Journal: PLoS ONE

Article Title: Pre-eclampsia is associated with altered expression of the renal sodium transporters NKCC2, NCC and ENaC in urinary extracellular vesicles

doi: 10.1371/journal.pone.0204514

Figure Lengend Snippet: Antibodies used in the study.

Article Snippet: Anti-NKCC2 , Rabbit polyclonal , Rat N-terminus aa 33–55 , Stress-Marq.

Techniques:

Journal: PLoS ONE

Article Title: Pre-eclampsia is associated with altered expression of the renal sodium transporters NKCC2, NCC and ENaC in urinary extracellular vesicles

doi: 10.1371/journal.pone.0204514

Figure Lengend Snippet: (A) Representative Western blot from normal controls (NC), women with normal pregnancies (NP) and with pre-eclampsia (PE) probed with antibodies against NKCC2, pNKCC2 (S130 and T101/105), and CD9. (B) Densitometric analysis of total NKCC2 normalised for CD9 in the three groups. Difference between groups by Kruskall-Wallis test p<0.002. (C and D) Densitometric analysis of total NKCC2 pS130 normalised for total NKCC2 (C) and CD9 (D) in the three groups. Difference between groups by Kruskall-Wallis test p<0.0001 for both comparisons. (E and F) Densitometric analysis of total NKCC2 pS130 normalised for total NKCC2 (E) and CD9 (F) in the three groups. Difference between groups by Kruskall-Wallis test p<0.01 for (E) and not significant for (F). Individual comparisons by Dunns’ test *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Anti-NKCC2 , Rabbit polyclonal , Rat N-terminus aa 33–55 , Stress-Marq.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Pre-eclampsia is associated with altered expression of the renal sodium transporters NKCC2, NCC and ENaC in urinary extracellular vesicles

doi: 10.1371/journal.pone.0204514

Figure Lengend Snippet: Detection of bands for phosphorylated forms of NKCC2 and NCC, and for ENaC proteins.

Article Snippet: Anti-NKCC2 , Rabbit polyclonal , Rat N-terminus aa 33–55 , Stress-Marq.

Techniques:

Journal: PLoS ONE

Article Title: Pre-eclampsia is associated with altered expression of the renal sodium transporters NKCC2, NCC and ENaC in urinary extracellular vesicles

doi: 10.1371/journal.pone.0204514

Figure Lengend Snippet: (A) NKCC2 pS130 vs urine protein/creatinine ratio, p = 0.014, n = 19. (B) γ-ENaC 50kD species vs urine protein/creatinine ratio, p = 0.001, n = 19. (C) γ-ENaC 50kD species vs urine protein/creatinine ratio, p = 0.002, n = 19.

Article Snippet: Anti-NKCC2 , Rabbit polyclonal , Rat N-terminus aa 33–55 , Stress-Marq.

Techniques:

Journal: PLoS ONE

Article Title: Pre-eclampsia is associated with altered expression of the renal sodium transporters NKCC2, NCC and ENaC in urinary extracellular vesicles

doi: 10.1371/journal.pone.0204514

Figure Lengend Snippet: Linear regression analysis of sodium transporter densitometry as a ratio with CD9.

Article Snippet: Anti-NKCC2 , Rabbit polyclonal , Rat N-terminus aa 33–55 , Stress-Marq.

Techniques:

