Journal: The Febs Journal

Article Title: Myeloid‐derived suppressor cells promote epithelial ovarian cancer cell stemness by inducing the CSF2/p‐STAT3 signalling pathway

doi: 10.1111/febs.15311

Figure Lengend Snippet: ALDH + EOC cells are EOC stem cells. (A) Sphere formation assays using ALDH + and ALDH ‐ ES2 and SKOV3 cells. Scale bar, 50 μm. (B) qRT‐PCR analysis of core stem cell molecule expression levels in ALDH + and ALDH ‐ ES2 and SKOV3 cells. (C) Sphere formation assays using ES2 and SKOV3 cells cultured with MDSCs from different groups of patients. Scale bar, 50 μm. (D, E) Statistical analysis of the number of ES2 and SKOV3 tumour spheres with MDSCs from different groups of patients. All data were analysed using Student’s t ‐test and are expressed as the mean ± SD. Experiments were performed in triplicate with MDSCs from three different patients. Symbols represent statistical significance (* P < 0.01; ** P < 0.001; and *** P < 0.0001).

Article Snippet: ES‐2, SKOV3 and HO8910 cells were incubated with the specific STAT3 phosphorylation inhibitor Stattic (20 μ m ) (Cat. HY‐13818; MCE, Monmouth Junction, NJ, USA) in 10% serum medium for 24 h. Control cells were treated with the same volume of DMSO.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture

Journal: The Febs Journal

Article Title: Myeloid‐derived suppressor cells promote epithelial ovarian cancer cell stemness by inducing the CSF2/p‐STAT3 signalling pathway

doi: 10.1111/febs.15311

Figure Lengend Snippet: MDSCs enhance the stemness of EOC cells. (A) Flow cytometry analysis of the ALDH + cells within the ES2 and SKOV3 cell populations cultured with MDSCs from different groups of patients. (B, C) Statistical analysis of the proportion of the ALDH+ cells within the ES2 and SKOV3 cell populations cultured with MDSCs from different groups of patients. (D) Colony formation assays using ES2 and SKOV3 cells cocultured with MDSCs. Scale bar, 50 μm. (E) qRT‐PCR analysis of core stem cell molecule expression in ES2 and SKOV3 cells cultured with MDSCs. (F, G) Western blot analysis of core stem cell molecule expression in ES2 and SKOV3 cells cultured with MDSCs. All data were analysed using Student’s t‐test and are expressed as the mean ± SD. Experiments were performed in triplicate with MDSCs from three different patients. Symbols represent statistical significance (* P < 0.01; ** P < 0.001; and *** P < 0.0001).

Article Snippet: ES‐2, SKOV3 and HO8910 cells were incubated with the specific STAT3 phosphorylation inhibitor Stattic (20 μ m ) (Cat. HY‐13818; MCE, Monmouth Junction, NJ, USA) in 10% serum medium for 24 h. Control cells were treated with the same volume of DMSO.

Techniques: Flow Cytometry, Cell Culture, Quantitative RT-PCR, Expressing, Western Blot

Journal: The Febs Journal

Article Title: Myeloid‐derived suppressor cells promote epithelial ovarian cancer cell stemness by inducing the CSF2/p‐STAT3 signalling pathway

doi: 10.1111/febs.15311

Figure Lengend Snippet: MDSCs stimulate CSF2 expression in EOC. (A) Microarray analysis of differentially expressed genes for SKOV3 cells based on coculture with MDSCs. (B) Scatter diagram analysis of discriminating genes for SKOV3 cells based on coculture with MDSCs. (C) qRT‐PCR analysis of CSF2, ICAM‐1, IL‐32, BIRC3 and TNFAIP3 expression in ES2 and SKOV3 cells cultured with or without MDSCs. (D, E) Western blot analysis of CSF2 expression in ES2 and SKOV3 cells cultured with or without MDSCs. All data were analysed using Student’s t‐test and are expressed as the mean ± SD. Experiments were performed in triplicate with MDSCs derived from three different patients, and statistically significant differences are presented as follows: ** P < 0.001 and *** P < 0.0001.

Article Snippet: ES‐2, SKOV3 and HO8910 cells were incubated with the specific STAT3 phosphorylation inhibitor Stattic (20 μ m ) (Cat. HY‐13818; MCE, Monmouth Junction, NJ, USA) in 10% serum medium for 24 h. Control cells were treated with the same volume of DMSO.

