Powerplex 16 hs system

The PowerPlex 16 HS System (Promega) was used to verify sample identity for #13 and #16 (monozygotic twins) and #5 and #4 (father and child, respectively). 0.5 and 1 ng genomic DNA was used as template for the PCR amplification according to the manufacturer’s protocol. Fragments were resolved on an ABI 3730xl DNA analyser (Thermo Fisher Scientific). Analysis was performed using the Applied Biosystems Microsatellite Analysis module on the Thermo Fisher Cloud.

The Calu-6 cell line was purchased from the American Type Culture collection (ATCC) and the H1650 cell line was obtained from Dr. Adi F. Gazdar (The University of Texas Southwestern Medical Center, Dallas, TX). The lung cancer cell lines were cultured in a 1:1 medium mix of Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F12 supplemented with 10% fetal bovine serum (FBS). Cells were maintained in humidified 5% CO₂ incubator. Cell lines used in the study were authenticated by short tandem repeat DNA fingerprinting using the PowerPlex 16 HS System (Promega) and the STR profiles were verified with MD Anderson fingerprint database. For experiments requiring serum starvation, serum containing cell culture media were removed, cells were then gently washed twice with 1x phosphate buffered saline (PBS) and then incubated in serum-free cell culture medium overnight. For analysis of autophagic flux, cells were cultured in serum-free medium containing Pepstatin A and EST (EMD Millipore).

Cells were screened for the absence of mycoplasma contamination using the Venor®GeM Classic kit (Minerva Biolabs). The identity of each cell line was checked before starting each experiment and after every genomic DNA extraction by PowerPlex® 16 HS System (Promega), through Short Tandem Repeats (STR) at 16 different loci (D5S818, D13S317, D7S820, D16S539, D21S11, vWA, TH01, TPOX, CSF1PO, D18S51, D3S1358, D8S1179, FGA, Penta D, Penta E, and amelogenin). Amplicons from multiplex PCRs were separated by capillary electrophoresis (3730 DNA Analyzer, Applied Biosystems) and analyzed using GeneMapper v 3.7 software (Life Technologies).

Human NB cell lines, SK-N-AS (MYCN single copy, chromosomal status of 1p36: loss of heterozygosity (LOH), derived from a 6 year old girl with a poorly differentiated NB) [55 (link)] and SK-N-DZ (MYCN-amplified, 1p status: no LOH, derived from a 2 year old girl with poorly differentiated NB) [55 (link)] were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in Dulbecco's Modified Eagle's medium (DMEM, Life Technologies, Carlsbad, CA) supplemented with 0.1 mM Non-Essential Amino Acids (NEAA, Life Technologies) and fetal calf serum to a final concentration of 10% fetal bovine serum (FBS, Life Technologies) at 37°C under 5% CO₂. Cell lines were tested for authenticity by using STR-PCR (PowerPlex 16 HS System, Promega, Madison, WI).

SH-SY5Y cells were cultured in DMEM/F-12 HEPES (1:1) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco^®, Carlsbad, CA, USA). Kelly cells were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-Glutamine (Gibco^®). Culture conditions were 37 °C in humid atmosphere with 5% CO₂. Cells were passaged when 90% confluency was reached. Routine check for mycoplasma contamination was conducted using the PCR Mycoplasma Test Kit I/C (PromoCell, Heidelberg, Germany) and STR profiling (PowerPlex 16HS System, Promega, Madison, WI, USA) was performed to verify cell lines.