Journal: Theranostics

Article Title: Membrane TLR9 Positive Neutrophil Mediated MPLA Protects Against Fatal Bacterial Sepsis

doi: 10.7150/thno.37139

Figure Lengend Snippet: The effect of MPLA mediated inflammatory preconditioning (InP) on cav-1 -/- mice. (A) Survival curves, n=8 mice per group. The mice were first given 0.1×10 8 CFUs E. coli , followed by the lethal dose of E. coli (3×10 8 CFUs) 2 h later,* vs. Saline. (B) Experimental procedures. (1) The WT mice were injected i.p. with 0.1×10 8 CFUs E. coli, and 2 h later were injected with 3×10 8 CFUs E. coli per mouse, and all 8 mice survived (InP). (2) C av-1 -/ - mice were injected i.p. with 0.1×10 8 E. coli, and 2 h later were injected with 3×10 8 CFUs E. coli, and all 8 mice died. (C) Intestine and liver tissues of mice stained with H&E. (D) ELISA of TNF-α/IL-6 in the supernatants of peripheral blood neutrophils in mice stimulated with 0.1×10 8 CFUs E. coli for 2 h. ** P <0.01 (Student's t test). (E) Western blot shows that InP did not promote translocation of TLR9 from cytosol to cell membrane in cav -/- mice. (F) Western blot shows significant reduction of endogenous Cav-1 by shRNA against Cav-1 (Cav-1 shRNA). (G) Immunoblot of TLR9, MyD88, TRAF3 and IRF3 in HL60 cells transfected Cav-1 shRNA and then stimulated with 0.1×10 8 CFUs E. coli for indicated times. Similar results were obtained in three independent experiments. (H) ELISA of TNF-α/IL-6 in the supernatants of HL60 cells transfected Cav-1 shRNA, stimulated with 0.1×10 8 CFUs E. coli for 2 h. ** P <0.01 (Student's t test).

Article Snippet: MyD88 homodimerization inhibitor peptide (NBP2-29328) was purchased from Novus Biologicals (Novus, USA).

Techniques: Saline, Injection, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Translocation Assay, Membrane, shRNA, Transfection

Journal: Theranostics

Article Title: Membrane TLR9 Positive Neutrophil Mediated MPLA Protects Against Fatal Bacterial Sepsis

doi: 10.7150/thno.37139

Figure Lengend Snippet: Expression of TLR9-Cav-1 signaling proteins in the neutrophils of patients with sepsis. (A) Membrane TLR9 expression. (B) Cav-1 expression. (C) ROC curve for mTLR9. P <0.05. (D) ROC curve for Cav-1. P <0.05. (E) Association of Cav-1 with surface TLR9 expression in neutrophils. r2 =0.5791. (F) MyD88 expression. (G) TRAF3 expression. (H) IRF3 expression.

Article Snippet: MyD88 homodimerization inhibitor peptide (NBP2-29328) was purchased from Novus Biologicals (Novus, USA).

Techniques: Expressing, Membrane

Journal: Advanced Science

Article Title: Embryo‐Derived Cathepsin B Promotes Implantation and Decidualization by Activating Pyroptosis

