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tgfβ2 (Proteintech)

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Proteintech tgfβ2
Tgfβ2, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 78 article reviews

tgfβ2 - by Bioz Stars, 2026-02

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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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

Effects of anterior chamber injection of hBMMSC sEVs on IOP reduction and antifibrotic activity in mice with ocular hypertension. ( A ) IOP trends over 28 days among three groups of mice ( n = 8). *Significant differences in IOP between the antifibrotic group and the fibrotic group. #Significant differences between the fibrotic and control groups. ( B ) Autofluorescence of Ad-TGFβ2 C226/228S in the anterior chamber angle observed in frozen sections using confocal microscopy. (The TM region is demarcated by yellow dashed circles .) ( C – E ) Immunofluorescence analysis of FN and α-SMA expression in the anterior chamber angle ( n = 8). The y axis represents the mean fluorescence integrated density per unit area ( IntDen/Area ) in the TM region (demarcated by white dashed circles ). Statistical significance: * P < 0.05; *** P < 0.001; **** P < 0.0001; ### P < 0.001; ns, not significant.

Journal: Investigative Ophthalmology & Visual Science

Article Title: Small Extracellular Vesicle Treatment of Trabecular Meshwork Fibrosis: 2D/3D In Vitro and In Vivo Analyses

doi: 10.1167/iovs.66.12.48

Figure Lengend Snippet: Effects of anterior chamber injection of hBMMSC sEVs on IOP reduction and antifibrotic activity in mice with ocular hypertension. ( A ) IOP trends over 28 days among three groups of mice ( n = 8). *Significant differences in IOP between the antifibrotic group and the fibrotic group. #Significant differences between the fibrotic and control groups. ( B ) Autofluorescence of Ad-TGFβ2 C226/228S in the anterior chamber angle observed in frozen sections using confocal microscopy. (The TM region is demarcated by yellow dashed circles .) ( C – E ) Immunofluorescence analysis of FN and α-SMA expression in the anterior chamber angle ( n = 8). The y axis represents the mean fluorescence integrated density per unit area ( IntDen/Area ) in the TM region (demarcated by white dashed circles ). Statistical significance: * P < 0.05; *** P < 0.001; **** P < 0.0001; ### P < 0.001; ns, not significant.

Article Snippet: The fibrotic cells were treated with 5 ng/mL, 10 ng/mL, or 20 ng/mL TGFβ2 (HY-P7119, MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Injection, Activity Assay, Control, Confocal Microscopy, Immunofluorescence, Expressing, Fluorescence

( A, B ) Five-day TGFβ2 treatment (1 ng/ml) significantly altered expression of TGFβ pathway effectors, cytoskeletal machinery, and canonical fibrotic markers. ( C ) TGFβ2 treatment significantly increased TRPV4 and PIEZO1 expression, but not TREK1 and TRPC1 expression. Mean ± SEM shown. N = 4–8 experiments, each gene tested in 3–7 different pTM strains (see ). Two-tailed one-sample t -test of TGFβ2-induced gene expression levels as a percent of control samples. ( D ) Isolation of membrane proteins from two separate pooled pTM samples suggests TGFβ2 treatment drives increased TRPV4 membrane insertion. N = 2 independent pooled samples, 3 pTM strains were pooled per sample. *p < 0.05, **p < 0.01. Figure 1—source data 1. Uncropped images of the membrane and HRP-signal for the western blots shown in (labelled). Figure 1—source data 2. Uncropped images of the membrane and HRP-signal for the western blots shown in .

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A, B ) Five-day TGFβ2 treatment (1 ng/ml) significantly altered expression of TGFβ pathway effectors, cytoskeletal machinery, and canonical fibrotic markers. ( C ) TGFβ2 treatment significantly increased TRPV4 and PIEZO1 expression, but not TREK1 and TRPC1 expression. Mean ± SEM shown. N = 4–8 experiments, each gene tested in 3–7 different pTM strains (see ). Two-tailed one-sample t -test of TGFβ2-induced gene expression levels as a percent of control samples. ( D ) Isolation of membrane proteins from two separate pooled pTM samples suggests TGFβ2 treatment drives increased TRPV4 membrane insertion. N = 2 independent pooled samples, 3 pTM strains were pooled per sample. *p < 0.05, **p < 0.01. Figure 1—source data 1. Uncropped images of the membrane and HRP-signal for the western blots shown in (labelled). Figure 1—source data 2. Uncropped images of the membrane and HRP-signal for the western blots shown in .

