Fig 1: Schematic of APN up‐regulating CXCL1/8 via promoting phosphorylation of AMPK and p38 MAPK in h‐JBMMSCs. APN increases chemotaxis of h‐JBMMSCs through APN‐AMPK/p38 MAPK‐CXCL1/8 pathway. The APN‐induced CXCL1/8 up‐regulation could be blocked by AMPK inhibitor, WZ4003, and the p38 MAPK inhibitor, SB203580.
Fig 2: The AMPK inhibitor, WZ4003, and the p38 MAPK inhibitor, SB203580, could inhibit the APN‐induced overexpression of CXCL1 and CXCL8 and h‐JBMMSCs chemotaxis. After treated with WZ4003 and SB203580, the expression of CXCL1 (A and B) and CXCL8 (C and D) in the cells and supernatants of h‐JBMMSCs treated with or without APN was evaluated by the ELISA assay (n = 3; *P < 0.05; **P < 0.01). (E) After treated with WZ4003 and SB203580, the chmotaxis of h‐JBMMSCs treated with or without APN was evaluated by the transwell assay (n = 3; *P < 0.05).
Fig 3: APN promoted h‐JBMMSCs chemotaxis through CXCL1/8 and activated the phosphorylation of AMPK and p38 MAPK in a time‐dependent manner. (A) SB225002 could inhibited h‐JBMMSCs chemotaxis in both the co‐culture and the monoculture transwell systems (n = 3; *P < 0.05). (B) The h‐JBMMSCs were cultured in 60‐mm dishes, then starved for 12 hrs after reaching 90% confluence and treated with 1 μg/ml APN at the indicated time‐points. Then, phospho‐p38 MAPK, total p38 MAPK, phospho‐AMPK, total AMPK and actin were examined by Western blotting. The protein concentration was also analysed using Image J software (n = 3; **P < 0.01).
Fig 4: APN promoted h‐JBMMSCs up‐regulating CXCL1 and CXCL8. (A) h‐JBMMSCs were cultured in osteoblast‐inducing conditional media with or without APN (1 μg/ml) for 1 week, and the total RNA was analysed using RNA‐seq. A total of 198 genes showing differential expression were identified, including 185 up‐regulated and 13 down‐regulated genes in the APN‐treatment group (P < 0.005). The CXCL1 and CXCL8 were selected as the target genes. To validate the results of RNA‐seq, real‐time PCR (B) and (C) ELISA were used to measure the expression of CXCL1 and CXCL8 in the control and APN‐treated groups (n = 3; *P < 0.05; **P < 0.01). (D) The exogenous CXCL1 or CXCL8 proteins were added in the lower compartment to verify its chemotaxis effect. The cell number was displayed as mean ± standard deviation (n = 3; *P < 0.05). (E) The Western blot was used to evaluate the expression of CXCR1 and CXCR2, the specific receptor of CXCL1 and CXCL8, after different treatments (n = 3; *P < 0.05; **P < 0.01).
Fig 5: SLC25A22 knockout abrogates recruitment and activation of MDSC in KRAS-mutant CRC via a CXCL1-CXCR2 axis.a Flow cytometry validation of isolated mouse MDSC (CD11b+ Gr-1+) and human MDSC (CD11b+CD33+) from the spleens of tumor-implanted syngeneic mice and PBMC humanized mice, respectively. b Conditioned medium from sgControl cells induced MDSC migration, which was impaired by SLC25A22 knockout (n = 4). Each dot represents an independent sample. c siCXCL1, but not siCXCL3, abrogated induction of MDSC migration in sgControl cell conditioned medium (n = 4). Each dot represents an independent sample. d Anti-Cxcl1 neutralizing antibody (0.25 µg/mL) suppressed MDSC migration in sgControl cell conditioned medium; but had no effect on SLC25A22 knockout cells (CT26: n = 3; Colo26: n = 4). Each dot represents an independent sample. e Recombinant Cxcl1 (1 ng/ml) rescued MDSC migration in CT26-Slc-KO and Colo26-Slc-KO conditioned medium (n = 4). Each dot represents an independent sample. f CXCR2 inhibitors SX682 (2 µM) or SB265610 (10 µM) abolished MDSC migration in sgControl cell conditioned medium (n = 4). Each dot represents an independent sample. g Tumoral CXCL1 positively correlated with MDSC infiltration in ApcMin/+KrasG12D/+ organoids (n = 10) in C57BL/6 mice, CT26 allografts (n = 9) in BALB/c mice, and DLD1 xenografts (sgControl, n = 15; SLC-KO1, n = 10) in PBMC humanized mice. Each dot represents an independent mouse. h qPCR of isolated MDSC from CT26 allografts showed that activation markers PD-L1, iNOS, and ARG1 were enhanced in intratumoral MDSC compared to that of splenic MDSC, and was abrogated in SLC25A22 knockout tumors (n = 3). Each dot represents an independent mouse. i Flow cytometry of MDSC from CT26 allografts mice showed that surface PD-L1 protein on MDSC were induced in tumors compared with spleen, which was impaired in SLC25A22 knockout tumors (spleen: n = 3; tumors: n = 10). Each dot represents an independent sample. j SB265610 suppressed the growth of CT26-sgControl allografts (n = 10) and k downregulated MDSC (n = 9), but had no effect on CT26-Slc-KO allografts in BALB/c mice. MDSC positively correlated with tumor weight (n = 36). Each dot represents an independent tumor. Data are shown as mean ± SD. Two-tailed one-way ANOVA (b—d, f, h–k). Two-tailed Student’s t test analysis for two-group comparison e. Spearman correlation test (g, k). Source data are provided as a Source Data file.
Supplier Page from Abcam for Human CXCL1 ELISA Kit (GRO alpha)