GRO alpha Antibody - #AF5403

FIGURE 4 BHS inhibits the CXCL1 cargo in EV-Apo and leads to decreased M2 polarization of Raw264.7 macrophages. (a) M2 polarization changes of Raw264.7 macrophages after treatments as indicated for 48 h were detected by an F4/80+/CD206+ population analysis. EVs were used at a concentration of 100 µg/mL. n = 3. (b) EV-Apo and EV-ApoBHS uptake by Raw264.7 macrophages quantified by flow cytometry. EVs were labelled with PKH67 (green). n = 3. (c) EV-Apo and EV-ApoBHS uptake by Raw264.7 macrophages visualized by immunofluorescence labelling. EVs were labelled with PKH67 (green), and Raw264.7 macrophages were labelled with Actin-Red (red) and DAPI (blue). (d) CXCL1 concentrations in EV-Apo and EV-ApoBHS detected by ELISA assay. n = 3. (e) Western blotting analysis of PD-L1 expression in Raw264.7 cells when treated as indicated for 48 h. CXCL1-NA concentration, 5 µg/mL. Recombinant CXCL1 (rCXCL1), 30 ng/mL. n = 3. (f) Immunofluorescence analysis of PD-L1 expression in Raw264.7 cells when treated as indicated for 48 h. n = 3. (g) QPCR analysis of the mRNA expression levels (left) and promoter activity of PD-L1 (right) in Raw264.7 cells when treated as indicated for 48 h. n = 3. **p < 0.01.

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

Fig. 2: TAMs/CXCL1 signaling promoted autophagy-mediated chemoresistance in breast cancer cells. A Cell counting assay was conducted to examine CXCL1 (20 ng/mL) and its combination with several chemotherapeutic drugs (Paclitaxel at 50 nM, Epirubicin at 1 μg/mL, Doxorubicin at 1 μg/mL, and 5-FU 5 μg/mL) on cell growth of MDA-MB-231 and MCF-7 cells after 48 h treatment. B Western blotting assay was applied to detect the expressions of ABCG2, SQSTM1/p62 and LC3 levels in the presence of CXCL1 ranging from 0 to 50 ng/mL after 24 h treatment. C Cell counting assay was used to detect the effect of CXCL1 neutralizing antibodies (20 ng/mL) on the chemosensitivity of MDA-MB-231 and MCF-7 in response to TAMs-CM. D Western blotting assay was performed on MDA-MB-231 and MCF-7 cells to determine the effects of TAMs-CM containing a CXCL1 neutralizing antibody (20 ng/mL) on ABCG2, SQSTM1/p62 and LC3 expression levels. E The mRFP-GFP-LC3 reporter assay was carried out to verify autophagic alternations in the presence of either CXCL1 (10–20 ng/mL) or TAMs-CM containing CXCL1 neutralizing antibodies (20 ng/mL) (n = 5, scale bars indicate 20 μm). Values are presented as Mean ± SD, n = 3 unless otherwise indicated, *P 

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 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

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.