Basement matrigel

The luciferase sequence was cloned into the pLVX‐mCherry vector (Addgene, USA). Transduction and viral infection were performed as previously described.^[46] Subsequently, luciferase‐expressing Mum2B and B16F10 cells were incubated with negative control, 100 × 10⁻⁶m NAC, 50 µg mL⁻¹ Cdots, 50 µg mL⁻¹ Cdots together with 100 × 10⁻⁶m NAC, or 200 µg mL⁻¹ Cdots for 24 h. For the subcutaneous xenograft model, the pretreated Mum2B cells were suspended in 100 µL of Basement Matrigel (BD Biosciences, USA) and injected into the subcutaneous tissue of BALB/c nude mice. For the intraocular xenograft model, pretreated B16F10 cells were directly injected into the left eye of BALB/c nude mice. The tumor volume was recorded twice weekly. The animals were imaged using a VivoVision Systems Lumazone imaging system (Mag Biosystems, Tucson, AZ, USA) at days 7 and 14 postinjection. The animals were sacrificed at day 14 and the tumor volume was calculated by the following formula: tumor volume = π/6(s1 × s2 × s2), where s1 was the largest tumor diameter and s2 was the smallest tumor diameter.

Cyst assays were performed as previously described^{7 (link)}. In brief, for ADPKD cystic cell lines, 2 human lines namely OX161-c1, SKI-001, and one mouse line F1-Pkd1^−/− were used. OX161 and SKI-001 were kept at 33 °C F1-Pkd1−/− were at 37 °C. Healthily growing cells were trypsinised and re-suspended in DMEM media containing 10% FBS (no antibiotics) and counted using a haemocytometer. Subsequently 2 × 10⁴ cells per well were mixed with basement Matrigel (354230, BD biosciences). Prior to use, the Matrigel was left to thaw on ice and once cells were added it was immediately loaded onto the 96 well plate, allowing it to polymerise. 100 μl of DMEM media supplemented with 10% FBS was added to each well and cells returned to the incubator for 24 hours to allow them to recover. After 24 hours, the media was aspirated and replaced with fresh media (DMEM + 10% FBS) supplemented with either vehicle (DMSO) or curcumin (Sigma). The cells were photographed before every media change and every two days, the media was replaced with fresh media containing either curcumin or DMSO.

BE(2)-C cells were transfected for 96 h with empty, MYCN and/or ELOVL2 vectors in culture, and SK-N-AS cells were transfected for 72 h with the negative control or shRNA targeting MYCN and/or ELOVL2 vectors in culture; then, 2 × 10⁶ viable cells suspended in 200 μl Basement Matrigel (BD Biosciences) were subcutaneously implanted in the flanks of 4-week-old female nude mice (n = 5 per study group). Tumor size was measured with a calliper every 3 or 4 days. After 24 days, the animals were sacrificed, and the tumor volume was calculated by π/6(s1 × s2 × s2), where s1 was the largest tumor diameter, and s2 was the smallest tumor diameter. Experiments conformed to the regulatory standards and were approved by the local ethics committee.