Journal: Nature

Article Title: MAPPING HUMAN HAEMATOPOIETIC STEM CELLS FROM HAEMOGENIC ENDOTHELIUM TO BIRTH

doi: 10.1038/s41586-022-04571-x

Figure Lengend Snippet: (a) UMAP analysis showing hemato-vascular populations (CDH5+ endothelium, RUNX1+HLF+ HSC and RUNX1+HLF- other hematopoietic cells) from CS14–15 AGM tissues (n=3 biologically independent samples). The contribution of cells in venous EC and non-HSC clusters was balanced. (b) UMAP feature plots displaying the expression of landmark genes for HSC emergence. HE was selected based on co-expression of RUNX1 and CDH5 and absence of PTPRC/CD45 (bottom, right; 66 cells). (c) “EHT scorecard” dot plot showing EHT landmark genes and genes co-regulated at different stages of EHT in each cluster (cl 0–8) and HE. Selected genes significantly enriched in HE compared to other populations, or up- or downregulated during transition to/from HE, were selected. (d) UMAP feature plots displaying pre-HE (IL33 and ALDH1A1) and HE (ALDH1A1 and KCNK17) markers. (e) Spatial transcriptomics of CS15d (5 weeks) embryo transverse sections. Upper panels, H&E staining of two sections between vitelline and umbilical arteries, focused on the dorsal aorta and surrounding region (red arrow: IAHC; green arrows: red blood cells, D=dorsal, V=ventral, bars=500μm). Lower panels showing the spatial expression of EHT genes, with the default color scale from Loupe browser, which represents the log2 expression from 0 to the maximum value in the spots. Each dot is 55 μm and shows combined expression of 1–10 cells. (f) H&E section (section #240) of CS15c (5 weeks) aorta at the intersection with vitelline artery (arrow: IAHC). Immunofluorescence staining of aorta for IL33, ALDH1A1, CD31 and DAPI (section #251), CXCR4, KCNK17 and DAPI (section #254) and SPINK2, PTPRC/CD45 and CD31/PECAM and DAPI (section #239). Bars=20μm. Individual antibody stainings were performed minimum three times in independent embryos with comparable staining pattern. (g) Schematic summarizing the model for EHT involving the specification of pre-HE and HE from arterial EC and HSC emergence. Stage-specific markers and signaling switches are shown. Created with BioRender.com.

Article Snippet: Primary antibodies rabbit anti-human PECAM-1/CD31 (Novus Biologicals, NB100–2284, 1:200, Lot# 2), mouse anti-human ALDH1A1 (SantaCruz Biotechnology, sc-374149, 1:200, Lot# L1719), mouse anti-human IL33 (Santa Cruz Biotechnology, sc-517600, 1:50, Lot# H2720), CXCR4 (Novus Biologicals, NB100–715, 1:100, Lot# MCX4–1019, MCX4–0920), mouse anti-human KCNK17 (SantaCruz Biotechnology, sc-390435, 1:100, Lot# D0113), rabbit anti-human SPINK2 (Sigma Aldrich, HPA026813, 1:100, Lot# B118804), mouse anti-human CD45 (Vector Laboratories, VP-V354CE, 1:50, Lot# 6009341) were diluted in antibody dilution buffer (PBS containing 1% BSA, 0.1% TritonX-100, 0.1% cold-water Fish Skin Gelatin, 0.05% Tween20) and samples were incubated overnight at 4°C.

