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. 2023 Mar 31;14:1794. doi: 10.1038/s41467-023-37515-8

Fig. 4. Transcriptome analysis shows a derivation of TAN development in N1/N2 TANs.

Fig. 4

A The arrows in the diffusion plot of RNA velocity analysis of the TAN dataset from Fig. 3A indicates that the Smad3-KO N1 population (cluster 2, green) is derived from the Smad3-WT N2 (cluster 3, red) as shown by: B expression of neutrophil maturation markers, C expression of N1/N2 markers, and D the cell line assignment (k-NN) MetaCell network graph showing the developmental relationship between Smad3-WT and KO TAN from Fig. 3A. E Dot plot showing the transcription gradient of neutrophil development gene expression from immature P1 to the mature P8 cluster. The size and color of the circles represent the value of log2 fold change and the direction of change in expression, respectively, of neutrophil development genes (lower panel) in each cluster. F Pseudotime analysis of the TAN scRNA-seq dataset clearly visualized an extended trajectory of the Smad-WT N2 (Ccl2+) phenotype towards the Smad3-WT N1 (Tnf+) phenotype within the LLC tumor, where G Smad3-KO TANs (397/429 cells = 92.5%) preserved an N1 phenotype compared to Smad3-WT cells (199/1111 cells = 17.9%). H RT-PCR analysis of BMDN stimulated LLC conditioned medium shows increased fold change of N1 markers (iNOS, Tnf-α, and Icam1) in Smad3-KO neutrophils, while Smad3-WT neutrophils showed increased levels of N2 markers (CD206, Vegf, and Arg1) (*P < 0.05, ***P < 0.001 vs. WT-BMDN, n = 3 independent samples, one-way ANOVA). H Data represents mean ± SEM from three independent experiments. The exact P values of WT-BMDN vs. KO-BMDN are 4H. P = 0.0001 (iNOS), P = 0.0001(Tnf-α), P = 0.0149(Icam1), P = 0.001(CD206), P = 0.0001(Vegf), and P = 0.0211(Arg1). Source data are provided as a Source Data file.