Fig. 5

The roles of interested hub immune gene CCL2 in clinical predicting, testicular immune microenvironment, testicular mast cell regulation and spermatogenic dysfunction. A ROC curves of CCL2 for predicting the success of TESE surgery (samples with JS/mJS ≥ 8 versus JS/mJS < 8). B CCL2-related immune PPI network in the testis. Different colors represented the gene categories summarized from the original immune gene list. C Heatmaps of spearman correlations between GSVA scores of mast cell-related signatures and CCL2 expression level in the discovery and both validation sets. Blue and red represented positive and negative correlations, respectively. The color intensity and spot diameter indicate |correlation coefficient|. *p < 0.05, **p < 0.01, ***p < 0.001. D Negatively enriched pathway (NES < 0) in GSEA of differently expressed genes compared between high versus low CCL2 groups in spermatogenic dysfunctional samples of the discovery set, validation set1 and validation set2. GSEA was based on c2.cp.kegg.v7.4.entrez.gmt. E In vitro colony formation experiments showing colony formation ability of GC-1 (up) and GC-2 cells (down) with (100 ng/ml) or without (0 ng/ml) Ccl2 treatment. F Bar plots showing CIP of the colony formation experiments for GC-1 (up) and GC-2 (down) cell lines. ns, not significant. G, H and I Ridge plots of the top 20 KEGG pathways (according to p values) enriched in GSEA analyses comparing high versus low CCL2 expression levels in samples with spermatogenic dysfunction (discovery set, validation set 1 and validation set 2, respectively). Note: there were only in total 15 pathways enriched in the discovery set. ROC receiver operating characteristic, TESE testicular sperm extraction, GSVA gene set variation analysis, CIP colony intensity percentage