SEARCH

SEARCH BY CITATION

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
STEM_746_sm_SuppFig1.tif2662KFigure S1. (Left) Lineage marker distributions as a function of ECM stiffness for NSCs differentiated in mixed media for 6 days shows that more undifferentiated, Nestin positive cells remain on stiff substrates, with an additional 10-20% marker negative cells on all stiffnesses. (Right) Immunofluorescence images of cells cultured on 100 Pa and 75000 Pa ECMs.
STEM_746_sm_SuppFig2.pdf33KFigure S2. RhoA-GTP and Cdc42-GTP levels of NSCs expressing DN and CA RhoA and Cdc42 cultured in proliferating conditions on laminin-coated tissue-culture polystyrene. Levels are normalized to control, as determined by GLISA assays of samples loaded with equivalent total protein levels. Error bars are 95% confidence intervals. *p < 0.05 (Student's unpaired twotailed t-test).
STEM_746_sm_SuppFig3.pdf161KFigure S3. 95% confidence bounds, (A, B) solid lines – control, dotted lines – (A) DN RhoA and (B) DN Cdc42, dashed lines – (A) CA RhoA and (B) CA Cdc42, generated from one-way analysis of covariance on log-transformed substrate elastic modulus and cell elastic modulus data shown in Figure 3, reveal lower stiffnesses of NSCs expressing DN RhoA and DN Cdc42 compared to control on substrates above 1000 Pa. Error bars for NSCs on thin gels are 95% confidence intervals for n = 17-50 cells. *p < 0.05 (ANOVA-TK) for comparisons to control on each ECM elastic modulus (control data previously shown in Figure 2A).
STEM_746_sm_SuppFig4.pdf128KFigure S4. 95% confidence bounds, (A, B) solid lines – control, dotted lines – (A) DN RhoA and (B) DN Cdc42, dashed lines – (A) CA RhoA and (B) CA Cdc42, generated from one-way analysis of covariance on log-transformed substrate elastic modulus and cell immunostaining data shown in Figure 3, show that (A) DN RhoA and (B) DN Cdc42 increase the proportion of neurons and decrease the proportion of astrocytes on stiffer ECMs compared to control after 6 days. In contrast, (A) CA RhoA and (B) CA Cdc42 decrease the proportion of neurons and increase the proportion of astrocytes on softer ECMs compared to control. (C) Higher power images of insets from Figure 3C-D.
STEM_746_sm_SuppFig5.pdf109KFigure S5. Rho GTPases modulate the effect of ECM elastic modulus on the proportions of neurons and astrocytes in survival conditions. Error bars are 95% confidence intervals, n = 5-6. *p < 0.05 for comparisons to control for each substrate elastic modulus (control data previously shown in Figure 1B) (ANOVA-TK). β-tubulin III (green), GFAP (red), DAPI (blue), MBP (white). 95% confidence bounds, (E, F) solid lines – control, dotted lines – (E) DN RhoA and (F) DN Cdc42, dashed lines – (E) CA RhoA and (F) CA Cdc42, generated from one-way analysis of covariance on log-transformed substrate elastic modulus and cell immunostaining data shown in (A) and (B). (G, H) Higher power images of insets from (A, B).
STEM_746_sm_SuppFig6.pdf22KFigure S6. Quantitative RT-PCR measurements of the increases in mRNA levels for neuronal and astrocytic markers in NSCs expressing CA or DN RhoA cultured on laminin-coated tissue culture polystyrene for 6 days in mixed conditions over the same NSCs cultured in self-renewal conditions (20 ng/ml FGF-2). Results show similar trends to immunostaining (Figure 4), with CA RhoA exhibiting a smaller increase in neuronal mRNA levels and a greater increase in astrocytic mRNA levels and DN RhoA exhibiting greater neuronal and smaller astrocytic increases in mRNA levels compared to control. Error bars are 95% confidence intervals, n = 3. *p < 0.05 for comparisons to control NSCs (ANOVA-TK).
STEM_746_sm_SuppFig7.pdf364KFigure S7. Rho GTPases modulate NSC lineage compositions on laminin-coated glass. Immunostaining against β-tubulin III (green bars), GFAP (red bars), and MBP (orange bars) of cells cultured on glass show that DN RhoA and Cdc42 increase the percentage of neurons and decrease the percentage of astrocytes while CA Cdc42 slightly increases the percentage of oligodendrocytes over control cells after 6 days of differentiation in (A) mixed conditions and (B) survival conditions. DN and CA Rac1 do not affect NSC lineage compositions in either media condition. Error bars are 95% confidence intervals, n = 5-6. *p < 0.05 for comparisons to control (ANOVA-TK). Representative immunofluorescence images of NSC lines after 6 days of differentiation in (C) mixed conditions and (D) survival conditions. β-tubulin III (green), GFAP (red), DAPI (blue), MBP (white). (E) Higher power images of insets from (D).
STEM_746_sm_SuppFig8.pdf272KFigure S8. Neither expression of DN and CA Rho GTPases nor variation of ECM stiffness compromises later stages of neuronal maturation and subtype marker expression, with GABAergic (GABA, red) and glutamatergic (VGlut1, green) neurons detectable on substrates of 700 and 75,000 Pa and all RhoA/Cdc42 genotypes (DAPI, blue). Cells were cultured in 1 μM forskolin and 5 μM all-trans retinoic acid for 6 days then switched to 20 ng/ml brain-derived neurotrophic factor with 1 μM forskolin for another 6 days.
STEM_746_sm_SuppFig9.pdf60KFigure S9. Inhibition of proteins that regulate cellular contractility reduces astrocytic differentiation in mixed conditions on soft and stiff ECMs. Error bars are 95% confidence intervals, n = 5-6. *p < 0.05 for comparisons to the same NSC population in control media conditions (control data previously shown in Figure 1A) (ANOVA-TK).
STEM_746_sm_SuppFig10.pdf143KFigure S10. (A) Inhibition of proteins that regulate cellular contractility rescues neuronal differentiation in survival conditions on soft and stiff ECMs. Error bars are 95% confidence intervals, n = 6. *p < 0.05 for comparisons to the same NSC population in control media conditions (ANOVA-TK). (B and C) Inhibition of proteins that regulate cellular contractility and adhesion do not appear to modulate the proportion of oligodendrocytes generated from NSCs. (B) mixed and (C) survival conditions. Error bars are 95% confidence intervals, n = 6. *p < 0.05 for comparisons to the same NSC population in control media conditions (ANOVA-TK).
STEM_746_sm_SuppInfo.pdf75KSupplementary Data

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.