doi: 10.1002/advs.202402299

Figure Lengend Snippet: CTSB induces rapid and reversible pyroptosis in mouse endometrial epithelial cells. A) Western blot analysis of pyroptosis‐associated protein levels after endometrial epithelial cells were treated with CTSB, CTSB and disulfiram, or disulfiram for 3 h. B) Western blot analysis of pyroptosis‐associated protein levels after endometrial epithelial cells were treated with CTSB, CTSB and CA‐074 Me, or CA‐074 Me for 3 h. C) Western blot analysis of pyroptosis‐associated protein levels after endometrial epithelial cells were treated with CTSB, CTSB and NLRP3‐IN‐21, or NLRP3‐IN‐21 for 3 h. D) NINJ1 immunofluorescence (Green) and PI fluorescence (red) at the implantation site and inter‐implantation site on day 4.5 of pregnancy. Arrowhead, blastocyst. E) NINJ1 immunofluorescence (green) and DAPI fluorescence (blue) after endometrial epithelial cells were treated with CTSB for 5 min. F) GSDMD‐N immunofluorescence (red), WGA fluorescence (green), and DAPI fluorescence (blue) after endometrial epithelial cells were treated with CTSB for 5 min. G) CHMP4B and CHMP3 immunofluorescence (green) and PI fluorescence (red) at the implantation site and inter‐implantation site on day 4.5 of pregnancy. Arrowhead, blastocyst. H) CHMP3 and CHMP4B immunofluorescence (green) and DAPI fluorescence (blue) after endometrial epithelial cells were treated with CTSB for 10 min. I) PI fluorescence (red) and DAPI fluorescence (blue) after endometrial epithelial cells were treated with CTSB for different time points. J) ELISA analysis of S100A8/S100A9 and LDH secretion after endometrial epithelial cells were treated with CTSB for 5 min ( n = 4 per group). Scale bar = 125 µm. Data were presented as mean ± SD. *** : p < 0.001, ns: not significant, by two‐tailed Student's t‐test.

Article Snippet: Cultured epithelial cells were treated with 5, 50 and 500 ng mL −1 mouse S100A9 (50284‐M07E, Sino Biological), 1, 10 and 100 ng mL −1 mouse TNF (410‐MT‐010, Bio‐Techne, Minnesota, USA), 10 ng mL −1 mouse IL‐18 (50073‐MNCE, Sino Biological), 100 ng mL −1 mouse IL‐18BP (50206‐M08H, Sino Biological), 10 ng mL −1 mouse IL‐1β (211‐11B, Peprotech), or 10 ng mL −1 mouse CTSB (50084‐M08H, Sino Biological) for 3 h. Cultured endometrial epithelial cells were also treated with mouse CTSB and disulfiram (pyroptosis inhibitor, PHR1690, Sigma–Aldrich), CTSB and CA‐074 Me (CTSB inhibitor, S7420, Selleck, Shanghai, China), CTSB and NLRP3‐IN‐21, disulfiram, NLRP3‐IN‐21 or CA‐074 Me for 3 h. Endometrial epithelial cells were treated with CTSB and PI, EthD‐III (HY‐D1723, MedChemExpress) or DRAQ7 (D15106, ThermoFisher, Waltham, MA, USA) for 0, 3, 5, 8, and 10 min. A fluorescence signal was detected.

Techniques: Western Blot, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Embryo‐Derived Cathepsin B Promotes Implantation and Decidualization by Activating Pyroptosis

doi: 10.1002/advs.202402299

Figure Lengend Snippet: Function of pyroptosis during embryo implantation. A) The number of implantation sites on day 5 of pregnancy after disulfiram, an inhibitor of pyroptosis, was injected into the uterine lumen of day 4 pregnant mice ( n = 6 per group). B) The number of implantation sites on day 5 of pregnancy after CA‐074 Me, an inhibitor of CTSB, was injected into the uterine lumen of day 4 pregnant mice ( n = 9 per group). C) The number of implantation sites on day 5 of pregnancy after IL‐18BP was injected into the uterine lumen of day 4 pregnant mice ( n = 6 per group). D) The number of implantation sites on day 5 of pregnancy after NLRP3‐IN‐21 was injected into the uterine lumen of day 4 pregnant mice ( n = 5 per group). Data were presented as mean ± SD. * : p < 0.05; ** : p < 0.01, by two‐tailed Student's t‐test.

