Kds310

Athymic BALB/c nude mice (male, 3- to 4-week-old; GemPharmatech Co., Ltd; Nanjing, China) were randomly divided into indicated groups. Luciferase-expressing cells (1 × 10⁶) were inoculated into the frontal lobe using a stereotactic apparatus (KDS310, KD Scientific; Holliston, MA, USA). Bioluminescence images were captured using an imaging system (PerkinElmer) every 5 days. When animals were displaying symptoms, such as apathy, decreased activity, severe hunchback posture, dragging legs, unkempt fur, or loss of body weight, they were killed by cervical dislocation. If necessary, animals were perfused with saline solution and 4% paraformaldehyde, and excised brains were prepared for further examination by HE and IHC staining.

Luciferase‐stable LN229 cells (5 × 10⁵) in a total volume of 10 μL of PBS were implanted into the frontal lobes of nude mice (female; 5 weeks old; Shanghai SLAC Laboratory Animal Co., Ltd, Shanghai, China) using a stereotactic device (KDS310; KD Scientific, Holliston, MA, USA). The burr hole was positioned 1 mm anterior and 2 mm lateral from the anterior fontanel, and the injection depth was adjusted to 2.0 mm. For the panobinostat treatment groups, panobinostat was administered intraperitoneally at a dose of 10 mg/kg three times a week for 4 weeks. At days 7, 14 and 21 after implantation, tumor growth was examined using bioluminescence imaging (IVIS Spectral In Vivo Imaging System, PerkinElmer; Hopkinton, MA, USA). Body weight was measured every 3 days, and the mice were sacrificed when they began to show symptoms of continuous discomfort. The brains of mice were removed, fixed, embedded in paraffin and sectioned for IHC staining.

U87MG cells were digested into single-cell and resuspended in PBS. 8-week-old nude male mice (bablc/nu, SPF level) (SLAC laboratory animal Center, Shanghai, China) (n = 12) were divided equally into 2 groups (n = 6 to each group): U87-NC and U87-sh-PHF14. Cells (3 × 10⁵) were implanted into mice brains using a stereotactic apparatus (KDS310, KD Scientific; Holliston, MA, USA). Animals were closely followed and euthanized by cervical dislocation when they exhibited symptoms, such as severe hunchback posture, apathy, decreased motion or activity, dragging legs, or drastic loss of body weight. Tumors were excised, formalin-fixed, paraffin-embedded, and sectioned for hematoxylin and eosin (HE) staining and IHC of Ki67 (Abcam, Cat# ab15580, 1:200).

Four weeks after ischemia, rats with severe learning and memory deficits were randomly assigned to 2 groups. In the transplanted group (grafted, n = 6), rats received bilateral grafts of olfactory stem cells (1,000,000 cells in total), while in the nontransplanted group (sham, n = 6), animals received an equal amount of culture medium without cells. Anesthetized rats were inserted in a stereotaxic frame, the skull surface was exposed, and holes were drilled at the appropriate site. Cell suspensions or vehicle was injected with a 1 μL Hamilton syringe connected to a stereotactic syringe pump (KDS 310, KD scientific, USA) into both hippocampi. Anteroposterior (AP), lateral (L), and vertical (V) coordinates for microinjection were taken relative to the bregma: (injection 1: AP—3.1; L ± 3; V—2.8), (injection 2: AP—3; L ± 2.4; V—3), and (injection 3: AP—3.8; L ± 2.6; V—3). The infused volume was 1 μL per injection site, and the rate of infusion was 0.5 μL/min. Rats were then allowed to recover 4 weeks until behavioral analysis.

For virus injection, mice at the age of 4–8 weeks were anaesthetized and placed in a stereotaxic frame (RWD Life Science). Viruses (the titres of all vectors>1.0 × 10¹² viral genome-containing particles per ml) were injected bilaterally into the insular cortex. The stereotaxic coordinates according to the mouse brain atlas64 were: anteroposterior, +0.98 mm; lateral, ±3.65 mm; dorsoventral, and –3.60 mm. One injection (1 μl) was made on each side of the insular cortex using microelectrodes connected with a microinjector pump (KDS 310, KD Scientific) at a rate of 0.1 μl min⁻¹. Microelectrodes were left in situ for an additional 10 min to allow the injectant to diffuse. Mice were allowed to recover for 4 weeks before behavioural analysis and the injection sites were examined at the end of the experiments. Brain slices from animals injected with the viruses were examined directly using fluorescent microscopy.

To establish an intracerebral glioma model, control and modified glioma cell lines were implanted into 4-week-old female nude mice (n = 32; Shanghai SLAC Laboratory Animal Co., Ltd, Shanghai, China). Animals were divided into four groups (eight mice per group) and inoculated into the frontal lobe using a stereotactic apparatus (KDS310, KD Scientific, Holliston, MA, USA) with one of the following cell populations (1 × 10⁶ cells): U251-NC, U251-shACTL6A, U87MG-NC, and U87MG-ACTL6A. Animals displaying symptoms, such as severe hunchback posture, apathy, decreased motion, or activity, dragging legs, unkempt fur, or drastic loss of body weight were killed by cervical dislocation. Excised tumor tissues were further examined through hemtoxylin and eosin, and IHC staining
For subcutaneous glioma model, nude mice (n = 40) were divided into eight groups (U87MG-NC, U87MG-ACTL6A, U87MG-ACTL6A + shcontrol, U87MG-ACTL6A + shYAP/TAZ, U251-NC, U251-sh-ACTL6A, U251-sh-ACTL6A + control, and U251-sh-ACTL6A + YAP-5SA/TAZ-4SA, five mice per group). Cells were resuspended in PBS/Matrigel (BD Biosciences) at a density of 10⁷ cells/ml and injected into the right shoulder of the nude mice. Tumor tissues were excised and weighed 30 days after inoculation.