Journal: Advanced Science

Article Title: Lnc‐HZ05 Suppresses Trophoblast Cell Migrasome Formation by Disrupting TGFβ2 Pathway in Unexplained Recurrent Miscarriage

doi: 10.1002/advs.202417558

Figure Lengend Snippet: Tgfβ2 signaling, migration/invasion, and migrasome formation were suppressed in placental tissues of a mouse miscarriage model. A) The scheme for BaP‐exposed mouse model with miscarriage. Pregnant mice were daily given 0, 0.05, or 0.2 mg kg −1 BaP in corn oil by oral gavage from D1 to D13 and were euthanized on D14 for collection of uterus (each n = 6). B) Embryo resorption (indicated by red arrows) in BaP‐exposed mice (scale bar, 1 cm). C) The average miscarriage rates (the ratios of embryo resorption) in BaP‐exposed mice (each n = 6). D,E,F) The protein levels of murine Tgfβ2, Tgfβr2, Smad3, pSmad3, Ndst1, and Tspan4 in placental tissues of BaP‐exposed mice and their relative quantification (each n = 6). G) The scheme for a mouse miscarriage intervention model. H,I) Embryo resorption (indicated by the red arrows) and miscarriage rates in 0.2 mg kg −1 BaP‐exposed mice supplemented with murine Tspan4. J) The protein levels of Ndst1 and Tspan4 in 0.2 mg kg −1 BaP‐exposed mice supplemented with Tspan4, with β‐Tubulin as internal standard (each n = 6). All results are representative data from three independent experiments. Data are presented as mean ± SD. The unpaired two‐tailed Student's t ‐test I,J) and one‐way ANOVA with the Tukey's multiple comparison test C,F). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant murine Tgfβ2 protein (HY‐ P70649 ) was from MedChemExpress (New Jersey, USA).

Techniques: Migration, Quantitative Proteomics, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Lnc‐HZ05 Suppresses Trophoblast Cell Migrasome Formation by Disrupting TGFβ2 Pathway in Unexplained Recurrent Miscarriage

doi: 10.1002/advs.202417558

Figure Lengend Snippet: Lnc‐HZ05 down‐regulated TGFβ2 signaling and suppressed trophoblast cell migration/invasion and migrosome formation. A) RT‐qPCR analysis of lnc‐HZ05 expression levels in HC and RM villous tissues (each n = 30). B) Lnc‐HZ05 levels in Swan 71 cells with lnc‐HZ05 overexpression or knockdown. C) Volcano plot analysis of the DEMs in lnc‐HZ05‐overexpressed Swan 71 cells versus control cells with difference >2‐fold and p < 0.05. D) KEGG analysis of the down‐regulated mRNAs in lnc‐HZ05‐overexpressed Swan 71 cells vs control cells. E) The mRNA levels of TGFβ2 in Swan 71 cells with lnc‐HZ05 overexpression or knockdown. F,G) The protein levels of TGFβ2, TGFβR2, Smad3, and pSmad3 in Swan 71 cells with lnc‐HZ05 overexpression or knockdown and their relative quantification. H,I) The nuclear cytoplasmic distribution of pSmad3 protein levels in Swan 71 with lnc‐HZ05 overexpression or knockdown, with Tubulin as cytoplasmic (C) indicator and H3 as nuclear (N) indicator, and its relative quantification. J) The protein levels of TGFβ2, Smad3, and pSmad3 in Swan 71 cells with co‐overexpression of lnc‐HZ05 and TGFβ2 and their relative quantification. K) Transwell assay analysis of the migration/invasion of Swan 71 cells with lnc‐HZ05 overexpression or knockdown, and their quantification. Scale bars, 100 µm. L) Transwell assay analysis of the migration/invasion of Swan 71 cells with co‐overexpression of lnc‐HZ05 and TGFβ2 and their relative quantification. Scale bars, 100 µm. M) The protein levels of NDST1 in Swan 71 or HTR/SVneo cells with lnc‐HZ05 overexpression and its relative quantification. N) Migrasome assays showed the formation of migrasome in Swan 71 cells overexpressing TSPAN4‐GFP and lnc‐HZ05, and their quantification (n = 100). Scale bar, 10 µm. All results are representative data from three independent experiments. Data are presented as mean ± SD. The unpaired two‐tailed Student's t ‐test A, B, E, G, I, K, M, and N) and one‐way ANOVA with the Tukey's multiple comparison test B, E, G, and I–L) were used for statistical analysis. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant murine Tgfβ2 protein (HY‐ P70649 ) was from MedChemExpress (New Jersey, USA).

