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. 2020 Sep 19;29(18):3107–3121. doi: 10.1093/hmg/ddaa183

Figure 3.

Figure 3

The effects of RAN downregulation on the integrity and function of the nuclear envelope. A53T hiPSC-derived MD NPCs and the isogenic corrected control were transduced with lentiviral vector harboring shRNA–RAN followed by 72 h induction with doxycycline. (A) Downregulation of RAN was confirmed by western-blot analysis using anti-RAN antibody (25 kDa) normalized to β-actin (42 kDa). (B) RAN protein levels were quantified in two independent western-blot experiments. Lower levels of RAN protein were detected in the A53T hiPSC-derived MD NPCs and the isogenic corrected control. (C) The levels of α-syn in the nucleus were measured by immunocytochemistry and no significant changes were observed upon downregulation of RAN protein in both the A53T hiPSC-derived MD NPCs and the isogenic corrected control. (D-M) The effect of downregulation of RAN on the integrity of the nuclear envelope was evaluated by immunocytochemistry for Lamin B1 (D-H) and Lamin A/C (I-M) in the A53T hiPSC-derived MD NPCs and the isogenic corrected control. The expression of shRNA-RAN was validated using dsRed reporter; thus, for these set of experiments, we used green fluorescent conjugated secondary antibody to detect the staining of Lamin B1 (D-G) and Lamin A/C (I-L). Percentages indicate the proportion of cells with folded/blebbed nuclear morphology, white arrows denote abnormal nuclei (I-L). (H) The nuclear circularity data are plotted as frequency distributions of for 250 cells (n = 2). (M) Percentages indicate the proportion of cells with folded/blebbed nuclear morphology. Bars represents mean ± SEM (n = 2). (A-B) *P < 0.05, **P < 0.001, Student’s t-test. Scale bars: 10 μm; (H) *P < 0.05, Kolmogorov–Smirnov test.