Real-time PCR

We used qPCR analysis to confirm the findings of the proteome analysis on the nuclear level and to identify the cellular source of the secreted CXCL8 and CCL2. We performed the analysis on independently cultured T3M4 and NHDF cells and co-cultures (n = 3 for each of treatments i, ii, iii). We applied the same conditions and cell numbers as per the proteome array. We performed qPCR reactions in triplicate. Relative gene expression was statistically compared using a Welch non-parametric t-test.

We extracted mRNA from single cultures of T3M4 and NHDF cells using primer sets for CXCL8 and CCL2, and generated cDNA for use in qPCR. We extracted total RNA from single cultures seeded at 4 × 10⁴ cells using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). 5 µg of total RNA was transcribed using a first-strand cDNA Synthesis Kit (Promega, Mannheim, Germany) following the manufacturer’s instructions. We performed qPCR reactions with 3 µL of cDNA (dilution 1:10) per reaction of SYBR Green, using the Light Cycler 480 SYBR Green I Master (Roche, Mannheim, Germany).

We used gene-specific primers (Supplementary Methods) and quantified relative gene expression using the 2^−∆∆Ct-method⁷⁵. We normalized the numbers of replication-cycles to gain stable fluorescence (Ct-value) for each gene to the 18 s housekeeping-gene (∆Ct-value). Subsequently, we calculated the relative fold enrichment of each gene in each cell-line by subtracting ∆Ct-values for single-cell-cultures from ∆Ct-values for co-cultures (∆∆Ct-value). Finally, the value of relative expression fold change was calculated as 2^−∆∆Ct.