Techniques: Expressing, Staining, Immunofluorescence

Journal: Nature

Article Title: MAPPING HUMAN HAEMATOPOIETIC STEM CELLS FROM HAEMOGENIC ENDOTHELIUM TO BIRTH

doi: 10.1038/s41586-022-04571-x

Figure Lengend Snippet: (a) tSNE plot documenting the main cell types in CS14–15 (4.5–5 weeks) AGM tissues (top, n=3 biologically independent samples). Feature plots displaying the expression of arterial (GJA5), pre-HE (IL33, ALDH1A1), HE (ALDH1A1, KCNK17), HSC (KCNK17 and SPINK2) and liver SPINK2 progenitor (SPINK, IL7R) markers in CS14–15 AGM samples (bottom). (b) First row, H&E staining of seven transverse sections, featuring dorsal aorta. Red arrows indicate intra-aortic hematopoietic cluster (IAHC) and green arrows red blood cells. Spatial sequencing plots showing the expression of arterial (GJA5), pre-HE (IL33, ALDH1A1), HE (ALDH1A1, KCNK17), HSC (KCNK17 and SPINK2) and liver SPINK2 progenitor (SPINK, IL7R) markers. The default color scale from Loupe browser was applied, which represents the log2 expression from 0 to the maximum value in the spots. Each dot is 55 μm and shows combined expression of 1–10 cells. White bars=250μm, black bars=1mm. (c) Immunofluorescence staining of CS15c (5 weeks) aorta for IL33, ALDH1A1, CD31/PECAM and DAPI (Section #251), CXCR4, KCNK17, CD31/PECAM and DAPI (section #254) and SPINK2, PTPRC/CD45, CD31/PECAM and DAPI (Section) #239. White bars=200μm, black bar=20μm. Individual antibody staining was performed minimum three times in independent embryos with comparable staining pattern.

Article Snippet: Primary antibodies rabbit anti-human PECAM-1/CD31 (Novus Biologicals, NB100–2284, 1:200, Lot# 2), mouse anti-human ALDH1A1 (SantaCruz Biotechnology, sc-374149, 1:200, Lot# L1719), mouse anti-human IL33 (Santa Cruz Biotechnology, sc-517600, 1:50, Lot# H2720), CXCR4 (Novus Biologicals, NB100–715, 1:100, Lot# MCX4–1019, MCX4–0920), mouse anti-human KCNK17 (SantaCruz Biotechnology, sc-390435, 1:100, Lot# D0113), rabbit anti-human SPINK2 (Sigma Aldrich, HPA026813, 1:100, Lot# B118804), mouse anti-human CD45 (Vector Laboratories, VP-V354CE, 1:50, Lot# 6009341) were diluted in antibody dilution buffer (PBS containing 1% BSA, 0.1% TritonX-100, 0.1% cold-water Fish Skin Gelatin, 0.05% Tween20) and samples were incubated overnight at 4°C.

Techniques: Gene Expression, Expressing, Staining, Sequencing, Immunofluorescence

Journal: Nature

Article Title: MAPPING HUMAN HAEMATOPOIETIC STEM CELLS FROM HAEMOGENIC ENDOTHELIUM TO BIRTH

doi: 10.1038/s41586-022-04571-x

Figure Lengend Snippet: (a) UMAP showing the contribution of each embryo/AGM (CS10-CS17) or YS (CS11) to CDH5+/RUNX1+ hemato-vascular cells (n=8 biologically independent samples). (b) Feature plots displaying EC, pre-HE, HE and HSC landmark genes. (c) UMAP plots highlighting HSPC (HLF+SPINK2+), HE (CDH5+RUNX1+and/orKCNK17+PTPRC-SPN-SPINK2-), pre-HE (CDH5+RUNX1-PTPRC-SPN-SPINK2-IL33+and/orALDH1A1+) and EC (remaining cells in HE-containing clusters) from early (CS10–11, blue) and HSC-forming (CS13–17, red) waves. (d) “Nascent HSC scorecard” genes in CS10 embryo HPC, CS11 YS HPC and CS13–17 AGM HSCs. (e) “HSPC waves scorecard” dot plot showing genes co-regulated in EC, pre-HE, HE and HSPC from distinct waves. Genes shown are identified through differential expression analysis and GO term enrichment between early HPC (CS10 embryo and CS11YS) vs HSCs. (f) “Endo waves scorecard” dot plot showing selected genes co-regulated in EC, pre-HE, HE and HSPC populations from the early and HSC-forming waves. Genes shown are identified through differential expression analysis and GO term enrichment between early HE (embryo CS10 and CS11, YS CS11) vs. HSC-forming HE (CS13–15). (g) UMAP feature plots displaying the expression of stage-specific markers.

