Matrigel mixture

Xenografts were generated as previously described.^{47 (link)–50 (no link found, link, link)} Briefly, DU145 EV or DU145 FAM84B cells (3 × 10⁶) in 0.1 ml culture media were mixed with Matrigel mixture (BD) at 1:1 (volume : volume), and implanted (one graft/mouse) subcutaneously (s.c.) into the flank of NOD/SCID mice (6-week-old males, n = 5 per group; The Jackson Laboratory). Tumor growth was monitored through observation and palpation; tumor size was measured every 5 days using calipers. Tumor volume was calculated as V = L × W² × 0.52. Animals were sacrificed when tumors reached a volume ⩾1000 mm³. Lung metastases were produced via tail vein injection of DU145 EV or DU145 FAM84B cells (10⁶) into NOD/SCID mice. Endpoints were defined by a weight loss ⩾10%. All animal experiments were performed according to the protocols approved by the McMaster University Animal Research Ethics Board (AUP#: 16-06-24).

Transwell assay was conducted with invasion chamber (8-μm pore size) (Corning Costar, Cambridge, MA, USA) coated with 40 µl Matrigel mixture (BD Biosciences, matrigel: serum-free culture medium=1:7) under the instructions of manufacturer. All colon cancer cell lines (1×10⁵ cells) were incubated with or without LDL-C (100 µg/mL), N-acetylcysteine (NAC, 4 mM) or SkQ1 (100 nM) for indicated time at 37 °C. The cells were stained with crystal violet, and observed under a phase-contrast microscope (Carl Zeiss, Axiovert-S100, Germany). The extents of invasion were analyzed by ImageJ software (NIH).

Animal studies were approved by the Institutional Animal Care and Use Committee of Sichuan Provincial People's Hospital, Chengdu, China. The athymic BALB/c nude (nu/nu) mice aged from 4 to 6 weeks were purchased from Beijing Huafukang Bioscience Co., Inc. (Beijing, China) and were used for the in vivo tumorigenesis experiments (N = 6 per group). 1 × 10⁷ PANC-1 cells with or without ZFP91 knockdown (NC and scramble) were suspended in a 200 μL PBS and Matrigel mixture (1:1, v/v; BD, USA) were injected subcutaneously into the right axillas of individual mice. When the tumor size reached at least 5 mm in diameter, the three groups received gemcitabine treatment (10 mg/kg/week, intraperitoneally (i.p.)), with or without co-injection of ginsenoside Rg3 (10 or 30 mg/kg/week). Tumor length and width were examined with calipers twice weekly until week 4. The tumor volume was calculated by the formula: (a x b²) x 0.5, where a and b were the longest and shortest diameters, respectively. The mice were sacrificed at the end of week 4 and the tumors were subsequently removed.

Generation of xenografts and lung metastasis was carried out according to our established procedure [36] (link), [37] (link), [38] (link). LNCaP (RPMI-1640), DU145 (MEM), and PC3 (F12) cells were resuspended in 0.1 ml cell culture medium/Matrigel mixture (BD) in their respective medium with 1:1 ratio, and implanted subcutaneously into the flanks of 6 weeks-old male NOD/SCID mice (The Jackson Laboratory). Tumors growth was measured weekly with calipers, and tumor volume (mm³) were calculated using the formula V = L × W² × 0.52 with L and W being the longest and shortest diameters, respectively. Generation of LNCaP cell-derived xenografts in intact and castrated mice was performed following our published procedure [39] (link). In brief, LNCaP tumor cells were implanted as described above. Blood was collected biweekly via facial vein to monitor serum PSA with ELISA (Abcam). When tumor reached approximately 150 mm³, mice received either castration (n = 5) or no surgery (n = 5). For the generation of lung metastasis, 10⁶ DU145 cells were resuspended into 0.3 mL of PBS and injected through the tail vein of 6 weeks-old NOD/SCID mice (n = 4). Lungs were harvested at 10 weeks post-injection. All animal work was carried out according to experimental protocols approved by the McMaster University Animal Research Ethics Board.