Journal: eLife

Article Title: Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium

doi: 10.7554/eLife.31274

Figure Lengend Snippet: ( A ) Representative SAβG staining in undifferentiated EnSCs (Day 0) or cells decidualized for the indicated time points with 8-bromo-cAMP and MPA. Scale bar = 100 µm. ( B ) SAβG activity, expressed in fluorescence intensity units (FIU), in undifferentiated EnSCs (day 0) or cells decidualized for the indicated time points. ( C ) Representative Western blot analysis of p53, p16, LMNB1, HMGB2, mH2A, H3K9me3 and H.H1 levels in undifferentiated EnSCs and cells decidualized for the indicated time points. β-actin served as a loading control. ( D ) Left panel: representative immunofluorescence staining for p16 expression in undifferentiated cells and cells decidualized for 8 days. Nuclei were counterstained with DAPI. Scale bar = 50 µm. Right panel: percentage of p16 + cells. ( E ) Left panel: representative confocal microscopy images of undifferentiated (Day 0) or decidualized (Day 8) EnSCs immune-probed for LMNB1, mH2A, H3K9me3 and H.H1. Scale bar = 10 µm. Right panel: nuclear size of undifferentiated EnSCs (n = 48) and of cells first decidualized for 8 days with 8-br-cAMP and MPA (C + M) (n = 48) was measured in three primary cultures. ( F ) Secretion of IL-8, GROα, and IL-6 was measured in the supernatant of primary EnSCs collected every 48 hr over an 8 day decidualization time-course. Data are mean ±SEM of 3 biological replicates unless stated otherwise. ** p < 0.01, *** p < 0.001. Different letters above the error bars indicate that those groups are significantly different from each other at p<0.05. 10.7554/eLife.31274.004 Figure 1—source data 1. Decidualization induces acute senescence in a subpopulation of EnSCs.

Article Snippet: ELISAs were performed exactly as per manufacturer’s instructions (DuoSet ELISA kits for IL-8 (D8000C), IL-15 (D1500), GROα (DY275) and IL-6 (DY206); Bio-Techne, Abingdon, UK).

Techniques: Staining, Activity Assay, Fluorescence, Western Blot, Immunofluorescence, Expressing, Confocal Microscopy

Journal: eLife

Article Title: Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium

doi: 10.7554/eLife.31274

Figure Lengend Snippet: ( A ) SAβG activity in EnSCs either undifferentiated, or decidualized for 8 days with 8-bromo-cAMP, MPA, or a combination. ( B ) Top left panel: FOXO1 mRNA levels in undifferentiated EnSCs and cells treated with 8-br-cAMP and MPA (C + M) following transfection with non-targeting (NT) or FOXO1 siRNA. Other panels: Secretion of IL-8, IL-6 and GROα was measured following FOXO1 knockdown in the supernatant of primary EnSCs every 48 hr over an 8 day decidualization time-course. ( C ) SAβG activity in EnSCs following transfection with NT or FOXO1 siRNA. The cultures either remain untreated or decidualized for 8 days. ( D ) SAβG activity in undifferentiated EnSCs treated for 8 days with increasing concentrations of recombinant IL-8 and in cells decidualized for 8 days in the presence of increasing concentrations of the CXCR2 antagonist, SB265610. ( E ) SAβG activity in EnSCs following transfection with IL-8 siRNA. The cultures either remain untreated or decidualized for 8 days. ( F ) PRL and IGFBP1 transcript levels in EnSCs following transfection with IL-8 siRNA. The cultures either remain untreated or decidualized for 8 days. ( G ) PRL and IGFBP1 expression in undifferentiated EnSCs, cells decidualized for 8 days, and upon withdrawal of 8-br-cAMP and MPA (C + M) for the indicated days. ( H ) Left panel: SAβG activity in undifferentiated EnSCs, cells decidualized for 8 days, and following withdrawal of C + M for the indicated days. Right panel: representative Western blot analysis of p53, p16, LMNB1 and HMGB2 levels in undifferentiated EnSCs, cells decidualized for 8 days, and following withdrawal of C + M for the indicated days. β-actin served as a loading control. Data are mean ±SEM of 3 biological replicates unless stated otherwise. *p < 0.05, **p < 0.01 and ***p < 0.005. Different letters above the error bars indicate that those groups are significantly different from each other at p<0.05. 10.7554/eLife.31274.009 Figure 3—source data 1. A FOXO1/IL-8 axis drives EnSC differentiation and senescence.