Techniques: Migration, Quantitative RT-PCR, Expressing, Over Expression, Knockdown, Control, Quantitative Proteomics, Transwell Assay, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Lnc‐HZ05 Suppresses Trophoblast Cell Migrasome Formation by Disrupting TGFβ2 Pathway in Unexplained Recurrent Miscarriage

doi: 10.1002/advs.202417558

Figure Lengend Snippet: Lnc‐HZ05 suppressed TGFβ2 mRNA transcription in human trophoblast cells. A) The mRNA levels of TGFβ2 in Swan 71 cells with FOXP3 overexpression or knockdown. B,C) The protein levels of TGFβ2 and FOXP3 in Swan 71 or HTR‐8/SVneo cells with FOXP3 overexpression or knockdown and their relative quantification. D–F) The mRNA and protein levels of FOXP3 in Swan 71 cells with lnc‐HZ05 overexpression or knockdown and its relative quantification. G) ChIP assay analysis of the levels of TGFβ2 promoter region enriched by FOXP3 in Swan 71 cells with lnc‐HZ05 overexpression. H) Dual‐luciferase reporter assay analysis of the transcription activity of FOXP3 at wild‐type (wt) or mutant (mut) promoter region of TGFβ2 in Swan 71 or HTR‐8/SVneo cells with lnc‐HZ05 overexpression. I,J) The protein levels of FOXP3, TGFβ2, Smad3, and pSmad3 in Swan 71 or HTR‐8/SVneo cells with co‐overexpression of FOXP3 and lnc‐HZ05 and their relative quantification. K) The mRNA levels of TGFβ2 and GAPDH in 10 µ m ActD‐treated Swan 71 cells within 10 h. All results are representative data from three independent experiments. Data are presented as mean ± SD. The unpaired two‐tailed Student's t‐test A–D and F) and one‐way ANOVA with the Tukey's multiple comparison test (A–D, and F–J) were used for statistical analysis. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant murine Tgfβ2 protein (HY‐ P70649 ) was from MedChemExpress (New Jersey, USA).

Techniques: Over Expression, Knockdown, Quantitative Proteomics, Luciferase, Reporter Assay, Activity Assay, Mutagenesis, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Lnc‐HZ05 Suppresses Trophoblast Cell Migrasome Formation by Disrupting TGFβ2 Pathway in Unexplained Recurrent Miscarriage

doi: 10.1002/advs.202417558

Figure Lengend Snippet: Lnc‐HZ05 promoted TGFβ2 protein autophagy degradation in human trophoblast cells. A) The protein levels of TGFβ2 in 10 µM CHX‐treated Swan 71 cells with lnc‐HZ05 overexpression or knockdown within 8 h and its relative quantification. B,C) The protein levels of TGFβ2 in lnc‐HZ05‐overexpressed and CHX‐treated Swan 71 cells with co‐treatment with 10 µ m MG132 or 50 µ m CQ and its relative quantification. D) IP assays using identical and limited TGFβ2 antibody showed the levels of poly‐ubiquitinated TGFβ2 (TGFβ2‐Ub) in Swan 71 cells with lnc‐HZ05 overexpression or 10 µ m MG132 treatment and its relative quantification, with the protein levels of TGFβ2 in cell lysates. E) The ratios of LC3II/LC3I protein and the protein levels of P62 in Swan 71 cells with lnc‐HZ05 overexpression or knockdown and their relative quantification. F–H) The formation of autophagosomes as indicated by the number of LC3‐GFP puncta (shown in green) in Swan 71 cells with lnc‐HZ05 overexpression or knockdown and their quantification (n = 10). Scale bar: 10 µm. All results are representative data from three independent experiments. Data are presented as mean ± SD. The unpaired two‐tailed Student's t‐test E, G, and H) and one‐way ANOVA with the Tukey's multiple comparison test (C–E) were used for statistical analysis. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant murine Tgfβ2 protein (HY‐ P70649 ) was from MedChemExpress (New Jersey, USA).