Article Snippet: Cultured epithelial cells were treated with 5, 50 and 500 ng mL −1 mouse S100A9 (50284‐M07E, Sino Biological), 1, 10 and 100 ng mL −1 mouse TNF (410‐MT‐010, Bio‐Techne, Minnesota, USA), 10 ng mL −1 mouse IL‐18 (50073‐MNCE, Sino Biological), 100 ng mL −1 mouse IL‐18BP (50206‐M08H, Sino Biological), 10 ng mL −1 mouse IL‐1β (211‐11B, Peprotech), or 10 ng mL −1 mouse CTSB (50084‐M08H, Sino Biological) for 3 h. Cultured endometrial epithelial cells were also treated with mouse CTSB and disulfiram (pyroptosis inhibitor, PHR1690, Sigma–Aldrich), CTSB and CA‐074 Me (CTSB inhibitor, S7420, Selleck, Shanghai, China), CTSB and NLRP3‐IN‐21, disulfiram, NLRP3‐IN‐21 or CA‐074 Me for 3 h. Endometrial epithelial cells were treated with CTSB and PI, EthD‐III (HY‐D1723, MedChemExpress) or DRAQ7 (D15106, ThermoFisher, Waltham, MA, USA) for 0, 3, 5, 8, and 10 min. A fluorescence signal was detected.

Techniques: Injection, Two Tailed Test

Journal: Materials Today Bio

Article Title: Injectable hypoxia-preconditioned cartilage progenitor cells-laden GelMA microspheres system for enhanced osteoarthritis treatment

doi: 10.1016/j.mtbio.2023.100637

Figure Lengend Snippet: Hypoxia-pretreated CGMs alleviate oxidative stress and inflammatory response and promote NRF2/HO-1 pathway activation in IL-1β-induced chondrocytes. (A) The level of malondialdehyde (MDA) in each group was detected after co-incubation for 48 ​h (n ​= ​3). (B) The levels of reactive oxygen species (ROS) in different groups were analyzed after co-incubation for 48 ​h (n ​= ​3). (C) Superoxide dismutase (SOD) activity levels were evaluated after different treatments after co-incubation for 48 ​h (n ​= ​3). (D) The concentrations of TNFα, IL6, and MMP13 were evaluated after co-incubation for 48 ​h by ELISA assay (n ​= ​3). (E–H) The mRNA expressions of the inflammatory factors ( Tnfα, Il6 , iNOS , and Cox2 ) in each group were detected after co-incubation for 48 ​h by PCR analysis (n ​= ​3). (I and J) The mRNA expressions of the antioxidant genes ( Sod1 and Sod2 ) were detected after co-incubation for 48 ​h by PCR analysis (n ​= ​3). (K) The mRNA expressions of the NRF2/HO-1 pathway-related genes ( Keap1 , Nrf2 , HO-1 , and NQO1 ) were detected after co-incubation for 48 ​h by PCR analysis (n ​= ​3). (L) The protein expressions of the NRF2/HO-1 pathway-related markers (KEAP1, NRF2, and HO-1) were assessed after co-incubation for 48 ​h by western blotting analysis. Data represent mean ​± ​S.D. ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001 versus the Control group. # P ​< ​0.05, ## P ​< ​0.01, ### P ​< ​0.001 versus the IL-1β group. $ P ​< ​0.05, $$ P ​< ​0.01, $$$ P ​< ​0.001 versus the norm-CGMs group.

Article Snippet: The primary antibodies Collagen2 (Abcam, USA), Aggrecan (Abcam, USA), GDF5 (Abcam, USA), PRG4 (Abcam, USA), SOX9 (Preoteintech, USA), ADAMTS5 (Abcam, USA), MMP13 (Abcam, USA), TNFa (Abcam, USA), IL6 (Abcam, USA), SOD1 (Proteintech, USA), KEAP1 (Abcam, USA), NRF2 (Abcam, USA), HO-1 (Abcam, USA), β-actin (Abcam, USA), and horseradish peroxidase (HRP)-conjugated secondary antibody were used.

