Overexpression of ANL, 5SANL, NHB + A, or NHB + 5SA in CTX and VM astrocytes results in direct reprogramming into dopaminergic neurons (iDANs), as demonstrated by immunocytochemistry (ICC). (A) Timeline for iDAN direct reprogramming. (B) The ICC results for iDAN direct reprogramming from rat CTX astrocytes on days 3, 5, 7, and 14. iDANs stained with anti-TH (red) and anti-TUJ1 (green) antibodies. When co-expressed, NURR1 enables direct iDAN reprogramming in rat CTX astrocytes. EGFP (ECL), ASCL1 + NURR1 + LMX1A (ANL), ASCL15SA + NURR1 + LMX1A (5SANL), NURR1 + SHH + BclXL + ASCL1 (NHB + A), and NURR1 + SHH + BclXL + ASCL15SA (NHB + 5SA). (C) Counts of TH+/TUJ1+ cells following direct reprogramming of rat CTX astrocytes on days 3, 5, 7, and 14. A significant increase in TH+/TUJ1+ cells is confirmed across all groups. In particular, the ANL group shows the greatest increase in TH cells on day 5 of differentiation, followed by a decline. (D) ICC of iDAN direct reprogramming from rat VM astrocytes on days 3, 5, 7, and 14. iDANs stained with anti-TH (red) and anti-TUJ1 (green) antibodies. Rat VM astrocytes can also directly undergo iDAN reprogramming when NURR1 is co-expressed. (E) On days 3, 5, 7, and 14 of direct reprogramming of rat VM astrocytes, the number of TH+ and TUJ1+ cells was determined. Although the increase in the number of TH+/TUJ1+ cells is lower than that of CTX astrocytes in all groups, they show a significant increase in the ANL and 5SANL groups. *P < 0.05. **P < 0.01. ***P < 0.001. The error bar depicts the standard error of the mean. Scale bar, 20 μm.
