Supplementary File Supplementary Methods

Supplementary File Supplementary Methods In vitro steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) NCI-H295R cells were incubated in 6-well plates in 1 ml of serum-free DMEM/Ham’s F-12 medium (Gibco, Thermo Fisher), supplemented with 1% penicillin-streptomycin and 1% ITS universal cell culture premix. Serum-free media was used, as serum itself contains steroids which may confound results. Media was collected after 48-hour incubation in silanized glass tubes and stored at -20oC. To extract steroids from the media, 20 l of serum steroid internal standard solution was transferred to each tube, followed by 3 ml Methyl tert-butyl ether (MTBE, Sigma-Aldrich). After vortexing, samples were frozen at -20oC for at least 1 hour. The top layer (liquid phase) was transferred to a 96-well plate using Pasteur pipettes. MTBE was evaporated to dryness at 55 oC and samples were reconstituted in 125 l of 1:1 H2O/methanol. Steroid metabolites were identified and quantified by LC-MS/MS, with reference to a linear calibration series and appropriate internal standards as described previously (AcquityTM Ultra Performance Liquid Chromatographer, Xevo TQ Mass Spectrometer) (1, 2). RNA sequencing – NCI-H295R NNT knockdown models RNA samples from KD siRNA, SCR SiRNA (72 hours post-transfection), KD shRNA and SCR shRNA cells (in triplicates) were prepared using the RNeasy Mini kit (Qiagen). NNT protein knockdown was confirmed independently by Western Blotting. Libraries were generated using the TruSeq Stranded mRNA Library Prep Kit (Illumina). RNA was quantified using Qubit® RNA BR Assay Kit and if necessary diluted to 25-100 ng in 12.5 l. This was followed by RNA quality check to establish the RIN (RNA Integrity Number) using the Agilent RNA ScreenTape system. RNA was then processed on the automated Illumina Neoprep 1

system following the Library prep TruSeq Stranded mRNA Library Prep Kit for NeoPrep. Libraries were normalized to 10 nM by the Neoprep. Each library quantity was checked again using Qubit DNA HS Assay Kit and 2 l of each library were pooled together into a single tube. This pool of 16 samples was checked on the Agilent High Sensitivity D1000 ScreenTape to ensure the libraries were the correct size at 300 bp. The pooled sample was diluted to 4 nM. The 4 nM library (containing the 16 pooled libraries) was sequenced on a NextSeq500 using a NextSeq® 500/550 High Output Kit v2 (150 cycles) with a 1% PhiX control spiked in. Data Quality control was performed with FastQC v0.11.4 (RRID:SCR_014583), revealing no appreciable technical biases. RNA-seq reads were mapped to the human genome (hg19, UCSC annotation) using STAR software v2.4.2a (RRID:SCR_015899) (3) supplied with default parameters. Counts per gene were calculated using the htseq-count tool from the HTSeq v0.6.1p1 package (RRID:SCR_005514) (4) with the

following parameters: --format=bam --minaqual=10 --

stranded=reverse --mode=union. Differentially expressed genes were identified using the DESeq2 v1.14.1 package (RRID:SCR_015687) (5) from Bioconductor release 3.3. Differentially expressed genes were called at a false discovery rate of 5%. Adjusted p-values for the KD-shRNA vs SCR-shRNA pairwise comparison were re-calculated using fdrtool (6). Pathway Analysis was carried out using GAGE (7) v2.22 package from Bioconductor release 3.3, referencing KEGG pathways (RRID:SCR_012773) and assessing gene sets towards both single directions (up-/down-regulated) and both directions simultaneously (bi-directional). Differentially regulated pathways were called at a p value of 20% or detected in less than 60% of the QC samples being removed (12). Univariate and multivariate data analysis and pathway enrichment analysis were performed in MetaboAnalyst 3.0 (RRID:SCR_015539) (13). This included Principal Components Analysis (PCA), Mann Whitney U tests or Kruskal-Wallis tests to identify metabolites demonstrating a statistically significant change in relative concentrations between two or three biological classes. Fold changes were calculated by division of the mean peak response for one biological class by the mean peak response of the second biological class.

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Supplementary Tables and Figures

Supplementary Figure 1. A, Western Blotting confirms efficient NNT protein silencing in NCI-H295R cells transfected with anti-NNT siRNA (KD siRNA), 72-166 hours post transfection. B, Real-time PCR confirms effective NNT silencing 72 hours post siRNA transfection in NCI-H295R cells. Two different siRNAs were tried, targeting different sequences on the NNT transcriptome (KD siRNA and KD siRNA2); KD siRNA2 was used to corroborate the effects observed with KD siRNA transfection on proliferation and apoptosis. Cells transfected with scrambled, non-sense siRNA were used as controls (SCR siRNA). ***p