Techniques: Over Expression, Knockdown, Quantitative Proteomics, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Lnc‐HZ05 Suppresses Trophoblast Cell Migrasome Formation by Disrupting TGFβ2 Pathway in Unexplained Recurrent Miscarriage

doi: 10.1002/advs.202417558

Figure Lengend Snippet: Lnc‐HZ05 impaired TGFβ2/TGFβR2 protein interactions in human trophoblast cells. A–C) IP assays using identical and limited TGFβ2 or TGFβR2 antibody showed the protein levels of the immunoprecipitated TGFβR2 or TGFβ2, respectively, in Swan 71 cells with lnc‐HZ05 overexpression or knockdown and their relative quantification, with the protein levels of TGFβ2 and TGFβR2 in cell lysates. D,E) IP assays using identical and limited TGFβR2 antibody showed the protein levels of the immunoprecipitated TGFβ2 in Swan 71 cells with lnc‐HZ05 overexpression and Rnase A treatment and its relative quantification, with the protein levels of TGFβ2 and TGFβR2 in cell lysates. F) RIP assays using identical and excessive TGFβ2 or TGFβR2 antibody showed that lnc‐HZ05 was pulled down by TGFβ2 or TGFβR2 in Swan 71 cells. G) The protein levels of TGFβ2 or TGFβR2 pulled down by biotin‐labeled lnc‐HZ05 in Swan 71 cells or HTR8/SVneo cells. H) Schematic diagram of RIP‐re‐RIP. In the first round of RIP, one antibody was used to immunoprecipitate proteins containing lnc‐HZ05; in the second round of RIP, the other antibody was used to immunoprecipitate the remaining proteins containing lnc‐HZ05. In both rounds, lnc‐HZ05 were separated and detected. I) The levels of lnc‐HZ05 enriched by TGFβ2 or TGFβR2 in the first and second rounds of RIP‐re‐RIP assays in Swan 71 cells. J) The levels of lnc‐HZ05 pulled down by Flag‐labeled wild‐type (wt) or mutant (mut) TGFβ2 or TGFβR2 in RIP assays. K) The schematic diagram that lnc‐HZ05 impaired the protein interactions between TGFβ2 and TGFβR2. All results are representative data from three independent experiments. Data are presented as mean ± SD. The unpaired two‐tailed Student's t ‐test (C) and one‐way ANOVA with the Tukey's multiple comparison test C, E, I, and J). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant murine Tgfβ2 protein (HY‐ P70649 ) was from MedChemExpress (New Jersey, USA).

Techniques: Immunoprecipitation, Over Expression, Knockdown, Quantitative Proteomics, Labeling, Mutagenesis, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Lnc‐HZ05 Suppresses Trophoblast Cell Migrasome Formation by Disrupting TGFβ2 Pathway in Unexplained Recurrent Miscarriage

