Fig. 3

Immunofluorescence staining changes during shPTB-induced astrocyte-to-neuron-like cell conversion. (A) Representative images of immunofluorescence staining of non-reactive astrocytes after Ptbp1 knockdown by shRNA after 1 week, with GFP (green), astrocyte-specific marker GFAP (indigo, Alexa Fluor 555), and neuron-specific marker Tuj1(red, Alexa Fluor 647). The lowest panels show the enlarged views of the regions outlined by the white dashed boxes in the shPTB-2 group panels. The arrowheads represent Tuj1-positive cells with slightly reduced GFAP expression after 1 week, in contrast to the Tuj1-negative cells (arrows). Scale bar: 100 μm. (B) Representative images of immunofluorescence staining of non-reactive astrocytes after Ptbp1 knockdown by shRNA for 2 weeks, with GFP (green), astrocyte-specific marker GFAP (indigo, Alexa Fluor 555), and neuron-specific marker Tuj1 (red, Alexa Fluor 647). The lowest panels show enlarged views of the regions outlined by the white dashed boxes in the shPTB-2 group panels. The arrowheads represent Tuj1-positive cells with noticeably reduced GFAP expression after 2 weeks, in contrast to the Tuj1-negative cells (arrows). Scale bar: 100 μm. (C) Quantification of Tuj1⁺ cells within GFP⁺ cells at 1, 2, 3, and 4 weeks following shPTB-1 and shPTB-2 transduction, compared to the shCtrl group. The proportion of Tuj1⁺ cells among GFP⁺ cells increased over time following transduction in both the shPTB-1 and shPTB-2 groups, reaching approximately 23% at four weeks. (D) Quantitative analysis of GFAP fluorescence intensity in GFP⁺Tuj1⁺ cells compared to GFP^-Tuj1^- cells at 1 and 2 weeks post-transduction in the shPTB-2 group (two-tailed Student’s t-test). (E) Quantitative analysis of the mean fluorescence intensity of GFAP in GFP⁺ cells from shPTB-1 and shPTB-2 group at 4 weeks, compared to the shCtrl group (one-way ANOVA followed by Bonferroni post hoc test). The expression levels of GFAP, a key element of the astrocyte cytoskeleton, significantly decreased in both the shPTB-1 and shPTB-2 groups after 4 weeks of transduction. Approximately 100 cells from each of three randomly selected non-overlapping fields were used for quantification per experiment. All data are presented as means ± SD. The experiments were performed three times. ***P < 0.001. n.d.: not detected; PTB: polypyrimidine tract-binding protein; GFAP: glial fibrillary acidic protein; Tuj1: tubulin beta 3; GFP: green fluorescent protein; w: week.