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: Respiratory Research

Article Title: Different expression levels of interleukin-35 in asthma phenotypes

doi: 10.1186/s12931-020-01356-6

Figure Lengend Snippet: Correlations between sputum IL-35 and clinical characteristics

Article Snippet: Quantitative detection of sputum IL-35 was performed using the commercial human interleukin-35 (IL-35) ELISA kit (Catalog Number CSB-E13126h, CUSABIO, China) in accordance with the manufacturer’s protocol.

Techniques: