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. 2019 Aug 8;10(8):598. doi: 10.1038/s41419-019-1837-1

Fig. 6. The transcriptional suppression of PI3Kγ and HRAS regulated by SAP18 attenuates ERK-mediated STAT3 activation and leads to an inhibition of mo-MDSCs accumulation.

Fig. 6

a The expression of ERK1/2, p-ERK1/2, STAT3, p-STAT3, and SAP18 in the HSPCs isolated from WT, CXCR2−/−, U0126-treated WT, and U0126-treated CXCR2−/− tumor-bearing mice was determined by western blot. b The mice were treated as described in a and the percentage of mo-MDSCs in the bone marrow was analyzed by flow cytometry. c The expression of ERK1/2, p-ERK1/2, STAT3, p-STAT3, and SAP18 in the 32D clone three cells transfected with the empty vector, CXCR2, CXCR2 administrated with U0126 or CXCR2 combined with SAP18. All cells were incubated with CXCL1 and CXCL2 for 4 h. d The cells were treated as described in c and cultured with CXCL1 and CXCL2 for 5 days in the presence of GM-CSF. The percentage of mo-MDSCs was evaluated from 32D clone three cells by flow cytometry. e The expression of ERK1/2, p-ERK1/2, STAT3, p-STAT3, and SAP18 was analyzed by Western blot in the HSPCs isolated from WT control mice, CXCR2−/− control mice, WT, SAP18 over-expressing WT, U0126-treated WT, CXCR2−/−, and shSAP18 CXCR2−/− tumor-bearing mice. f The mice were treated as described in e and the percentage of mo-MDSCs was analyzed in the bone marrow by flow cytometry. g Quantitative analyses of the data described in f. h ChIP analysis using anti-SAP18 antibodies in HSPCs. HSPCs were sorted from both WT and CXCR2−/− tumor-bearing mice. i ChIP analysis using an anti-mSinA antibody in HSPCs. HSPCs were sorted from WT and SAP18 over-expressing WT tumor-bearing mice. j The expression of HRAS, PI3Kγ, ERK1/2, p-ERK1/2, STAT3, p-STAT3, and SAP18 in the HSPCs was analyzed by Western blot. HSPCs were sorted from WT control mice, CXCR2−/− control mice, WT, CXCR2−/−, SAP18 over-expressing WT and shSAP18 CXCR2−/− tumor-bearing mice. Quantitative analyses of the protein in Fig. 6 were conducted by Image J software. SAP18, HRAS and PI3Kγ were normalized over β-actin. p-ERK1/2 was normalized over ERK1/2, and p-STAT3 was normalized over STAT3. Bars represent the mean ± SD of five independent experiments. A one-way ANOVA with repeated measures followed by a Dunnett’s post hoc test or a two-way ANOVA followed by a Holm-Sidak’s post hoc test were used to evaluate the level of statistical significance (*p < 0.05; **p < 0.01; and ***p < 0.001; ns, not significant)