Fig. 6

The different states of astrocytes following Ptbp1 knockdown resulted in distinct outcomes in astrocyte-to-neuron-like cell conversion. (A) Representative images of immunofluorescence staining of non-reactive astrocytes and LPS-induced reactive astrocytes after Ptbp1 knockdown by shPTB-1 for 4 weeks, with GFP (green), astrocyte-specific marker GFAP (indigo, Alexa Fluor 555), and the neuron-specific marker Tuj1(red, Alexa Fluor 647). Notably, after LPS induction, distinct from non-reactive astrocytes (yellow arrows), reactive astrocytes (yellow arrowheads) exhibited significant morphological changes, including enlarged cell bodies and web-like structures. While most cells displayed GFP fluorescence in the LPS group, only a small fraction stained positively for Tuj1, without exhibiting a typical neuronal morphology. Scale bar: 100 μm. (B) Quantification of Tuj1⁺ cells within GFP⁺ cells at 4 weeks following Ptbp1 knockdown by shPTB-1 in non-reactive astrocytes, compared to LPS-induced reactive astrocytes (two-tailed Student’s t-test). (C) Representative images of immunofluorescence staining of LPS-induced reactive astrocytes (LPS) and DEX-reversed reactive astrocytes (LPS + DEX), with Ptbp1 knockdown by siRNA-PTB-1 after 3 weeks, or Lucid control, showing Ptbp1 (green, Alexa Fluor 488) and neuron-specific marker Tuj1(red, Alexa Fluor 647). Scale bar: 50 μm. (D) Quantification of Tuj1⁺ cells within Ptbp1^- cells at 3 weeks after transfection by siRNA-PTB-1 in DEX-reversed reactive astrocytes (LPS + DEX), compared to the LPS group (two-tailed Student’s t-test). All data are presented as means ± SD. The experiments were performed three times. ***P < 0.001. LPS: lipopolysaccharide; DEX: dexamethasone; ns: no significance; PTB: polypyrimidine tract-binding protein; Ptbp1: polypyrimidine tract-binding protein; GFP: green fluorescent protein; GFAP: glial fibrillary acidic protein; Tuj1: tubulin beta 3.