SEM).</p

Validation of NRP1 and NRP2 knockdown effects on SC cluster formation.

<p>(A) mRNA levels of NRP1 were decreased in SCs after transfection with the siRNA pool or either of the 4 single siRNAs. Expression levels were ranging from 9 to 66% of endogenous expression measured in the siGlo condition. Knockdown was significant for all conditions (p<0.001) except for siNRP1 single 1 (p<0.12). The value presented for the siNRP1 pool is the result of 4 different experiments. The values for the siNRP1 singles are the result of 1 experiment with 4 technical replicates. The siGlo condition presented in the graph is the control for the single siRNA conditions. (B) Three single siRNAs confirm the significant decrease in the number of clusters which is observed with the siNRP1 pool (p<0.01). (C) Two of the single siRNAs confirm the increase of cluster size observed with the siNRP1 pool (p<0.001). All values are normalized to the siGlo condition. The presented values represent the average of 3 separate experiments. The differential effects of siNRP1 single 2 and 3 may be the result of off-target effects (D) mRNA levels of NRP2 were decreased in SCs after transfection with the siRNA pool or either of the 4 single siRNAs. Expression levels were ranging from 18 to 64% of endogenous expression measured in the siGlo condition. Knockdown was significant for all conditions (p<0.05). The value presented for the siNRP2 pool is the result of 4 different experiments. The values for the siNRP1 singles are the result of 1 experiment with 4 technical replicates. The siGlo condition presented in the graph is the control for the single siRNA conditions. (E) All 4 single siRNAs confirm the significant decrease in the number of clusters which is observed with the siNRP2 pool (p<0.01). The presented values represent the average of 3 separate experiments. Error bars indicate the SEM.</p

Knockdown of NRP1 increases while knockdown of NRP2 decreases formation of Schwann cell clusters.

<p>(A–C) Representative SC clusters when co-cultured with MCs for 5 days after a transfection with siGlo, siNRP1 pool or siNRP2 pool respectively. SCs are shown in grey (S100 labeling). MCs are not labeled. Valid cell clusters were outlined with a blue line. Invalid clusters were outlined with an orange line. Scalebar = 250 µm. (D) The total area of SC clusters per well after knockdown of NRP1 or NRP2. There was a significant increase in total area of SC clusters after knockdown of NRP1 (137%) while in contrast, there was a strong decrease of total area of SC clusters after knockdown of NRP2 (54%). (E) The number of valid clusters per well significantly decreases to 77% after knockdown of NRP1 and 53% after knockdown of NRP2. (F) Cluster size after knockdown of NRP1 increases strongly to 183% (p<0.001) when compared to the siGlo condition, while the average SC cluster size after knockdown of NRP2 does not change. Values are normalized to the siGlo condition and represent the averages of all wells of 6 separate experiments. Error bars indicate the SEM.</p