doi: 10.1002/advs.202417558

Figure Lengend Snippet: Lnc‐HZ05‐S1 impaired TGFβ2/TGFβR2 protein interactions and also suppressed trophoblast cell migration/invasion. A) Lnc‐HZ05 was divided into three segments: lnc‐HZ05‐S1, lnc‐HZ05‐S2, and lnc‐HZ05‐S3. B) RIP assay analysis of the levels of lnc‐HZ05‐S1, lnc‐HZ05‐S2, and lnc‐HZ05‐S3 that was pulled down by TGFβ2 or TGFβR2 in Swan 71 cells treated with RNase T1. C) RNA pulldown assay analysis of the protein levels of TGFβ2 or TGFβR2 that was pulled down by biotin‐labeled various lnc‐HZ05 in Swan 71 cells or HTR‐8/SVneo cells. D) The levels of lnc‐HZ05‐S1, lnc‐HZ05‐S2, or lnc‐HZ05‐S3 pulled down by Flag‐labeled wild‐type (wt) or mutant (mut) TGFβ2 or TGFβR2 in RIP assays. E) The schematic diagram that lnc‐HZ05‐S1 impaired the protein interactions between TGFβ2 and TGFβR2. F) IP assays using identical but limited TGFβ2 antibody showed the protein levels of the immunoprecipitated TGFβR2 in Swan 71 cells with overexpression of lnc‐HZ05, lnc‐HZ05‐S1, lnc‐HZ05‐S2, or lnc‐HZ05‐S3 and its relative quantification, with the protein levels of TGFβ2 and TGFβR2 in cell lysates. G) The protein levels of TGFβ2, Smad3, pSmad3, NDST1, and TSPAN4 in Swan 71 with overexpression of lnc‐HZ05, lnc‐HZ05‐S1, lnc‐HZ05‐S2, or lnc‐HZ05‐S3, and their relative quantification. H) Transwell assay analysis of the migration/invasion of Swan 71 cells with co‐overexpression of lnc‐HZ05, lnc‐HZ05‐S1, lnc‐HZ05‐S2, or lnc‐HZ05‐S3 and their relative quantification. Scale bars, 100 µm. I) Migrasome assays showed the formation of migrasome in Swan 71 cells with overexpression of lnc‐HZ05, lnc‐HZ05‐S1, lnc‐HZ05‐S2, or lnc‐HZ05‐S3 (n = 100). Scale bar, 10 µm. All results are representative data from three independent experiments. Data are presented as mean ± SD. The one‐way ANOVA with the Tukey's multiple comparison test B, D, F–H). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant murine Tgfβ2 protein (HY‐ P70649 ) was from MedChemExpress (New Jersey, USA).

Techniques: Migration, Labeling, Mutagenesis, Immunoprecipitation, Over Expression, Quantitative Proteomics, Transwell Assay, Comparison

Journal: Advanced Science

Article Title: Lnc‐HZ05 Suppresses Trophoblast Cell Migrasome Formation by Disrupting TGFβ2 Pathway in Unexplained Recurrent Miscarriage

doi: 10.1002/advs.202417558

Figure Lengend Snippet: Lnc‐HZ05 and TGFβ2 expression levels in HC and RM villous tissues. A) The protein levels of DNMT1 and FOXP3 in HC and RM villous tissues (n = 12) and their relative quantification. B) MS‐PCR analysis of the methylation (M) and unmethylation (UM) levels in lnc‐HZ05 promoter region in HC and RM villous tissues (n = 12) and its relative quantification. C) ChIP assay analysis of the levels of lnc‐HZ05 promoter region enriched by FOXP3 in HC and RM villous tissues (each n = 6), with IgG as negative control. D–F) The pearson correlation analysis of the relative levels of lnc‐HZ05 with the protein levels of TGFβ2, TGFβR2, Smad3, pSmad3, MMP‐2, NDST1 and TSPAN4 in HC (blue) and RM (pink) groups (n = 12). G) ChIP assay analysis of the levels of TGFβ2 promoter region enriched by FOXP3 in HC and RM villous tissues (each n = 6), with IgG as negative control. H) The pearson correlation analysis of the relative levels of lnc‐HZ05 with that of FOXP3 protein in HC (blue) and RM (pink) groups (each n = 12). I) RIP assay using identical but excessive TGFβ2 or TGFβR2 antibody showed the levels of lnc‐HZ05 enriched by TGFβ2 or TGFβR2 in HC and RM villous tissues (each n = 12). J) IP assay using identical but limited TGFβ2 antibody showed the protein levels of TGFβR2 immunoprecipitated by TGFβ2 in HC and RM villous tissues (each n = 6) and its relative quantification. All results are representative data from three independent experiments. Data are presented as mean ± SD. The unpaired two‐tailed Student's t ‐test (A, B, and H) and one‐way ANOVA with the Tukey's multiple comparison test (C, E, and G) were used for statistical analysis. Pearson analysis was used for the correlation analysis (D and F). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant murine Tgfβ2 protein (HY‐ P70649 ) was from MedChemExpress (New Jersey, USA).