Techniques: Activation Assay, Incubation, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Materials Today Bio

Article Title: Injectable hypoxia-preconditioned cartilage progenitor cells-laden GelMA microspheres system for enhanced osteoarthritis treatment

doi: 10.1016/j.mtbio.2023.100637

Figure Lengend Snippet: Hypoxia-pretreated CGMs ameliorate cartilage inflammatory response and oxidative stress and activate NRF2/HO-1 pathway in subacute OA rat. (A and B) Synovial inflammation in each group was determined by H&E staining and scored (n ​= ​5). (C) Immunohistochemical staining inflammatory factors (IL-1β, IL6, and TNFα) in different groups (n ​= ​5). Scale bar, 25 ​μm. (D–F) Quantification of IL-1β-positivity, IL6-positivity, and TNFα-positivity in chondrocytes after different treatments in subacute OA rat (n ​= ​5). (G) Immunohistochemical staining of antioxidant enzyme SOD1 and the NRF2/HO-1 pathway-associated markers (NRF2, HO-1, and NQO1) in different groups (n ​= ​5). Scale bar, 25 ​μm. (H) Quantification of SOD1-positive chondrocytes after different treatments (n ​= ​5). (I–K) Quantification of NRF2-positivity, HO-1-positivity, and NQO1-positivity in chondrocytes in different groups (n ​= ​5). Data represent mean ​± ​S.D. ∗∗∗P ​< ​0.001 versus the Sham group. # P ​< ​0.05, ### P ​< ​0.001 versus the PBS group. $$ P ​< ​0.01, $$$ P ​< ​0.001 versus the norm-CGMs group.

Article Snippet: The primary antibodies Collagen2 (Abcam, USA), Aggrecan (Abcam, USA), GDF5 (Abcam, USA), PRG4 (Abcam, USA), SOX9 (Preoteintech, USA), ADAMTS5 (Abcam, USA), MMP13 (Abcam, USA), TNFa (Abcam, USA), IL6 (Abcam, USA), SOD1 (Proteintech, USA), KEAP1 (Abcam, USA), NRF2 (Abcam, USA), HO-1 (Abcam, USA), β-actin (Abcam, USA), and horseradish peroxidase (HRP)-conjugated secondary antibody were used.

Techniques: Staining, Immunohistochemical staining

Journal: Materials Today Bio

Article Title: Injectable hypoxia-preconditioned cartilage progenitor cells-laden GelMA microspheres system for enhanced osteoarthritis treatment

doi: 10.1016/j.mtbio.2023.100637

Figure Lengend Snippet: Hypoxia-pretreated CGMs alleviate cartilage inflammation and oxidative stress and promote the activation of NRF2/HO-1 pathway in chronic OA rat. (A and B) Synovial inflammation in each group was determined by H&E staining and scored (n ​= ​5). (C) Immunohistochemical staining inflammatory factors (IL-1β, IL6, and TNFα) in articular cartilage (n ​= ​5). Scale bar, 25 ​μm. (D–F) Quantification of IL-1β-positivity, IL6-positivity, and TNFα-positivity in chondrocytes after different treatments in chronic OA rat (n ​= ​5). (G) Immunohistochemical staining of antioxidant enzyme SOD1 and the NRF2/HO-1 pathway-related markers (NRF2, HO-1, and NQO1) in different groups (n ​= ​5). Scale bar, 25 ​μm. (H) Quantification of SOD1-positive chondrocytes in each group (n ​= ​5). (I–K) Quantification of NRF2-positivity, HO-1-positivity, and NQO1-positivity in chondrocytes in different groups (n ​= ​5). Data represent mean ​± ​S.D. ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001 versus the Sham group. # P ​< ​0.05, ## P ​< ​0.01, ### P ​< ​0.001 versus the PBS group. $$ P ​< ​0.01 versus the norm-CGMs group.

Article Snippet: The primary antibodies Collagen2 (Abcam, USA), Aggrecan (Abcam, USA), GDF5 (Abcam, USA), PRG4 (Abcam, USA), SOX9 (Preoteintech, USA), ADAMTS5 (Abcam, USA), MMP13 (Abcam, USA), TNFa (Abcam, USA), IL6 (Abcam, USA), SOD1 (Proteintech, USA), KEAP1 (Abcam, USA), NRF2 (Abcam, USA), HO-1 (Abcam, USA), β-actin (Abcam, USA), and horseradish peroxidase (HRP)-conjugated secondary antibody were used.

