Fig 1: 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.
Fig 2: CD200R signaling regulates chemokine expression in TME(A) Differential gene expression analysis of the scRNA-seq data revealed significantly altered overall expression of chemokine genes in NB9464D tumors from CD200R−/− mice versus tumors from WT mice.(B) Violin plots for the expression of chemokine genes that show significant differences between tumors from WT and CD200R−/− mice. ∗∗∗P < 1e−100, ∗∗P < 1e−10 by Wilcoxon rank-sum test.(C and D) qPCR analyses for the expression of chemokine genes in NB9464D (C) and Yummer1.7 (D) tumors from WT and CD200R−/− mice. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-sided student t test.(E) ELISA assay for production of chemokines in NB9464D tumor lysates. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-sided student t test.(F) Featured plot data of scRNA-seq suggests that CCL24 and CCL8 were mainly expressed by TAMs, while CCL3, CXCL2 and CXCL3 were mainly expressed by neutrophils, but also by TAMs.(G) Differential gene expression analysis of the scRNA-seq data revealed altered expression of chemokine genes in TAMs and Neutrophils.(H) qPCR was used to quantify expression of chemokine genes in sorted CD11b+Ly6G− and CD11b+Ly6G+ cells from NB9464D tumors from WT and CD200R−/− mice.
Fig 3: Cxcr2 inhibition blocks eLC wound repopulation. (A) Heatmap of normalized log2 fold change of chemokine receptor gene expression from homeostatic epithelial (KTC) and Langerhans cells (LC). Cells isolated through FACS and sequenced through bulk RNA‐seq. Each column represents an independent sample, and each row is assigned to a specific gene. Red indicates maximum expression and blue indicates minimum expression. n = 4 mice (B) Experimental design for drug treatment. Starting on wound induction day, drug was injected once a day intradermally at the ear near the wound site. Control mice received vehicle (1% DMSO) injections. Wounds were imaged at wound closure (5 days after wound induction), and candidate drugs were further analyzed for migration dynamics through time‐lapse at 2 days after wound induction. Revisit imaging (camera icon) was performed on Day 0 and 5. Timelapse imaging (video icon) was performed on Day 2. (C) Mean LC number comparing cell density at the wound in response to drug treatment. Imaging was performed 5 days after wound induction. LC density normalized to the individual mouse wound area was quantified. Data analyzed using unpaired one‐way ANOVA; n = 6 control, n = 4 BX471, n = 4 INCB3344, n = 4 DAPTA, n = 3 Cenicriviroc, n = 5 Danirixin, n = 5 SB225002, and n = 4 CXCR3 antagonist‐treated mice.; data are mean ± s.d. with each dot representing individual mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) In vivo microscopy images show x‐y view of epithelial cells (red nuclei) and LCs (green) in the epidermis 5 days after wound induction. Top: control mouse (1% DMSO). Middle: CXCR2‐inhibited mouse (Danirixin). Bottom: CXCR2‐inhibited mouse (SB225002). Right: zoomed view of the wound center matching the image on the left. Dashed line indicates initial wound boundary. Representative images are shown. n = 6 control mice and n = 5 mice per drug‐treated group. Scale bars, 100 µm. (E) Confocal immunofluorescent images of CXCR2 expression at the epidermis during homeostasis and 2 days after wound induction. Images show x‐y view of LCs (green, MHC‐II), CXCR2 (red), and cell nuclei (blue, DAPI). Right: zoomed view in composite, green channel only, and red channel only. Dashed line indicates initial wound boundary. Representative images from 3 mice. Scale bars, 25 µm. (F) qRT‐PCR gene expression analysis of CXCR2 ligands in the skin during homeostasis (control) and 2 days after wound induction. Data analyzed using multiple unpaired two‐tailed t‐test; n = 5 mice; data are mean ± s.d. with each dot representing individual mice. **p < 0.01. (G) ELISA assay of CXCL1 present on wounds treated with CXCR2‐inhibitor (Danirixin) compared to control (DMSO) and homeostasis. (H) ELISA assay of CXCL2 present on wounds treated with CXCR2‐inhibitor (Danirixin) compared to control (DMSO) and homeostasis. (I) ELISA assay of CXCL3 present on wounds treated with CXCR2‐inhibitor (Danirixin) compared to control (DMSO) and homeostasis. (J) ELISA assay of CXCL5 present on wounds treated with CXCR2‐inhibitor (Danirixin) compared to control (DMSO) and homeostasis. (K) ELISA assay of CXCL7 present on wounds treated with CXCR2‐inhibitor (Danirixin) compared to control (DMSO) and homeostasis. (G–K) Data analyzed using unpaired one‐way ANOVA; n = 4 mice; data are mean ± s.d. with each dot representing individual mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Supplier Page from Abcam for Mouse GRO gamma ELISA Kit (CXCL3)