Gene expression analysis
Data for analyses and comparison of DLG2 expression between the different NB patient subgroups was imported from the R2 platform (http://r2.amc.nl). The independent NB cohorts and cell line datasets; 1): SEQC GSE49710 (microarray), 2): Versteeg GSE16476 (microarray) 3): Maris GSE89413 (RNA-Seq), 4): Versteeg GSE28019 (microarray), 5), Versteeg GSE16478 (microarray), 6): Maris GSE3960 (microarray), 7): Westermann GSE73517 (microarray), 8): Westermann GSE73515 (Methylation array), 9): Fisher GSE120650 (Methylation array); and human embryogenesis dataset 10): Yi GSE15744 (microarray). The microarray data was downloaded as the centered log2 fold change. Methylation data was downloaded as raw values.
Cell Lines and tissue culture
Human NB cell line SKNAS and NB69 were obtained from ATCC Cell Line Collection. The cell lines were maintained in RPMI 1640 supplemented with 10% FBS, 1% L-Glutamine, 1% HEPES solution and 1% sodium pyruvate. Cells were maintained at 37 °C with 5% CO2.
Plasmids, siRNAs and transfections
DLG2 (NM_001364) expression plasmids on a backbone of pCMV6-AC-GFP (catalogue # PS100010) vector were purchased from Origene Technologies. siRNA targeting DLG2 (s4122) or Silencer™ Select Negative control No. 1 siRNA (4390843) was purchased from Ambion (Thermo Fischer Scientific). SKNAS and NB69 cells were grown to 80% confluence and subsequently transfected with; DLG2 plasmid, empty vector “mock” (pCMV6-AC-GFP), si-DLG2 or scrambled control “mock”. 100 ng of plasmid DNA or 10 pmol siRNA was complexed with 0.3 µl of Lipofectamine 2000 according to the Lipofectamine 2000 reagent forward transfection protocol (Invitrogen; Thermo Fisher Scientific).
Cell viability, proliferation and cell cycle analysis
100 µl cell suspension of SKNAS and NB69 (1 × 104 cells/well) was seeded in 96-well culture plates (Corning Incorporated). After culturing to 80% confluence the supernatant was removed and transfection media was added to the cells. 48 hours post transfection, cells were counted using a 60 µm sensor for the Scepter handheld cell counter (Millipore) as per the manufactures instructions (27). Cell proliferation was measured using the MTS/MPS Cell Titer 96® One solution Reagent (Promega) and detecting the color variation (FLUOstar Omega, BMG Labtech) as per the manufacturer’s recommendations. The absorbance values were normalized to the mock transfection and expressed as a percentage. All experiments were repeated three times.
Cell cycle analysis was performed using the Cell-clock cell cycle assay (Biocolor). Images were subsequently analyzed using Image J image analysis as per the manufacturer’s instructions. The data presented is the average of three biological replicates. Each experiment series was repeated in triplicate.
Fly Strains and crosses
Commercially available control white (w1118) flies, UAS-dMYC and UAS-RNAi-dlg1 flies were crossed with da-GAL4 driver strain to ubiquitously force or silence gene expression, all strains were obtained from the Bloomington stock center (Bloomington). 20 female da-GAL4 flies were crossed with 10 UAS-transgenic flies or control flies and the progeny incubated at 25 °C on standard fly media. Five of the progeny were harvested after 72 hours during the third instar larvae phase. Each cross was controlled using the inverse cross using 20 UAS-transgenic female flies crossed with 10 da-GAL4 male flies.
Protein analysis by Western blot
Protein was extracted from the transfected cells in 96 well plates (1 × 104 cells/well), by aspirating the media and incubating on ice for 5 minutes then adding ice cold RIPA buffer (Thermo-fisher Scientific, 89901). For fly protein extractions 5 larvae were homogenized in RIPA buffer, followed by centrifugation. Western blot analysis was performed using a Mini-PROTEAN® TGX™ 8–20% gradient gel (Bio-Rad), protein was blotted onto LF-PVDF membrane (8 minutes, 25V and 2.5A) using a Trans-Blot® Turbo™ Transfer System (Bio-Rad). Blots were subsequently blocked for 1 hour in 5% milk in TBST buffer (0.1% Tween-20 and 150 mM NaCl in 10 mM Tris–HCL, pH 7.4) as per the manufacturer’s recommendations. Blots were probed overnight at 4 °C with antibodies diluted in PBST (0.1% Tween-20 in PBS). Primary antibodies; dmDLG (4F3, anti-discs large, Goodman, C. ), Cyclin A (A12, Lehner, C.F.), Cyclin B (F2F4, O’Farrell, P.H.) and α-tubulin (12G10, Frankel, J. / Nelsen, E.M. ) were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. All primary antibodies were diluted to 0.5 µg/ml in PBST 0.1%. The secondary antibody used for detection was Starbright goat anti-mouse 1:5000 (12004159, Bio-Rad). All wash stages were 3 × 10 minutes in TBST 0.1%. Secondary antibodies were incubated for 1 hour at room temperature. Image detection was performed on ChemiDoc MP (Bio-Rad).
Quantitative PCR analysis
RNA from NB cell lines was extracted with RNeasy Kit® (Qiagen) according to manufacturer’s protocol. RNA was quantified by NanoDrop (NanoDrop Technologies) and 2 µg of RNA was reverse transcribed into double stranded cDNA on a T-professional Basic Gradient thermal cycler (Biometra) using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). cDNA corresponding to 20 ng of RNA was used for each qPCR reaction. qPCR was performed on a Pikoreal qPCR System (Thermo Fischer Scientific) in triplicate for TaqMan target transcripts; DLG2 (Hs00265843_m1) and dmDLG (Dm01799281_g1) using TaqMan™ Gene Expression Master Mix (4369016, Applied Biosystems). Quantitative gene expression data were normalized to the expression levels of the human reference genes GAPDH (Hs02758991_m1), GUSB (Hs99999904_m1) and fly reference gene Rpl32 (Dm02151827_g1).
Gene set enrichment analysis
To identify the pathways to which DLG2 expression is correlated with, the previously described independent datasets; 2, 3 and 10 were selected from the R2 platform (http://r2.amc.nl). A gene expression list was derived based on the correlation to DLG2 expression, normalized to z-score. To further investigate enriched pathways, the non-NB developmental dataset 10 was selected for additional comparison. The top 10 enriched KEGG pathways were subsequently shown for each dataset using the R2 platform. Data was corrected for multiple corrections using the false discovery rate (FDR). Concordance between the datasets was shown using Venn diagrams produced with the geneVenn tool (genevenn.sourceforge.net).
All data presented are plotted as Tukeys box and whisker plots showing IQR, line at the median, + at the mean with whiskers ± 1.5-fold of interquartile range from at least 3 independent experiments. For all multi-group analyses, differences were determined by one way ANOVA test followed by Holm-Sidak’s multiple comparison test. For comparisons between two groups a Mann-Whitney U test was used: *p < 0.05, **p < 0.01, ***p < 0.001. All analyses were conducted using GraphPad Prism version 8.0.1 for Windows, (GraphPad Software, www.graphpad.com).