Techniques: Activation Assay, Staining, Immunohistochemical staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Embryo-Derived Cathepsin B Promotes Implantation and Decidualization by Activating Pyroptosis.

doi: 10.1002/advs.202402299

Figure Lengend Snippet: Figure 2. Blastocyst-derived CTSB triggers pyroptosis. A) CTSB immunofluorescence (green) and PI fluorescence (red) at the implantation site on day 5 of pregnancy. Arrowhead, blastocyst. B) CTSB immunofluorescence of CTSB (green) and DAPI fluorescence (blue) in mouse blastocyst collected on day 4 of pregnancy. C) ELISA analysis of CTSB protein in the cultured medium after blastocytes were cultured in KSOM medium for 6 h (n = 4 per group). D) Western blot analysis and quantification of CTSB protein levels in cultured medium after dormant blastocysts were treated with different concentrations of estradiol-17𝛽for 12 h (n = 3 per group). Coomassie bright blue staining was used as a loading control. E) CTSB immunofluorescence (green) and DAPI fluorescence (blue) in dormant and dormant blastocysts treated with 1 nM estradiol-17𝛽for 12 h. F) Western blot analysis and quantification of uterine ASC, NLRP3, CASPASE 1, cleaved CASPASE 1, GSDMD-N, GSDMD, IL-18, and IL-1𝛽protein levels after CTSB was injected into uterine lumen of day 4 pseudopregnant mice (n = 3 per group). G) The blue bands of attachment response after CTSB-soaked beads were transferred into the uterine lumen of day 4 pseudopregnant mice for 24 h (n = 6 per group). H) GSDMD immunofluorescence (green) and PI fluorescence (red) after CTSB-soaked beads were transferred into the uterine lumen of day 4 pseudopregnant mice. Arrowhead, CTSB-soaked beads. Scale bar = 125 μm. Data were presented as mean ± SD. *: p < 0.05; **: p < 0.01; ns: not significant, by two-tailed Student’s t-test.

Article Snippet: Cultured epithelial cells were treated with 5, 50 and 500 ng mL−1 mouse S100A9 (50284-M07E, Sino Biological), 1, 10 and 100 ng mL−1 mouse TNF (410- MT-010, Bio-Techne, Minnesota, USA), 10 ng mL−1 mouse IL-18 (50073- MNCE, Sino Biological), 100 ng mL−1 mouse IL-18BP (50206-M08H, Sino Biological), 10 ng mL−1 mouse IL-1β (211-11B, Peprotech), or 10 ng mL−1 mouse CTSB (50084-M08H, Sino Biological) for 3 h. Cultured endometrial epithelial cells were also treated with mouse CTSB and disulfiram (pyroptosis inhibitor, PHR1690, Sigma–Aldrich), CTSB and CA-074 Me (CTSB inhibitor, S7420, Selleck, Shanghai, China), CTSB and NLRP3-IN-21, disulfiram, NLRP3-IN-21 or CA-074 Me for 3 h. Endometrial epithelial cells were treated with CTSB and PI, EthD-III (HY-D1723, MedChemExpress) or DRAQ7 (D15106, ThermoFisher, Waltham, MA, USA) for 0, 3, 5, 8, and 10 min. A fluorescence signal was detected.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Staining, Control, Injection, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Embryo-Derived Cathepsin B Promotes Implantation and Decidualization by Activating Pyroptosis.

