Supplementary Material for: Nox4-Generated Superoxide Drives Angiotensin II-Induced Neural Stem Cell Proliferation

Reactive oxygen species (ROS) have been reported to affect neural stem cell self-renewal and therefore may be important for normal development and may influence neurodegenerative processes when ROS activity is elevated. To determine if increasing production of superoxide, via activation of NADPH oxidase (Nox), increases neural stem cell proliferation, 100 nM angiotensin II (Ang II) - a strong stimulator of Nox - was applied to cultures of a murine neural stem cell line, C17.2. Twelve hours following a single treatment with Ang II, there was a doubling of the number of neural stem cells. This increase in neural stem cell numbers was preceded by a gradual elevation of superoxide levels (detected by dihydroethidium fluorescence) from the steady state at 0, 5, and 30 min and gradually increasing from 1 h to the maximum at 12 h, and returning to baseline at 24 h. Ang II-dependent proliferation was blocked by the antioxidant N-acetyl-L-cysteine. Confocal microscopy revealed the presence of two sources of intracellular ROS in C17.2 cells: (i) mitochondrial and (ii) extramitochondrial; the latter indicative of the involvement of one or more specific isoforms of Nox. Of the Nox family, mRNA expression for one member, Nox4, is abundant in neural stem cell cultures, and Ang II treatment resulted in elevation of the relative levels of Nox4 protein. SiRNA targeting of Nox4 mRNA reduced both the constitutive and Ang II-induced Nox4 protein levels and attenuated Ang II-driven increases in superoxide levels and stem cell proliferation. Our findings are consistent with our hypothesis that Ang II-induced proliferation of neural stem cells occurs via Nox4-generated superoxide, suggesting that an Ang II/Nox4 axis is an important regulator of neural stem cell self-renewal and as such may fine-tune normal, stress- or disease-modifying neurogenesis.