Article Snippet: Primary antibodies rabbit anti-human PECAM-1/CD31 (Novus Biologicals, NB100–2284, 1:200, Lot# 2), mouse anti-human ALDH1A1 (SantaCruz Biotechnology, sc-374149, 1:200, Lot# L1719), mouse anti-human IL33 (Santa Cruz Biotechnology, sc-517600, 1:50, Lot# H2720), CXCR4 (Novus Biologicals, NB100–715, 1:100, Lot# MCX4–1019, MCX4–0920), mouse anti-human KCNK17 (SantaCruz Biotechnology, sc-390435, 1:100, Lot# D0113), rabbit anti-human SPINK2 (Sigma Aldrich, HPA026813, 1:100, Lot# B118804), mouse anti-human CD45 (Vector Laboratories, VP-V354CE, 1:50, Lot# 6009341) were diluted in antibody dilution buffer (PBS containing 1% BSA, 0.1% TritonX-100, 0.1% cold-water Fish Skin Gelatin, 0.05% Tween20) and samples were incubated overnight at 4°C.

Techniques: Activity Assay, Quantitative Proteomics, Expressing

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: Nuclear localization of the IL-33 and reduced with the onset of labor. The sections fixed paraffin-embedded of human myometrium were immune-stained with anti-IL-33 antibody. (A) Immunohistochemistry results showed that whether in TNL or PNL tissue, IL-33 mostly located in the nuclear while reduced sharply and emerged in the cytoplasm with the initiation of labor as shown in figure TL and PTL. Quantification of IL-33 expression within the nucleus region showed apparent differences between labor and non-labor tissue (n=4). (B) The sections of human myometrium were visualized by an Alexa Flour 594 secondary antibody labeled with IL-33 (Red), and nuclei were stained with DAPI (Blue). Analysis of nuclei was performed by confocal microscopy, fluorescence signals of IL-33 and nuclei were superimposed. Immunofluorescence staining results reflected the same phenomenon. (C) From Western blots, we also can see that non-labor groups had more nuclear expression and less cytoplasmic expression of IL-33 compared to labor groups while the total IL-33 had no obvious differences between groups. QT-PCR discovered no apparent alteration in the levels of IL-33 mRNA. Each value represents the mean ± standard deviation (SD) of three independent experiments. ** P<0.01, *** P<0.001, bar 50 μm.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Staining, Immunohistochemistry, Expressing, Labeling, Confocal Microscopy, Fluorescence, Immunofluorescence, Western Blot, Standard Deviation

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: Dynamic changes of IL-33 position during LPS stimulation. (A) Localization analysis of confocal laser scanning microscopy images of IL-33 with the stimulation of 10 μg/ml LPS, we could see that IL-33 in the nucleus declined sharply compared with the control group, especially in 3 hours. However, with the longer time of the LPS treatment, the expression of IL-33 commenced to rise again from 6 hours and reached the peak at 12 hours. The lower panel is the percentage of cells of which IL-33 expressed mainly in the nucleus (n=3). (B) After the primary cells were loaded with LPS for different times, cytoplasmic and nuclear proteins were isolated. In the cytoplasmic and nucleus fraction IL-33 were quantified by Western blotting, the experiment was performed as described for . Each experiment was performed at least three times while shown are representative results (n=3). From the blots, we known that whether cytoplasmic or nucleus fraction IL-33 levels presented the same change trend as immunofluorescence. * P<0.05, ** P<0.01, bar 50 μm.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Confocal Laser Scanning Microscopy, Expressing, Isolation, Western Blot, Immunofluorescence

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 silencing enhanced LPS-induced expression of calcium channels and the intracellular calcium concentration. Primary myometrium cells were transfected with IL-33 or non-targeting (as control) siRNAs and treated with 10 μg/ml LPS or PBS (as control) for 6 hours. (A) Immunofluorescence staining analysis performed with fluo-3AM (green), nuclei were stained with DAPI (blue) (n=3). (B) Western blot analysis for expression levels of Cav 3.1 and Cav 3.2 in stably transfected and non-transfected myometrium cells with treatment as indicated (n=5). * P<0.05, ** P <0.01, bar 50 μm.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Expressing, Concentration Assay, Transfection, Immunofluorescence, Staining, Western Blot, Stable Transfection