doi: 10.1002/advs.202402299

Figure Lengend Snippet: Figure 3. CTSB induces rapid and reversible pyroptosis in mouse endometrial epithelial cells. A) Western blot analysis of pyroptosis-associated protein levels after endometrial epithelial cells were treated with CTSB, CTSB and disulfiram, or disulfiram for 3 h. B) Western blot analysis of pyroptosis- associated protein levels after endometrial epithelial cells were treated with CTSB, CTSB and CA-074 Me, or CA-074 Me for 3 h. C) Western blot analysis of pyroptosis-associated protein levels after endometrial epithelial cells were treated with CTSB, CTSB and NLRP3-IN-21, or NLRP3-IN-21 for 3 h. D) NINJ1 immunofluorescence (Green) and PI fluorescence (red) at the implantation site and inter-implantation site on day 4.5 of pregnancy. Arrowhead,

Article Snippet: Cultured epithelial cells were treated with 5, 50 and 500 ng mL−1 mouse S100A9 (50284-M07E, Sino Biological), 1, 10 and 100 ng mL−1 mouse TNF (410- MT-010, Bio-Techne, Minnesota, USA), 10 ng mL−1 mouse IL-18 (50073- MNCE, Sino Biological), 100 ng mL−1 mouse IL-18BP (50206-M08H, Sino Biological), 10 ng mL−1 mouse IL-1β (211-11B, Peprotech), or 10 ng mL−1 mouse CTSB (50084-M08H, Sino Biological) for 3 h. Cultured endometrial epithelial cells were also treated with mouse CTSB and disulfiram (pyroptosis inhibitor, PHR1690, Sigma–Aldrich), CTSB and CA-074 Me (CTSB inhibitor, S7420, Selleck, Shanghai, China), CTSB and NLRP3-IN-21, disulfiram, NLRP3-IN-21 or CA-074 Me for 3 h. Endometrial epithelial cells were treated with CTSB and PI, EthD-III (HY-D1723, MedChemExpress) or DRAQ7 (D15106, ThermoFisher, Waltham, MA, USA) for 0, 3, 5, 8, and 10 min. A fluorescence signal was detected.

Techniques: Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Embryo-Derived Cathepsin B Promotes Implantation and Decidualization by Activating Pyroptosis.

doi: 10.1002/advs.202402299

Figure Lengend Snippet: Figure 4. Function of pyroptosis during embryo implantation. A) The number of implantation sites on day 5 of pregnancy after disulfiram, an inhibitor of pyroptosis, was injected into the uterine lumen of day 4 pregnant mice (n = 6 per group). B) The number of implantation sites on day 5 of pregnancy after CA-074 Me, an inhibitor of CTSB, was injected into the uterine lumen of day 4 pregnant mice (n = 9 per group). C) The number of implantation sites on day 5 of pregnancy after IL-18BP was injected into the uterine lumen of day 4 pregnant mice (n = 6 per group). D) The number of implantation sites on day 5 of pregnancy after NLRP3-IN-21 was injected into the uterine lumen of day 4 pregnant mice (n = 5 per group). Data were presented as mean ± SD. *: p < 0.05; **: p < 0.01, by two-tailed Student’s t-test.

Article Snippet: Cultured epithelial cells were treated with 5, 50 and 500 ng mL−1 mouse S100A9 (50284-M07E, Sino Biological), 1, 10 and 100 ng mL−1 mouse TNF (410- MT-010, Bio-Techne, Minnesota, USA), 10 ng mL−1 mouse IL-18 (50073- MNCE, Sino Biological), 100 ng mL−1 mouse IL-18BP (50206-M08H, Sino Biological), 10 ng mL−1 mouse IL-1β (211-11B, Peprotech), or 10 ng mL−1 mouse CTSB (50084-M08H, Sino Biological) for 3 h. Cultured endometrial epithelial cells were also treated with mouse CTSB and disulfiram (pyroptosis inhibitor, PHR1690, Sigma–Aldrich), CTSB and CA-074 Me (CTSB inhibitor, S7420, Selleck, Shanghai, China), CTSB and NLRP3-IN-21, disulfiram, NLRP3-IN-21 or CA-074 Me for 3 h. Endometrial epithelial cells were treated with CTSB and PI, EthD-III (HY-D1723, MedChemExpress) or DRAQ7 (D15106, ThermoFisher, Waltham, MA, USA) for 0, 3, 5, 8, and 10 min. A fluorescence signal was detected.

