Fig. 4

CCL5-dependent fibroblast recruitment is mediated by SLC25A24 in fibroblasts. a Heatmap of the differentially expressed genes in the transcriptome sequencing of CCD-18Co before and after 40 ng/ml CCL5 stimulation for 24 h. Data, n = 3. b Relative expression of SLC25A24 mRNA in CCD-18Co and human primary normal colorectal fibroblasts (NF1, NF2, NF3, NF4, NF5, NF6, NF7, NF8, NF9) before and after 40 ng/ml CCL5 stimulation for 24 h. Data, mean ± SD; n = 3. c Representative α-SMA and SLC25A24 IF images at the invasive front of human CRC tissue without (the upper panel) or with (the lower panel) tumor budding trend. Data, n = 10. Scale bar, 100 μm. d Representative SLC25A24 IHC staining images of mouse subcutaneous tumors with the injection of SW620/shCtrl or SW620/shCCL5. Data, n = 3. Scale bar, 50 μm. e Model diagram of recruitment assay of 40 ng/ml CCL5 to SLC25A24 knockdown fibroblasts. f Immunoblots for SLC25A24 protein expression of CCD-18Co/siCtrl and CCD-18Co/siSLC25A24, NF4/siCtrl and NF4/siSLC25A24, NF7/siCtrl and NF7/siSLC25A24. g Quantification of cell numbers of CCD-18Co/siCtrl and CCD-18Co/siSLC25A24, NF4/siCtrl and NF4/siSLC25A24, NF7/siCtrl and NF7/siSLC25A24 recruited by 40 ng/ml CCL5. Data, mean ± SD; n = 5. h The differentially expressed genes in the transcriptome sequencing were analyzed by KEGG enrichment analysis. i Immunoblots for SLC25A24, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR protein expression in CCD-18Co before and after different concentration of CCL5 stimulation for 24 h (the left panel) and those proteins in CCD-18Co/siCtrl and CCD-18Co/siSLC25A24 after 40 ng/ml CCL5 stimulation for 24 h (the right panel). SLC, SLC25A24; p-PI3K, phosphorylated PI3K; p-Akt, phosphorylated Akt; p-mTOR, phosphorylated mTOR. ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Student’s t test (b, g)