Supplementary Fig. 2 A B ** newly generated ISL1+ motor neurons (%)

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Supplementary Fig. 2 A B ** newly generated ISL1+ motor neurons (%) Proportion of Surviving pre-existing motor neurons (ISL1+/BrdU- cells) Wildtype hSOD1 (G93A) Wildtype hSOD1 (G93A)

Supplementary Fig. 2. Methylcobalamin treatment had no effect on motor neuron production in our in vitro ALS model. (A) Mouse ESC-derived motor neurons were co-cultured with wild-type or hSOD1 (G93A)-expressing astrocytes and 100 nM methylcobalamin or vehicle in the presence of BrdU for 2 days. Cells were fixed and stained with anti-ISL1 and anti-BrdU antibodies. (A) BrdU+/ISL1+ newly generated (black bar) or BrdU-/ISL1+ pre-existing motor neurons (white bar) were calculated as a percentage of total ISL1+ cell number in each condition. (B) Graphs showing the actual number of surviving -ISL1+/BrdU- pre-existing motor neurons. Each experiment was performed in duplicate as three independent experiments (n = 3). Data represent mean ± standard error of the mean. Asterisk (**) indicate statistical significance (P < 0.01), which was determined using Student’s t-test.