GRO alpha Antibody - #AF5403

Fig. 1| XIAOPI formula suppresses the polarization of M2 macrophages and CXCL1 expression.e XIAOPI formula dose-dependently inhibited expression of CXCL1 in M2 phenotype RAW264.7 cells. (The results were obtained from triplicate experiments and were represented as mean values ± SD.,*P < 0.05, **P < 0.01 as compared with control, ##P < 0.01, comparison between three time points of 24 h, 48 h, and 72 h)

Fig. 2 ADQ inhibits M2 phenotype polarization and CXCL1 secretion by TAMs in vitro and in vivo. a Flow cytometry assay was conducted to investigate the effect of ADQ treatment (0.7 or 1.4 g/kg/day) on TAM infiltration and polarization within the TME of breast tumors. b The expression levels of CD206 (red) and CXCL1 (green) in breast tumor tissues were detected by the tissue immunofluorescence assay. Scale bar = 5 μm. c 40 ng/ml IL-4 and IL-13 were used to induce the transformation of Raw264.7 macrophages into M2-like macrophages (TAMs). Their phenotype validation was conducted by flow cytometry. d The cell viability changes of TAMs when treated with ADQ (20–200 μg/ml) for 12–48 h were detected using the CCK-8 assay. e–f The phenotype changes of Raw264.7-derived TAMs when treated with 20–200 μg/ml ADQ for 24 h were detected by flow cytometry. G–i Raw264.7-derived TAMs were treated with ADQ (20–200 μg/ml) for 24 h. The protein expression, secretion as well as mRNA transcription levels of CXCL1 in Raw264.7-derived TAMs were detected by Western blot, ELISA, and qPCR assays, respectively. j Raw264.7-derived TAMs were treated with ADQ (20–200 μg/Ml) for 24 h. The promoter activity changes of CXCL1 gene in Raw264.7-derived TAMs were detected by the double luciferase reporter gene assay. N = 3. *p < 0.05. **p < 0.01

Fig. 2 ADQ inhibits M2 phenotype polarization and CXCL1 secretion by TAMs in vitro and in vivo. a Flow cytometry assay was conducted to investigate the effect of ADQ treatment (0.7 or 1.4 g/kg/day) on TAM infiltration and polarization within the TME of breast tumors. b The expression levels of CD206 (red) and CXCL1 (green) in breast tumor tissues were detected by the tissue immunofluorescence assay. Scale bar = 5 μm. c 40 ng/ml IL-4 and IL-13 were used to induce the transformation of Raw264.7 macrophages into M2-like macrophages (TAMs). Their phenotype validation was conducted by flow cytometry. d The cell viability changes of TAMs when treated with ADQ (20–200 μg/ml) for 12–48 h were detected using the CCK-8 assay. e–f The phenotype changes of Raw264.7-derived TAMs when treated with 20–200 μg/ml ADQ for 24 h were detected by flow cytometry. G–i Raw264.7-derived TAMs were treated with ADQ (20–200 μg/ml) for 24 h. The protein expression, secretion as well as mRNA transcription levels of CXCL1 in Raw264.7-derived TAMs were detected by Western blot, ELISA, and qPCR assays, respectively. j Raw264.7-derived TAMs were treated with ADQ (20–200 μg/Ml) for 24 h. The promoter activity changes of CXCL1 gene in Raw264.7-derived TAMs were detected by the double luciferase reporter gene assay. N = 3. *p < 0.05. **p < 0.01

FIGURE 7 |Total flavonoids from sea buckthorn fruit (TFSB) treatment suppressed the expression of cytokines, chemokines, and MUC5AC in bronchial tissues in lipopolysaccharide/cigarette smoke extract (LPS/CS)‐exposed mice. After LPS/CS exposure, mice were treated with TFSB (100, 200, and 500 mg/kg) or dexamethasone (0.25 mg/kg). After treatment of TFSB, lung tissues were used to detect the expression of (a) IL‐1β,(b) IL‐6, (c) COX2, (d) MUC5AC, and (e) CXCL1 through immunohistochemical method. (f) The quantitative results of the expression of IL‐1β, IL‐6,COX2, MUC5AC, and CXCL1 were shown, respectively. Bar = 50 μm. *p < .05, **p < .01, ***p < .001, ****p < .0001, versus LPS/CS alone, n = 4

Fig. 1 High CXCL1 expression predicts a poor prognosis in HCC, and is positively related to macrophage enrichment. A Overall survival plot of CXCL1 expression in HCC patients was analysed through the GEPIA database (n = 364). B The expression of CXCL1 in tumour tissues and adjacent nontumor tissues was measured by IHC. Arrows indicate positive staining. Scale bar, 100 μm (up) and 50 μm (down). C–E CD68 and CXCL1 staining and representative images of the same tissue. D CXCL1, CD68, and CD206 staining in the same tissues. Arrows indicate positive staining. Scale bar, 200 μm. E The relationship between CXCL1 expression and the CD206+/CD68+ cell ratio. Data are expressed as the mean ± SEM. **P 

FIGURE 3 | XIAOPI formula (XPS) inhibits M2 phenotype polarization, C-X-C motif chemokine ligand 1 (CXCL1) expression and secretion of tumor-associated macrophages (TAMs).(D–E) Western blot and QPCR results further verified that XIAOPI formula treatment for 48 h could dramatically inhibit CXCL1 protein expression levels (D) and CXCL1 mRNA transcription levels (E) in both human and mouse TAMs. (F) Double luciferase reporter gene assay suggested that XPS treatment for 48 h could suppress the promoter activity of CXCL1 gene in Raw264.7-derived TAMs. All values are presented as the mean ± SD, n = 3, **P < 0.05.

FIGURE 5 | XIAOPI formula (XPS) suppresses breast tumor growth and breast cancer stem cells (CSCs) activity in vivo. (D–E) XPS administration significantly decreased the infiltration degree of macrophages as well as their M2 phenotype polarization and C-X-C motif chemokine ligand 1 (CXCL1) expression in the 4T1-Luc xenografts in vivo. n = 3.

FIGURE 4.| Effect of autophagy on PMN recruitment and morphology. (A-C) The mRNA levels of CXCL-1 in corneas of C57BL/6 mice after 3-MA, CQ, or rapamycin treatment at 3 days p.i.; (D-F) The protein level of CXCL-1 in corneas of C57BL/6 mice after 3-MA, CQ, or rapamycin treatment at 3 days p.

Figure 3. CXCL1exo-dead 404 induces macrophage M2 polarization by activating PD-L1 expression. (A) Chemokine array assay was conducted to characterize the differences in chemokine content between exo-dead and exo-alive. An ELISA was conducted to compare the relative CXCL1 content in exo-dead and exo-alive. (B) Changes in M2 phenotype polarization of Raw264.7 macrophages when treated with 10 ng/ml murine CXCL1, 50 μg/ml exo-dead, 50 μg/ml exo-deadshCXCL1 408 , 5 μg/ml CXCL1 neutralizing antibody (NA), or exo-dead and CXCL1-NA combination for 48 h. (C) Representative images of Transwell assay. Raw264.7 macrophages were treated as indicated for 48 h and then co-cultured with 4T1 cells. Scale bar: 200 μm. (D–F) Expression changes of CXCL1 and PD-L1 in Raw264.7 macrophages when treated as indicated for 48 h. Scale bar: 10 μm. (G) The results of flow cytometry assay suggested that 50 μg/ml exo-dead treatment for 48 h induced the M2 polarization of Raw264.7 macrophages by activating PD-L1 expression;