Figure 2.

Effect of Oxygen Tension on AP⁺GSCs in a Serum-Free Culture System

(A) Colony morphology and AP activity (in blue) of mouse AP⁺GSCs under normoxia (21% O₂) and hypoxia (5% and 3% O₂). Cluster of mesenchymal-like cells under 1% O₂ hypoxia (arrowhead). Scale bar, 100 μm.

(B) Quantification of GSC colony number and size distribution. One-way ANOVA.

(C) Bromodeoxyuridine incorporation assay of AP⁺GSCs under hypoxia (5% O₂). The arrowheads indicate the hypoxic effect on the cell morphological transition to AP⁺-migrating GSC cells. Scale bars, 100 μm.

(D) Quantification of the percentage of the GSC colony with migratory cells. One-way ANOVA.

(E) Pie charts representing the GO secretome analysis of mouse AP⁺GSCs under normoxia (21% O₂) and hypoxia (5% O₂). The secretome analysis was performed using LC-MS/MS. #, proteins expressed under hypoxia only. (a) Cellular component. (b) Protein class; classes accounting for more than 10% of the total classes are labeled I–V. (c) Signaling pathway. Intrigued classifications are labeled VI–XV.

(F) Secretome protein network of AP⁺GSCs under hypoxia. Protein levels that were higher in the hypoxia than in the normoxia are shown.

(G) Validation of the representative LC-MS/MS-determined differentially expressed proteins in an AP⁺GSC colony using qRT-PCR.

Student’s t test. For all quantifications, data are the means ± SEM of at least three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. See also Figure S1 and Tables S1–S4.