Article Snippet: ELISAs were performed exactly as per manufacturer’s instructions (DuoSet ELISA kits for IL-8 (D8000C), IL-15 (D1500), GROα (DY275) and IL-6 (DY206); Bio-Techne, Abingdon, UK).

Techniques: Activity Assay, Transfection, Recombinant, Expressing, Western Blot

Journal: eLife

Article Title: Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium

doi: 10.7554/eLife.31274

Figure Lengend Snippet: ( A ) PRL and IGFBP1 transcript levels in EnSCs following transfection with FOXO1 siRNA. The cultures either remained untreated or were decidualized for 8 days. ( B ) Left panel: SAβG staining in undifferentiated EnSCs that remained untreated (control) or were incubated with recombinant IL-8 (30 μM) for 8 days. SAβG staining was also performed in parallel cultures decidualized with 8-bromo-cAMP and MPA (C + M) in the absence or presence of the CXCR2 antagonist SB265610 (10 μM). Right panel: IL-8 concentration in conditioned media from decidualized EnSCs following siRNA-mediated CXCL8 (IL-8) knockdown. ( C ) SAβG staining (left panel) and activity (right panel) in undifferentiated (day 0) and decidualized (day 8) EnSCs in the presence of the mTOR inhibitor rapamycin. FIU: fluorescence intensity units. ( D ) PRL and IGFPB1 transcripts in undifferentiated EnSCs and cells decidualized for 8 days in the presence or absence of rapamycin (100 nM). All data are mean ±SEM of 3 biological replicates. Different letters above the error bars indicate that those groups are significantly different from each other at p<0.05. Scale bars = 100 μm.

Article Snippet: ELISAs were performed exactly as per manufacturer’s instructions (DuoSet ELISA kits for IL-8 (D8000C), IL-15 (D1500), GROα (DY275) and IL-6 (DY206); Bio-Techne, Abingdon, UK).

Techniques: Transfection, Staining, Incubation, Recombinant, Concentration Assay, Activity Assay, Fluorescence

Journal: eLife

Article Title: Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium

doi: 10.7554/eLife.31274

Figure Lengend Snippet: ( A ) Pearson’s correlation analysis of SAβG activity in 75 matched undifferentiated primary cultures and cultures decidualized for 8 days. ( B ) Representative SAβG staining in undifferentiated (Day 0) and decidualizing EnSCs (Day 8) following 4 days of pretreatment with vehicle, dasatinib (250 nM) or palbociclib (1 μM). Scale bar = 100 µm. ( C ) PRL and IGFBP1 mRNA expression in response to pretreatment with vehicle, dasatinib or palbociclib. The cultures then remained undifferentiated or were decidualized for 8 days. ( D ) IL-8, IL-6 and GROα secretion was measured every 48 hr in the supernatant of primary EnSCs decidualized for the indicated time-points following pretreatment with vehicle, dasatinib or palbociclib. ( E ) Colony forming unit (CFU) activity in paired EnSC cultures that either remain undifferentiated (Day 0) or were decidualized for 8 days (n = 10). ( F ) Left panel: representative clonogenic assays established from EnSC cultures first pretreated with vehicle, dasatinib or palbociclib and then decidualized for 8 days. Right panel: CFU activity in EnSC cultures first pretreated with vehicle, dasatinib or palbociclib and then decidualized for 8 days. Data are mean ±SEM of 3 biological replicates unless stated otherwise. *p < 0.05, **p < 0.01 and ***p < 0.001. Different letters above the error bars indicate that those groups are significantly different from each other at p<0.05. 10.7554/eLife.31274.012 Figure 4—source data 1. Functions of senescent decidual cells.

