Transport parameters and shape aspect ratio for Halo-tagged WT and SFPQY527A. (A) Left: Expression levels of Halo-tagged WT and SFPQY527A in CB compartment from neurons simultaneously infected with shRNA against endogenous SFPQ. Neurons were grown in Campenot compartmented cultures and CB lysate was collected. Actin serves as loading control (Ctrl). Right: Relative Halo-SFPQ (normalized to actin) is quantified. *P = 0.0149; data are from three independent experiments; data represent mean ± SEM. (B) Percentage of time spent in stationary, anterograde, or retrograde phase for WT and SFPQY527A. (C) Average anterograde velocity and run length for WT and SFPQY527A. (D) Average retrograde velocity and run length for WT and SFPQY527A. ****P < 0.0001. Data from B–D are analyzed from 258 granules for WT control and 202 granules for SFPQY527A from at least 30 axons across at least four independent microfluidic experiments. (E) Shape aspect ratio (maximum diameter/minimum diameter) of SFPQY527A granules calculated for stationary and moving granules, categorized with their instantaneous velocities as indicated. Data are calculated from 15 SFPQ granules from five axons from three independent microfluidic experiments for time points in stationary (n = 14), ≤0.5 (n = 14), 0.5–1 (n = 28), 1–2 (n = 48), 2–3 (n = 24), and >3 µm/s (n = 6). ****P < 0.0001; **P = 0.0061 by one-way ANOVA. (F) Comparison of shape aspect ratio between WT SFPQ and SFPQY527A granules moving in anterograde (top) or retrograde transport (bottom). Data for WT anterograde granules are calculated from six SFPQ granules from five axons from five independent microfluidic experiments for time points in ≤0.5 (n = 5), 0.5–1 (n = 12), 1–2 (n = 12), 2–3 (n = 13), and >3 µm/s (n = 9). Retrograde: Data are calculated from 15 SFPQ granules from five axons from five independent microfluidic experiments for time points in ≤0.5 (n = 28), 0.5–1 (n = 17), 1–2 (n = 16), 2–3 (n = 12), and >3 µm/s (n = 2). Data for SFPQY527A anterograde granules are calculated from six SFPQ granules from two axons from two independent microfluidic experiments for time points in ≤0.5 (n = 8), 0.5–1 (n = 12), 1–2 (n = 22), 2–3 (n = 13), and >3 µm/s (n = 3). Retrograde: Data are calculated from eight SFPQ granules from three axons from three independent microfluidic experiments for time points in ≤0.5 (n = 6), 0.5–1 (n = 16), 1–2 (n = 26), 2–3 (n = 11), and >3 µm/s (n = 3).
Figure S4.

Transport parameters and shape aspect ratio for Halo-tagged WT and SFPQY527A. (A) Left: Expression levels of Halo-tagged WT and SFPQY527A in CB compartment from neurons simultaneously infected with shRNA against endogenous SFPQ. Neurons were grown in Campenot compartmented cultures and CB lysate was collected. Actin serves as loading control (Ctrl). Right: Relative Halo-SFPQ (normalized to actin) is quantified. *P = 0.0149; data are from three independent experiments; data represent mean ± SEM. (B) Percentage of time spent in stationary, anterograde, or retrograde phase for WT and SFPQY527A. (C) Average anterograde velocity and run length for WT and SFPQY527A. (D) Average retrograde velocity and run length for WT and SFPQY527A. ****P < 0.0001. Data from B–D are analyzed from 258 granules for WT control and 202 granules for SFPQY527A from at least 30 axons across at least four independent microfluidic experiments. (E) Shape aspect ratio (maximum diameter/minimum diameter) of SFPQY527A granules calculated for stationary and moving granules, categorized with their instantaneous velocities as indicated. Data are calculated from 15 SFPQ granules from five axons from three independent microfluidic experiments for time points in stationary (n = 14), ≤0.5 (n = 14), 0.5–1 (n = 28), 1–2 (n = 48), 2–3 (n = 24), and >3 µm/s (n = 6). ****P < 0.0001; **P = 0.0061 by one-way ANOVA. (F) Comparison of shape aspect ratio between WT SFPQ and SFPQY527A granules moving in anterograde (top) or retrograde transport (bottom). Data for WT anterograde granules are calculated from six SFPQ granules from five axons from five independent microfluidic experiments for time points in ≤0.5 (n = 5), 0.5–1 (n = 12), 1–2 (n = 12), 2–3 (n = 13), and >3 µm/s (n = 9). Retrograde: Data are calculated from 15 SFPQ granules from five axons from five independent microfluidic experiments for time points in ≤0.5 (n = 28), 0.5–1 (n = 17), 1–2 (n = 16), 2–3 (n = 12), and >3 µm/s (n = 2). Data for SFPQY527A anterograde granules are calculated from six SFPQ granules from two axons from two independent microfluidic experiments for time points in ≤0.5 (n = 8), 0.5–1 (n = 12), 1–2 (n = 22), 2–3 (n = 13), and >3 µm/s (n = 3). Retrograde: Data are calculated from eight SFPQ granules from three axons from three independent microfluidic experiments for time points in ≤0.5 (n = 6), 0.5–1 (n = 16), 1–2 (n = 26), 2–3 (n = 11), and >3 µm/s (n = 3).

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