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 silencing highlighted LPS-induced endoplasmic reticulum stress response. Based on the discovery that calcium ions affected endoplasmic reticulum stress, Western blot and QT-PCR were we further to explore the expression of endoplasmic reticulum stress in tissues from protein and mRNA level. (A) The protein levels of P-IRE1α and XBP1s in the TL and PTL groups were higher than those in the TNL and PNL groups while there was no alteration in the level of GRP78 protein(n=6). (B) The mRNA level of XBP1s in the PTL groups was higher than that in the PNL groups (n=6). Furthermore, in order to illustrate the protein level changes of endoplasmic reticulum stress during labor, LPS was used to stimulate primary uterine smooth muscle cells for different time course and then Western blot was used to detect the alteration of endoplasmic reticulum stress. (C) It was found that the protein level of pIRE1α reached its peak at 10 minutes while XBP1s at 15 minutes, while the protein level of GRP78 did not change significantly (n=5). We also detected apparent alteration in endoplasmic reticulum stress protein during cells stimulated based knockdown experiments targeting IL-33. (D) It revealed that the endoplasmic reticulum stress response in the siRNA-based group was more obvious compared with the LPS stimulated directly especially at 30 minutes (n=5). * P<0.05, ** P<0.01, *** P<0.005, ****P<0.001

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Western Blot, Expressing

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 siRNA and ER stress response affected COX-2 expression in myometrium cells. (A) In the process of studying whether IL-33 affects COX-2, we found that the COX-2 expression in the siRNA-mediated group was significantly increased compared with the LPS alone group (n=5). (B) Western blot analyses showing protein expression of COX-2 in myometrium cells was decreased following treatment with LPS for 12 hours (n=5). * P<0.05, ** P<0.01. Each value represents the mean ± standard deviation (SD) of three independent experiments.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 knockdown enhanced LPS-induced NF-κB and p38/MAPK signaling pathways. (A) Relative levels of p-P38, P38, p-NF-κB and NF-κB were assessed by western blot analysis at the indicated time point after LPS (10 μg/ml) stimulation. Phosphorylation levels of P38 and NF-κB increased gradually with LPS stimulation and peaked at 15 minutes and 1 hour, respectively(n=5). (B) Western blot analysis of p-P38, P38, p-NF-κB and NF-κB expression in cells transfected with siRNA targeting IL-33 after treatment with LPS for 30minutes. Compared with the LPS group, the protein expression of phosphorylated P38 and NF-κB were increased in the LPS + siRNA IL-33 group(n=5). (C) The protein level of COX-2 was decreased when SB-202190 and JSH-23 blocked p38/MAPK and NF-KB signaling pathway, respectively(n=5). * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Western Blot, Expressing, Transfection

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 siRNA and cytoplasmic calcium influence the expression of IL-8 and IL-6. Compared with the LPS group, the expression of IL-8 and IL-6 was increased in the LPS+IL-33 siRNA group and decreased in the LPS+BAPTA-AM group (n=5). * P <0.05.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Expressing

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: Model for the role of IL-33 in the myometrium participates in maintaining a uterine quiescent state at the tissue-to-cellular level during late pregnancy.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques:

Journal: Biomedical Journal

Article Title: Effect of three clinical therapies on cytokines modulation in the hip articular cartilage and bone improvement in rat early osteonecrosis of the femoral head

doi: 10.1016/j.bj.2022.11.004

Figure Lengend Snippet: The expression of IL33 and its ST2 receptor. Immunohistochemical staining was performed for (A) IL33 and (B) ST2 in the articular cartilage of the left femoral head (left) and the data was calculated after treatments (right). Scale bar = 50 μm. ∗∗∗ p < 0.001 was by comparing with ONFH group and ### p < 0.001 was by comparing with ESWT group among the treatment groups. N = 6 for all groups.

Article Snippet: The slides were probed with specific proteins using anti-rat IL5 (1:100, LS-B14355, LifeSpan BioSciences Inc., USA), IL6 (1:50, ab9324, Abcam, USA), IL17A (1:200, PA5-79470, Invitrogen, USA), IL33 (1:200, orb6205, Biorbyt, USA), and ST2 (1:150, 11920-1-AP, Proteintech, USA) antibodies to recognise proteins in the articular cartilage of the femoral head of rats.

Techniques: Expressing, Immunohistochemical staining, Staining