Techniques: Injection, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Embryo-Derived Cathepsin B Promotes Implantation and Decidualization by Activating Pyroptosis.

doi: 10.1002/advs.202402299

Figure Lengend Snippet: Figure 6. IL-18 promotes decidualization through activating epithelial ADAM12 and EREG. A) qPCR analysis on effects of IL-18 on Prl8a2 mRNA level under in vitro decidualization for 48 h (n = 4 per group). B) qPCR analysis on effects of IL-1𝛽on Prl8a2 mRNA level under in vitro decidualization for 48 h (n = 4 per group). C) qPCR analysis on Prl8a2 mRNA level in stromal cells after the co-culture of epithelial and stromal cells were treated with IL-1𝛽 for 48 h (n = 4 per group). D) qPCR analysis on Prl8a2 mRNA level in stromal cells after the co-culture of epithelial and stromal cells were treated with IL-18 for 48 h (n = 4 per group). E) qPCR analysis on Prl8a2 mRNA level after the co-culture of epithelial and stromal cells were treated with CTSB, CTSB and IL-18BP, CTSB and NLRP3-IN-21, IL-18BP, or NLRP3-IN-21 for 48 h (n = 4 per group). F) Western blot analysis and quantification of AREG, HB-EGF, and EREG protein levels after endometrial epithelial cells were treated with IL-18 for 3 h (n = 3 per group). G) Western blot analysis and quantification of ADAM12 and EREG protein levels after endometrial epithelial cells were treated with IL-18, IL-18 and IL-18BP, or IL-18BP for 3 h (n = 3 per group). H) Western blot analysis and quantification of ADAM12 and EREG protein levels after endometrial epithelial cells were treated with IL-18, IL-18 and ADAM12 siRNA, or ADAM12 siRNA for 3 h (n = 3 per group). I) qPCR analysis on Prl8a2 mRNA level after stromal cells were treated with EREG under in vitro decidualization for 48 h (n = 4 per group). J) qPCR analysis on Prl8a2 mRNA level after the co-culture of epithelial and stromal cells under in vitro decidualization was treated with IL-18, ADAM12 siRNA, IL-18BP, IL-18 and ADAM12 siRNA, or IL-18 and IL-18BP for 48 h (n = 4 per group). EP, treatment with estradiol-17𝛽and progesterone for in vitro decidualization. Data were presented as mean±SD. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant, by two-tailed Student’s t-test.

Article Snippet: Cultured epithelial cells were treated with 5, 50 and 500 ng mL−1 mouse S100A9 (50284-M07E, Sino Biological), 1, 10 and 100 ng mL−1 mouse TNF (410- MT-010, Bio-Techne, Minnesota, USA), 10 ng mL−1 mouse IL-18 (50073- MNCE, Sino Biological), 100 ng mL−1 mouse IL-18BP (50206-M08H, Sino Biological), 10 ng mL−1 mouse IL-1β (211-11B, Peprotech), or 10 ng mL−1 mouse CTSB (50084-M08H, Sino Biological) for 3 h. Cultured endometrial epithelial cells were also treated with mouse CTSB and disulfiram (pyroptosis inhibitor, PHR1690, Sigma–Aldrich), CTSB and CA-074 Me (CTSB inhibitor, S7420, Selleck, Shanghai, China), CTSB and NLRP3-IN-21, disulfiram, NLRP3-IN-21 or CA-074 Me for 3 h. Endometrial epithelial cells were treated with CTSB and PI, EthD-III (HY-D1723, MedChemExpress) or DRAQ7 (D15106, ThermoFisher, Waltham, MA, USA) for 0, 3, 5, 8, and 10 min. A fluorescence signal was detected.

Techniques: In Vitro, Co-Culture Assay, Western Blot, Two Tailed Test