Fig 1: CXCL2 in omental adipocytes is critical for GC cell growth/migration and in vitro angiogenesis.Each graph represents the mean ± SE from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. a Gene expression of GRO family in omental adipocyte. Bar graph represents the relative ratio of each RNA expression to CXCL2 expression level, using 2-??Ct, where ?Ct indicates the difference in Ct values between each gene and ß-actin [?Ct = Ct (target gene) - Ct (ß-actin)]. Mean, 0.41 (CXCL1), 1.0 (CXCL2), 0.18 (CXCL3). b Western blotting. c Efficiency of siRNA. Omental preadipocytes were transfected with non-targeting siRNA and CXCL2. The left graph represents RNA gene expression of CXCL2 in the OmAd cells. Bar graph represents the relative ratio of siNT and siCXCL2 to control, using 2-??Ct, where ?Ct indicates the difference in Ct values between each gene and ß-actin [?Ct = Ct (target gene) - Ct (ß-actin)]. The right graph represents protein concentration of CXCL2 in the OmAd-CM. Mean, RNA level: 1.0 (control), 1.0 (siNT), 0.27 (siCXCL2); protein level: 11.0 (control), 11.0 (siNT), 1.3 (siCXCL2). d Cell growth. Shown are the relative ratios of absorbance under each condition of OmAd-CM to those under control media (n = 5). Mean, AGS: 1.0 (control), 1.2 (control OmAd-CM), 1.3 (siNT OmAd-CM), 1.1 (siCXCL2 OmAd-CM); IM95: 1.0 (control), 1.3 (control OmAd-CM), 1.2 (siNT OmAd-CM), 1.0 (siCXCL2 OmAd-CM). e Representative images of migration assay (×100). f Quantification of migration assay. Migrated GC cells were counted from averages at four microscopic fields, and each result was presented as the mean of at least three independent experiments. Each value represents the mean relative ratio of migrated GC cells under each type of OmAd-CM to those under control media. Mean, AGS: 1.0 (control), 3.8 (control OmAd-CM), 4.3 (siNT OmAd-CM), 1.7 (siCXCL2 OmAd-CM); IM95: 1.0 (control), 2.5 (control OmAd-CM), 2.9 (siNT OmAd-CM), 0.7 (siCXCL2 OmAd-CM). g Representative images of EC recruitment assay (×100). h Quantification of EC recruitment assay. Migrated HMVECs were counted from averages at four microscopic fields, and each result was presented as the mean of at least three independent experiments. Each value represents the mean relative ratio of migrated HMVECs co-cultured with GC cells treated with each type of OmAd-CM to those co-cultured with GC cells treated with control media. Mean, AGS: 1.0 (control), 1.4 (control OmAd-CM), 1.6 (siNT OmAd-CM), 0.7 (siCXCL2 OmAd-CM); IM95: 1.0 (control), 1.8 (control OmAd-CM), 2.2 (siNT OmAd-CM), 1.0 (siCXCL2 OmAd-CM). i Representative images of tube-formation assay (×100). j Quantification of tube-formation assay. Each value represents the mean number of branched tubes under each condition. Mean, AGS: 4.0 (control), 9.0 (siNT OmAd-CM), 3.3 (siCXCL2 OmAd-CM); IM95: 2.0 (control), 7.0 (siNT OmAd-CM), 2.8 (siCXCL2 OmAd-CM). k Semi-comprehensive analysis for angiogenic factors. RNA was extracted from both AGS and IM95 cells before and after OmAd-stimulation. Each bar represents the relative ratios of each gene expression in OmAd-treated GC cells to those in non-treated GC cells. l VEGFA expression in GC cells. Relative ratios are expressed with 2-??Ct, where ?Ct indicates the difference in Ct values between each gene and ß-actin. Mean, AGS: 1.0 (control), 1.8 (control OmAd-CM), 2.1 (siNT OmAd-CM), 1.4 (siCXCL2 OmAd-CM); IM95: 1.0 (control), 2.1 (control OmAd-CM), 2.4 (siNT OmAd-CM), 1.9 (siCXCL2 OmAd-CM). m AKT phosphorylation and HIF1a expression in GC cells. Each protein extracted from GC cells incubated with control media, OmAd-CM, siNT OmAd-CM or siCXCL2 OmAd-CM for 24 h was immunoblotted with anti-phospho-AKT, anti-AKT and anti-HIFa antibodies. Each band density was quantified with Image J. ß-actin is shown as a loading control.
Fig 2: SLC25A22 knockout abrogates secretion of CXCL1 and CXCL3 in vitro and in vivo.a RNA-seq of SLC25A22 knockout DLD1 cells and gene set enrichment analysis (GSEA) for the identification of common differentially regulated pathways in SLC-KO1 and SLC-KO2 cells (n = 4). b, c GSEA enrichment scores for differentially regulated gene sets unveiled the cytokine-cytokine receptor interaction signaling pathway as the top pathway depleted in SLC25A22 knockout cells. d Inflammatory Response and Autoimmunity PCR array showed that CXCL1, CXCL3 and IL1B were induced in ApcMin/+KrasG12D/+Villin-Cre mice tumors, but were down-regulated by SLC25A22 knockout (FC > 2). e qPCR validated that SLC25A22 knockout inhibited CXCL1/3 mRNA in DLD1, CT26 and Colo26 cells (n = 3). Each dot represents an independent sample. f Antibody array showed SLC25A22 knockout down-regulated cytokine secretion in DLD1 cells. g Densitometry showed CXCL1 and CXCL1/2/3 were top-down-regulated cytokines. h ELISA confirmed SLC25A22 knockout impaired CXCL1/3 secretion in DLD1 (72 h), CT26 (24 h) and Colo26 (24 h) (n = 3). Each dot represents an independent sample. i Detection of CXCL1/3 in serum and tumors of mice implanted with CT26 allografts (left, n = 5) and ApcMin/+KrasG12D/+ organoid allografts (right, n = 10) with or without SLC25A22. Each dot represents an independent mouse. j SLC25A22 mRNA correlates with CXCL1/2/3 mRNA in TCGA CRC (COADREAD) cohort (n = 677). Each dot represents an independent patient. Data are shown as mean ± SD (e, h, i). Two-tailed one-way ANOVA (e, h, i). Two-tailed Student’s t test analysis for two-group comparison i. Pearson correlation test j. Source data are provided as a Source Data file.
Supplier Page from Abcam for Human CXCL3 ELISA Kit