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: bioRxiv

Article Title: HSP70 is a chaperone for IL-33 secretion and function in chronic airway disease

doi: 10.1101/2025.01.30.635799

Figure Lengend Snippet: A) SDS-PAGE gel demonstrating co-purification of a high molecular weight band (>65 kDa, MW markers as indicated) that co-purified with all IL-33 isoforms on tandem anti-FLAG, NiNTA affinity purification. B) Mass Spectrometry analysis of contaminating band isolated from Expi293 expression cells and HBE-1 airway cell line identified as HSP70 (and HSC70) with coverage and relative # peptides reported. C) Recombinant purified human IL-33 Δ34 and HSP70 interact by Co-Immunoprecipitation of biotinylated IL-33 protein compared to oxidation resistant Cys->Ser x4 mutant IL-33 Δ34SX4 . D) Intracellular interaction of these proteins verified based on overexpression of miniTurboID-IL-33 Δ34 proximity ligase fusion protein with robust biotinylation of HSP70 in HBE-1 cells but not in U937 cells. E) Differential proximity ligation comparison for IL-33 Δ34 and IL-33 Δ34SX4 miniTurboID fusions in HBE-1 cells. Heatmap of top 20 most abundant proteins and additional heat shock intermediates of relevance. Peptide number indicated by color mapping blue (0), white (25), red (300), replicates 1-3 as shown.

Article Snippet: Non-biotinylated IL-33 Δ34 (500 ng/ml) +/− HSP70 (500 ng/mL) were bound at room temp for 30 mins, plates washed 3x and bound IL-33 Δ34 was detected using R&D DuoSet Human IL-33 biotinylated detection antibody following manufacturer protocols and developed using streptavidin HRP (R&D Systems) and TMB peroxidase substrate (SeraCare).

Techniques: SDS Page, Copurification, High Molecular Weight, Purification, Affinity Purification, Mass Spectrometry, Isolation, Expressing, Recombinant, Immunoprecipitation, Mutagenesis, Over Expression, Ligation, Comparison

Journal: bioRxiv

Article Title: HSP70 is a chaperone for IL-33 secretion and function in chronic airway disease

doi: 10.1101/2025.01.30.635799

Figure Lengend Snippet: A) Phosphatidylserine (PS) binding for IL-33 Δ34(SX4) mutant (C208S, C228S, C232S, C259S) and B) phosphatidylcholine (PC) for IL-33 Δ34 binding ELISAs under conditions of HSP70 (500 ng/ml) and GW4869 (20 μM) in competition. Detection was performed using biotinylated anti-IL-33 secondary antibody (R&D Systems) and Streptavidin HRP, and TMB substrate followed by A450 absorbance measurements. C, D) Secreted and total Flag-IL-33 Δ34 -His and endogenous HSP70 in HBE-1 cells under conditions of GW4869 (20 μM) and GGA (1 μM) treatment, detection using anti-IL-33 and HSP70 Duosets (R&D Systems) and TMB substrate. E) Data points displayed with mean ± SEM. P -value limits: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Non-biotinylated IL-33 Δ34 (500 ng/ml) +/− HSP70 (500 ng/mL) were bound at room temp for 30 mins, plates washed 3x and bound IL-33 Δ34 was detected using R&D DuoSet Human IL-33 biotinylated detection antibody following manufacturer protocols and developed using streptavidin HRP (R&D Systems) and TMB peroxidase substrate (SeraCare).

Techniques: Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: HSP70 is a chaperone for IL-33 secretion and function in chronic airway disease

doi: 10.1101/2025.01.30.635799

Figure Lengend Snippet: A and B) Protein levels for IL-33 and HSP70 in lung tissue and lavage fluid measured by ELISA. C) HSP70 and HSP90 protein levels measured by ELISA in SPIROMICS BAL specimens. D) Schematic for EV isolation by size exclusion chromatography from bronchial wash (BW) and volcano plots for differential protein expression in COPD IV and COPD I-III specimens compared to nonsmoker control. Proteins annotated are associated with heat shock or proteostasis systems and were biotinylated by IL-33 in miniTurboID experiment . Inclusion in bronchial wash EV proteome was demonstrated for HSP70, HSC70 and HSP90. E) Transcript expression of molecular chaperonin TCP1 subunits CCT5 , CCT7 and CCT8 in lung tissue displayed as fold-change by ΔΔCt method with normalization to GAPDH . Data points displayed with mean ± SEM. P -value limits: * P < 0.05, ** P < 0.01, *** P < 0.001, *** P < 0.0001.

Article Snippet: Non-biotinylated IL-33 Δ34 (500 ng/ml) +/− HSP70 (500 ng/mL) were bound at room temp for 30 mins, plates washed 3x and bound IL-33 Δ34 was detected using R&D DuoSet Human IL-33 biotinylated detection antibody following manufacturer protocols and developed using streptavidin HRP (R&D Systems) and TMB peroxidase substrate (SeraCare).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Size-exclusion Chromatography, Expressing, Control