Figure 4

Prazosin induces SQSTM1 puncta and MAP1LC3B lipidation in hESC-derived motor neurons
(A) Representative immunocytochemistry images showing ISLET1 and TuJ1 staining in control hESCs-derived motor neurons (MNs), either untreated (NT) or treated with 10 μM prazosin for 24 h. The percentage of ISLET1+ cells is presented as a graph based on data obtained through automated image acquisition and analysis. Scale bars, 50 μm. Data are presented as mean ± SD from two independent differentiations from MN progenitors.

(B and C) Representative immunocytochemistry images showing ISLET1+ nuclei and SQSTM1 puncta staining in hESC-derived MNs after 24-h treatment with 10 μM prazosin. The number of SQSTM1 puncta per cell was quantified using automated microscope acquisition and analysis. Data are presented as mean ± SD from two independent differentiations from MN progenitors. Scale bars, 10 μm.

(D and E) Western blot analysis of MAP1LC3B modification and ACTB expression in control hiPSC-derived MNs treated with 10 μM prazosin for 24 h and bafilomycin (BAF) 400nM, 4 h before prazosin treatment ending. The displayed image represents one of the three independent differentiations from MN progenitors. Data are presented as mean ± SEM from three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons post hoc test. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, and ^∗∗∗p ≤ 0.001.