Techniques: Expressing, Microarray, Quantitative RT-PCR, Cell Culture, Western Blot, Derivative Assay

Journal: The Febs Journal

Article Title: Myeloid‐derived suppressor cells promote epithelial ovarian cancer cell stemness by inducing the CSF2/p‐STAT3 signalling pathway

doi: 10.1111/febs.15311

Figure Lengend Snippet: MDSCs enhance EOC stemness via CSF2. (A) qRT‐PCR analysis of CSF2 expression in ES2 and SKOV3 cells transfected with the shControl or shCSF2 plasmid. (B) Western blot analysis of CSF2 expression in ES2 and SKOV3 cells transfected with the ShControl or ShCSF2 plasmid. (C, D) Sphere formation assays with ES2 and SKOV3 cells with dysregulated CSF2 expression cultured with or without MDSCs. Scale bar, 50 μm. (E, F) Colony formation assays with ES2 and SKOV3 cells with knocked down CSF2 expression cultured with or without MDSCs. (G, H) Flow cytometry analysis of the ALDH + cells in ES2 and SKOV3 cell populations transfected with the shControl or shCSF2 plasmid and cultured with MDSCs. All data were analysed using Student’s t‐test and are expressed as the mean ± SD. Experiments were performed in triplicate with MDSCs derived from three different patients. Symbols represent statistical significance (* P < 0.01; ** P < 0.001; and *** P < 0.0001; ns, P > 0.05).

Article Snippet: ES‐2, SKOV3 and HO8910 cells were incubated with the specific STAT3 phosphorylation inhibitor Stattic (20 μ m ) (Cat. HY‐13818; MCE, Monmouth Junction, NJ, USA) in 10% serum medium for 24 h. Control cells were treated with the same volume of DMSO.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Western Blot, Cell Culture, Flow Cytometry, Derivative Assay

Journal: The Febs Journal

Article Title: Myeloid‐derived suppressor cells promote epithelial ovarian cancer cell stemness by inducing the CSF2/p‐STAT3 signalling pathway

doi: 10.1111/febs.15311

Figure Lengend Snippet: MDSCs induce epithelial ovarian CSCs through CSF2/p‐STAT3 signalling pathway. (A) qRT‐PCR analysis of NANOG, c‐MYC, SOX2 and STAT3 expression levels in ES2 and SKOV3 cells with knocked down CSF2 expression cultured with or without MDSCs. Experiments were performed in triplicate with MDSCs derived from three different patients. (B) Western blot analysis of NANOG, c‐MYC, CSF2, STAT3 and p‐STAT3 expression in ES2 and SKOV3 cells with knocked down CSF2 expression cultured with or without MDSCs. (C, D) Statistical analysis of NANOG, c‐MYC, CSF2, STAT3 and p‐STAT3 intensity in ES2 and SKOV3 cells with knocked down CSF2 expression cultured with or without MDSCs. (E, F, G) Sphere formation assays with ES2 and SKOV3 cells with dysregulated CSF2 expression or the p‐STAT3 inhibitor Stattic cultured with or without MDSCs. Scale bar, 100 μm. Data were analysed using a nonparametric test, and statistically significant differences are presented as follows: * P < 0.01; ** P < 0.001; and *** P < 0.0001.

Article Snippet: ES‐2, SKOV3 and HO8910 cells were incubated with the specific STAT3 phosphorylation inhibitor Stattic (20 μ m ) (Cat. HY‐13818; MCE, Monmouth Junction, NJ, USA) in 10% serum medium for 24 h. Control cells were treated with the same volume of DMSO.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Derivative Assay, Western Blot

Journal: The Febs Journal

Article Title: Myeloid‐derived suppressor cells promote epithelial ovarian cancer cell stemness by inducing the CSF2/p‐STAT3 signalling pathway

doi: 10.1111/febs.15311

Figure Lengend Snippet: CSF2 expression is significantly correlated with clinical stage in EOC. (A, B) Flow cytometry analysis of the ALDH + cells in ES2 cells transfected with the shControl or shCSF2 plasmid or treated with p‐STAT3 inhibitor Stattic cocultured with or without MDSCs. (C, D) Flow cytometry analysis of the ALDH + cells in SKOV3 cells with the shControl or shCSF2 plasmid or treated with p‐STAT3 inhibitor Stattic and cultured with or without MDSCs. (E, F) Immunohistochemical analysis of CSF2 expression in normal ovary, EOC (Stage I–II) and EOC (Stage III–IV) tissue samples. Data were analysed using a nonparametric test, and statistically significant differences are presented as follows: * P < 0.01; ** P < 0.001; and *** P < 0.001. Scale bar, 100 μm.

Article Snippet: ES‐2, SKOV3 and HO8910 cells were incubated with the specific STAT3 phosphorylation inhibitor Stattic (20 μ m ) (Cat. HY‐13818; MCE, Monmouth Junction, NJ, USA) in 10% serum medium for 24 h. Control cells were treated with the same volume of DMSO.

Techniques: Expressing, Flow Cytometry, Transfection, Plasmid Preparation, Cell Culture, Immunohistochemical staining