Techniques: Expressing, Quantitative Proteomics, Methylation, Negative Control, Immunoprecipitation, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Lnc‐HZ05 Suppresses Trophoblast Cell Migrasome Formation by Disrupting TGFβ2 Pathway in Unexplained Recurrent Miscarriage

doi: 10.1002/advs.202417558

Figure Lengend Snippet: The levels of TGFβ2 protein and lnc‐HZ05 in serum might predict miscarriage risk. A) The levels of TGFβ2 protein in HC and RM serum samples (each n = 30). B) The reference range of TGFβ2 protein levels in HC and RM serum samples (each n = 30). C) The percentage of RM women in total women in each range of serum TGFβ2 protein levels. D) Multivariate logistic regression analysis of TGFβ2 protein levels in HC and RM serum samples by adjusting for all these variables. E) The ROC curve of the serum TGFβ2 protein levels. F) The levels of lnc‐HZ05 absolute copy number in HC and RM serum samples (each n = 30). G) The reference range of lnc‐HZ05 absolute copy number in HC and RM serum samples (each n = 30). H) The percentage of RM women in total women in each range of lnc‐HZ05 absolute copy number. I) Multivariate logistic regression analysis of lnc‐HZ05 absolute copy number in HC and RM serum samples by adjusting for all these variables. J) The ROC curve of lnc‐HZ05 absolute copy number in serum. All results are representative data from three independent experiments. Data are presented as mean ± SD. Unpaired two‐tailed Student's t ‐test (A and F) were used for statistical analysis. ROC curves were plotted using survival ROC package. ROC AUC (shortly AUC) is calculated as the area under the ROC curve (E and J). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant murine Tgfβ2 protein (HY‐ P70649 ) was from MedChemExpress (New Jersey, USA).

Techniques: Two Tailed Test

Journal: Advanced Science

Article Title: Lnc‐HZ05 Suppresses Trophoblast Cell Migrasome Formation by Disrupting TGFβ2 Pathway in Unexplained Recurrent Miscarriage

doi: 10.1002/advs.202417558

Figure Lengend Snippet: Miscarriage treatment by supplement with murine Tgfβ2 protein in the mouse miscarriage model. A) The scheme for a mouse miscarriage treatment model. Pregnant mice treated with 0 or 0.2 mg kg −1 d −1 BaP were intraperitoneally injected with saline or recombinant murine Tgfβ2 protein once per three days from D1 to D13 (each n = 6). B) The curves of body weight of 0 or 0.2 mg kg −1 d −1 BaP‐exposed pregnant mice that were intraperitoneally injected with saline or recombinant murine Tgfβ2 protein once per three days from D1 to D13. C) Embryo resorption (indicated by red arrows) in BaP‐exposed mice with Tgfβ2 supplement (scale bar, 1 cm). D) The average miscarriage rates (embryo resorption ratios) in BaP‐exposed mice with Tgfβ2 supplement (each n = 6). E,F) The protein levels of murine Tgfβ2, Tgfβr2, Smad3, pSmad3, Ndst1, and Tspan4 in placental tissues of BaP‐exposed mice with Tgfβ2 supplement and their relative quantification (each n = 6). All results are representative data from three independent experiments. Data are presented as mean ± SD. The one‐way ANOVA with the Tukey's multiple comparison test (B, D,E). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant murine Tgfβ2 protein (HY‐ P70649 ) was from MedChemExpress (New Jersey, USA).

Techniques: Injection, Saline, Recombinant, Quantitative Proteomics, Comparison