Article Snippet: ELISAs were performed exactly as per manufacturer’s instructions (DuoSet ELISA kits for IL-8 (D8000C), IL-15 (D1500), GROα (DY275) and IL-6 (DY206); Bio-Techne, Abingdon, UK).

Techniques: Activity Assay, Staining, Expressing

Journal: Theranostics

Article Title: Cancer-associated Fibroblast-derived IL-6 Promotes Head and Neck Cancer Progression via the Osteopontin-NF-kappa B Signaling Pathway

doi: 10.7150/thno.22182

Figure Lengend Snippet: Fibroblasts contribute to the up-regulation of OPN in HNC cells. (A) Immunohistochemical analysis of OPN protein expression in HNC tissues and matched adjacent normal tissues (Scale bar: 25 μm). Higher staining of OPN was observed in tumor cells of the edge of bulk tumors. (B) The mRNA and protein levels of OPN were determined in NFs, CAFs and cancer cells (derived from 4 HNC patients) using real-time PCR and western blotting. (C) OPN at the protein and mRNA level and in the supernatant was detected in 8 representative HNC cell lines and normal oral epithelial cells (titled normal) using western blotting, real-time PCR and ELISA. (D) The protein level, supernatant concentration and mRNA level of OPN were determined in CAL-27 and SCC-25 cells after co-culture with NFs. (E) The protein level, supernatant concentration and mRNA level of OPN were determined in NFs after co-culture with CAL-27 and SCC-25 cells. (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001)

Article Snippet: The supernatant OPN and IL-6 concentrations and the plasma OPN and IL-6 levels in patients with HNC were assessed using the Human OPN ELISA kit (Boster, China) and human IL-6 ELISA kit (Boster, China).

Techniques: Immunohistochemical staining, Expressing, Staining, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Co-Culture Assay

Journal: Theranostics

Article Title: Cancer-associated Fibroblast-derived IL-6 Promotes Head and Neck Cancer Progression via the Osteopontin-NF-kappa B Signaling Pathway

doi: 10.7150/thno.22182

Figure Lengend Snippet: Effects of stromal IL-6-induced OPN on promoting tumor growth and metastasis in vivo . (A) OPN overexpression in CAL-27 cells facilitated the xenograft tumor growth in nude mice. (B) Plasma OPN levels of the tumor-bearing mice from the normal group, CAL-27 group and CAL-27-OPN group. (C) Subcutaneous injection of OPN antibody (10 μg per tumor nodule) or IL-6 antibody (10 μg per tumor nodule) around the tumor partly inhibited NF-mediated tumor growth, and the combination of OPN antibody (5 μg per tumor nodule) and IL-6 antibody (5 μg per tumor nodule) exhibited a more powerful antitumor activity. (D) Plasma OPN levels of the tumor-bearing mice from the normal group, IgG treatment group, OPN antibody treatment group, IL-6 antibody treatment group and the combination group. (E) Overexpression of OPN in Rca-T cells promoted the formation and growth of metastatic nodules in nude mice (Scale bar, left: 5 mm; right: 100 μm). (F) Western blot analysis confirmed the exogenous expression of OPN in Rca-T cells, and plasma OPN levels of the mice involved in experimental metastasis were measured using ELISA. (G) Kaplan-Meier analyses of overall survival. (H, I and J) ROC curve analysis of the mRNA panel of OPN and IL-6 stratified by different groups in the validation set. ROC plots for the mRNA panel of OPN and IL-6 discriminated the five-year survival group from the death group (H), the TNM stage I group from the healthy controls (I), and the metastasis group from the non-metastasis group (J). AUC, area under the curve. (K) A proposed model illustrating the modulatory role of stromal IL-6-induced neoplastic OPN in controlling tumor growth and metastasis. (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001)

Article Snippet: The supernatant OPN and IL-6 concentrations and the plasma OPN and IL-6 levels in patients with HNC were assessed using the Human OPN ELISA kit (Boster, China) and human IL-6 ELISA kit (Boster, China).

Techniques: In Vivo, Over Expression, Clinical Proteomics, Injection, Activity Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Biomarker Discovery