Lumina lt

BLI was performed at Vivoptic (UMS 3767—University of Bordeaux) using either a NightOWL II-LB 983 system (Berthold Technologies, Bad-Wildbad, Germany) or a Lumina LT (Perkin Elmer Inc., Boston, MA, USA). Mice received an intra-peritoneal injection of d-luciferin (Promega, 2.9 mg in 100 µL PBS) and were sedated 5 min later, and images were taken at 8 min. Brains, removed from euthanized animals approximately 20 min after d-luciferin injection, were placed in cold phosphate buffered saline (PBS) on a glass slide and imaged. For in vitro study, U87-iRFP+-Fluc+ cells were plated 48 h before imaging in 24-well culture plates. The culture medium was replaced by d-luciferin (6.10–4 M in 100 µL of PBS), and images were taken 5 min after adding the substrate. Bioluminescence images (1 min, 4 × 4 binning) and photographs (100 ms) were acquired successively. The bioluminescence signal was analyzed using IndiGO 2 software for NightOWL II-LB 983 apparatus and Living Image software for Lumina LT apparatus.

Female BALB/c nude mice (6 weeks old) were purchased from Ling Chang Biotechnology (Shanghai, China). The mice were randomly divided into two groups (n = 10). Briefly, 2 × 10⁷/mL shSCUBE3 Bel7404 cells or negative control Bel7404 cells were subcutaneously injected into the flanks of nude mice. The tumour volume was monitored every 3 days for 15 days by measuring the width and length of each tumour. Live animals were imaged in an IVIS 100 imaging system (Lumina LT, PerkinElmer) after using isoflurane anaesthesia on day 25. Then, the animals were euthanized with 2.5 L/min CO₂ until breathing stopped (approximately 5–8 min), and tumour samples were collected, and the weight of each tumour was determined. All the animals were treated according to the guidelines of the Institutional Animal Care and Use Committee of Chongqing Medical University.

Animal experiments was conducted in accordance with guidelines and protocols for animal care and protection. Twenty 4-week-old male BALB/c nude mice (SLAC Laboratory, Shanghai, China) were housed at pathogen-free condition and randomly divided into two groups (shCtrl and shCD52, n=5 for each group) before experiments. A549 cells (200 μL, 1×10⁷ cells/ml) were injected subcutaneously into the right forelimb axillary in nude mice and monitored the tumor formation in real time. During this period, data on mouse body weight and tumor volume (π/6×L×W×W, where L represents the long diameter and W represents the short diameter) were recorded at 5-day intervals (days 8, 13, 18, 23, 30). On the last day of breeding (30th day), the mice were anesthetized via intraperitoneal injection of 0.7% pentobarbital sodium (10 μL/g) and tumor burden was assessed under the multi-spectral living imaging system (Lumina LT, Perkin Elmer, Massachusetts, USA). Finally, the mice were sacrificed by cervical vertebrae and the tumor tissues were subjected to hematoxylin and eosin (H&E) and IHC staining for detection of Ki67 expression (anti- Ki67, 1:200, Abcam, ab16667).

A suspension of 200 µL SW1990 cells (1×10⁷ cells/mL) was subcutaneously injected into the right forelimb axillary of four weeks old Bagg’s Albino/c (BALB/c) female nude mice (Shanghai Jiesijie Experimental Animal Co., Ltd., Shanghai, China). The mice were randomly divided into two groups: shCtrl (n ═ 5) vs shCCNI2 (n ═ 5). Following tumor formation, the mice’s body weight and tumor dimensions, its long and short diameters, were measured every 3 days using digital calipers, during the experimental period. Tumor volume was calculated as π/6×L ×W ×W, where L represents the long diameter and W represents the short diameter of the tumor. On the last day of breeding (22th day), the mice were anesthetized via intraperitoneal injection of 0.7% pentobarbital sodium at a dose of 10 µL/g. Tumor burden of the mice was assessed in a flat box under the multi-spectral living imaging system (Lumina LT, Perkin Elmer, MA, USA). All mice were subsequently euthanized by cervical dislocation, and tumor tissues were surgically excised. Before storage at −80 ^∘C with liquid nitrogen, the tumor volumes were assessed with a caliper and the tumor weights were recorded. Finally, the tumor tissues were sectioned and subjected to immunohistochemical (IHC) experiments, as previously described, to examine the expression of the proliferation marker KI67 (1:200, Abcam, USA, Cat. # ab16667).