2.14 shRNA transfection
The Gal-3 knockdown lentiviruses (Hanheng China) were transiently transfected into the cells using a transfection reagent (Beyotime China) following the manufacturer’s instructions. Transfection efficiency was evaluated by harvesting the cells after 3 days. The sequence of Gal-3 shRNA and control is presented in Table 2. Stable HT22 cell lines with Gal-3 knockdown were established through lentivirus infection and subsequent selection with 5ug/ml puromycin (Sigma, China) for approximately one week. Finally, the stability of the generated cell lines was confirmed by Western blot analysis and qPCR.
Mice were anesthetized with intraperitoneal injection of chloral hydrate (400 mg/kg, Sigma) and secured onto a stereotactic frame (RWD, Shenzhen, China). The rectal temperature was maintained at 37.5°C using a heating blanket. A midline scalp incision was performed to fully expose the bregma and lambda. For intracortical injections, a borehole was created on the left side using a high-speed drill at the following coordinates relative to bregma (X=1.0 mm, Y=0.80 mm). The syringe was connected to a microinjection pump and the needle was inserted into the brain through the burr hole (Z=1.5 mm from the bone surface) to infuse 1μl Gal-3 knockdown adeno-associated virus(AAV) (Hanheng China)44. The Gal-3 shRNA sequence and control are presented in Table 2. Following the surgery, the cranial defect was sealed using bone wax, and the incision was closed with sutures.