Fig. 3
RvD1 regulates PMN gene expression and activity during co-incubation with cancer cell lines. a Scheme depicting in vitro PMN/cancer cell co-incubations and treatment. b Heatmap view (Morpheus, https://software.broadinstitute.org/morpheus) of gene expression levels (mean of RPKM values of n = 4–6 biological replicates pooled in 2–3 samples, respectively) of blood-derived PMN, treated with vehicle or RvD1, alone (PMN) or during co-incubation with HeLa cells (PMN + HeLa), as determined by RNAseq analysis. Color code is reported at the bottom. c Violin plot (SPSS Statistics) of gene expression patterns reporting RvD1 up- (yellow) or down-regulated (blue) genes in PMN with a fold-change cut-off of 1.5 (corresponding to 0.58 in the log2 scale reported in the figure) as compared to vehicle-treated PMN. PMN: 241 transcripts up- and 6018 down-regulated by RvD1. PMN + HeLa: 74 up, 2813 down-regulated by RvD1. d RvD1-regulated genes and significantly associated biological functions in PMN (IPA analysis). Adhesion of neutrophils p-value 4.99E-04, activation z-score − 3.057. Homing of PMN p-value 9.64E-03, activation z-score − 5.222. Chemotaxis of PMN p-value 1.45E-02, activation z-score − 5.222. Green symbols: down-regulated genes; red symbols: up-regulated genes. Blue dotted lines: expression leading to inhibition; yellow dotted lines: expression leading to activation; grey dotted lines: expression leading to unpredictable effect of function. Blue boxes: inhibited functions. e RvD1-regulated genes and biological functions related to cancer in PMN during co-incubations with HeLa cells (IPA analysis). Invasion of tumor, p-value 3.94E-02, activation z-score − 1.983. Growth of tumor, p-value 2.57E-02, activation z-score − 1.877. See above for symbol codes. f Relative HeLa cell growth during co-incubations with vehicle- or 100 nM RvD1-treated blood-derived PMN, as determined by crystal violet staining after 18 h of coincubation. HeLa cells treated with vehicle or RvD1 w/o PMN are shown for comparison. Data are expressed as fold over HeLa cells treated with vehicle n = 12. *, P < 0.05 (Mann Whitney test). g Relative growth of HeLa cells during co-incubations with freshly isolated blood-derived human PMN treated with vehicle or RvD1 (100 nM) and analyzed with impedance-based real time cell analysis (ACEA). Data are expressed as relative HeLa cell growth normalized at the time of addition of vehicle- or RvD1-treated PMN. n = 3. ****, P < 0.0001 (Wilcoxon matched-pairs signed rank test). h Relative growth of human HPV-positive head and neck cells UM-SCC-104, human lung A549 cells, and murine HPV-positive C3 cells, co-incubated with freshly isolated blood-derived human or bone marrow-derived mouse PMN. Data are expressed as fold over cancer cell lines co-incubated with vehicle-treated PMN, indicated in the graph with a dashed line, and determined by crystal violet staining after 18 h of PMN-cancer cells coincubations. n = 4 (UM-SCC-104), 8 (A549), and 3 (C3). *, P < 0.05; **, P < 0.01 (one-sample t test)