2B and C), implying a role for mAKAPα signalosomes in neuronal survival. Notably, loss of mAKAP expression caused a significant decrease in RGC viability in culture ( Fig. Two days after electroporation with mAKAP-specific siRNA, mAKAP mRNA expression was suppressed ~ 80% relative to control siRNA-treated cells ( Fig. In vitro culture and transfection of RGCs induces stress, resulting in ongoing cell death even in neurotrophic factor-rich growth media, and serves as a useful approach to mimic in vivo injury. ), RGCs were initially cultured in growth media containing BDNF, CNTF, and forskolin (to activate adenylyl cyclases and elevate intracellular cAMP levels) in this study. 2.7 Intravitreal Injection and Optic Nerve Crush Over 300 cells per condition for each of three biological replicates were assayed in each experiment a fourth experiment with unexplained low survival in all conditions was excluded from analysis. After fixation in 4% PFA for 30 min and washing with PBS 3 times, RGCs were stained for βIII-tubulin and analyzed using ImageJ Neurite Tracer or by Cellomics automated microscopy to quantify neurite length. For neurite outgrowth assays, RGCs were cultured in growth media for 3 days. RGC survival was expressed as the ratio of total calcein-positive cells to calcein- and sytox-positive cells. For survival assays, RGCs were cultured for up to 3 days in growth media and then stained with live cell marker calcein (C3100MP, Life Technologies), dead cell marker sytox (S11368, Life Technologies), and Hoechst nuclear dye (H1399, Life Technologies). To investigate the role of mAKAP in various neurotrophic signaling pathways we also cultured siRNA transfected RGCs in growth media containing either forskolin (5 μM) alone or forskolin with BDNF (50 ng/mL), CNTF (10 ng/mL), EGF (10 ng/mL), or FGF (20 ng/mL). RGCs were then plated at low density (3000–5000 cells/well) on 48-well plates in growth media containing forskolin, BDNF and CNTF. The Lancet Regional Health – Western Pacific. The Lancet Regional Health – Southeast Asia.The Lancet Gastroenterology & Hepatology.
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