Extended Data Fig. 5: Heterogeneity of neutrophils and validation using in vitro models.
a, Colony formation assay, in which control dHL-60 cells, NFKB1-overexpressing dHL-60 cells, and STAT4-overexpressing dHL-60 cells were cultured in the upper chamber, and SW480 (left upper) or HC116 cells (left bottom) were cultured in the lower chamber. Images are representative of n = 3 experiments.Bar plot showing the number of SW480 cells (right upper) or HC116 cells (right bottom) in the colony formation assay. p values were calculated using an unpaired two-sides t-test, n=3 biological replicate.b, Migration assay, SW480 cells or HCT116 cells co-cultured with control dHL-60 cells, NFKB1-overexpressing dHL-60 cells, and STAT4-overexpressing dHL-60 cells for 3 days. Their migration capacity was assessed using the Transwell assay. Images are representative of n = 3 experiments. p values were calculated using an unpaired two-sides t-test, biological replicate=3. c, Cytokine assays showing the relative concentration of cytokines in cancer cells co-cultured with NFKB1-overexpressing dHL-60 cells or STAT4-overexpressing dHL-60 cells (biological replicate=3) error bars represent mean +/- SD; P values were determined using an unpaired two-sided Wilcoxon test. d, Their migration capacity was assessed using the migration assay. HCT116 cells co-cultured with control neutrophils, NFKB1-overexpressing neutrophils, and STAT4-overexpressing neutrophils for 3 days. Images are representative of n = 3 experiments. Bar plots showing the migration cells number of HCT116 cells (biological replicate=3). Error bars represent mean +/- SD; P values were determined using an unpaired two-sided t-test. e, Colony formation assay, in which control neutrophils, NFKB1-overexpressing neutrophils, and STAT4-overexpressing neutrophils were cultured in the upper chamber, and HCT116 cells were cultured in the lower chamber. Images are representative of n = 3 experiments.(Empty vector VS copGFP-NFKB1 p = 0.0097; Empty vector VS copGFP-STAT4 p = 0.0353) p values were calculated using an unpaired two-sides t-test, biological replicate=3.f, The gene expression of NLRP3 and TNFSF10 in neutrophils in scRNA-seq analysis. g, qPCR for assessing the activation status of two transcription factors. Relative expression of NFKB1 and STAT4 in two sorted neutrophils and control neutrophils without sorted(biological replicate=4); Error bars represent mean +/- SD; P values were determined using an unpaired two-sided t-test. h, Top panel showing HCT116 cells (left panel) and SW480 cells (right panel) co-cultured with control neutrophils, TNFSF10 + NLRP3- neutrophils, and NLRP3 + TNFSF10- neutrophils for 3 days. Their migration capacity was assessed using the Transwell assay (middle panel). Images are representative of n = 3 experiments. p values were calculated using an unpaired two-sides t-test, biological replicate=3.Bar plot showing the comparison of number of cell migration and colonies among three groups (bottom panel). Plots represent 10 validations, and error bars represent mean +/- SD. p values were calculated using an unpaired two-sides t-test. Data are presented as mean values +/- SD.