Article Snippet: Lentiviral stock for TGFβ2 (C226,228S) was purchased from VectorBuilder Inc (Chicago, IL) (VB170816-1094fnw, pLV[Exp]-CMV> {hTGFB2[ NM_003238.3 ](C226,228S)}) ( ).

Techniques: Expressing, Two Tailed Test, Gene Expression, Control, Isolation, Membrane, Western Blot

( A ) Five-day TGFβ2 treatment (1 ng/ml) increased TRPV4 agonist-induced (GSK101, 10 nM) Ca 2+ influx in primary trabecular meshwork (pTM) cells compared to serum-free media alone treated cells tested on the same day ( N = 5 pTM strains, n = 3–5 slides/condition/day, individual data points over mean ± SEM). Two-tailed one-sample t -test of TGFβ2-treated cell average GSK101 response as a percent of control samples from the same pTM strain on the same day. ( B ) Violin plots showing the distribution of GSK101-induced Ca 2+ responses for each pTM strain tested in A. Thick dashed line indicates mean, while light dashed line indicates quartiles. ( C ) Representative traces showing TRPV4 agonist-induced Ca 2+ influx (seen as an increase in F 340 / F 380 ) in pTM (mean ± SEM of 4 representative cells/group), alongside example Fura-2-loaded pTM cells before ( i ), during ( ii ), and after ( iii ) GSK101 application. Scale bar = 50 µm. **p < 0.01.

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A ) Five-day TGFβ2 treatment (1 ng/ml) increased TRPV4 agonist-induced (GSK101, 10 nM) Ca 2+ influx in primary trabecular meshwork (pTM) cells compared to serum-free media alone treated cells tested on the same day ( N = 5 pTM strains, n = 3–5 slides/condition/day, individual data points over mean ± SEM). Two-tailed one-sample t -test of TGFβ2-treated cell average GSK101 response as a percent of control samples from the same pTM strain on the same day. ( B ) Violin plots showing the distribution of GSK101-induced Ca 2+ responses for each pTM strain tested in A. Thick dashed line indicates mean, while light dashed line indicates quartiles. ( C ) Representative traces showing TRPV4 agonist-induced Ca 2+ influx (seen as an increase in F 340 / F 380 ) in pTM (mean ± SEM of 4 representative cells/group), alongside example Fura-2-loaded pTM cells before ( i ), during ( ii ), and after ( iii ) GSK101 application. Scale bar = 50 µm. **p < 0.01.

Article Snippet: Lentiviral stock for TGFβ2 (C226,228S) was purchased from VectorBuilder Inc (Chicago, IL) (VB170816-1094fnw, pLV[Exp]-CMV> {hTGFB2[ NM_003238.3 ](C226,228S)}) ( ).

Techniques: Two Tailed Test, Control

( A ) TGFβ2 treatments for 24 hr at 1 ng/ml ( N = 6 pTM strains, n = 3–5 slides/condition/day) or 5 ng/ml ( N = 5 pTM strains, n = 3–5 slides/condition/day) did not show potentiation of GSK101-evoked TRPV4 Ca 2+ influx and were significantly lower than cells treated with TGFβ2 for 5 days at 1 ng/ml (5 days TGFβ2 results from ). Individual data points over mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test, statistics for individual 1 day treatment groups compared to control groups shown in . ( B ) Representative traces for GSK101 response following 24 hr TGFβ2 treatment, traces show mean ± SEM of 3–4 cells. ( C ) Average current density in response to GSK101 (24 hr control: n = 11 cells, 24 hr TGFβ2: n = 10 cells) shows generally increased current in TGFβ2-treated cells. Data show mean ± SEM ( D, E ). Violin plots of individual cell strains shown in A . Thick dashed line indicates mean, while light dashed line indicates quartiles. ** p < 0.01, *** p < 0.001.

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A ) TGFβ2 treatments for 24 hr at 1 ng/ml ( N = 6 pTM strains, n = 3–5 slides/condition/day) or 5 ng/ml ( N = 5 pTM strains, n = 3–5 slides/condition/day) did not show potentiation of GSK101-evoked TRPV4 Ca 2+ influx and were significantly lower than cells treated with TGFβ2 for 5 days at 1 ng/ml (5 days TGFβ2 results from ). Individual data points over mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test, statistics for individual 1 day treatment groups compared to control groups shown in . ( B ) Representative traces for GSK101 response following 24 hr TGFβ2 treatment, traces show mean ± SEM of 3–4 cells. ( C ) Average current density in response to GSK101 (24 hr control: n = 11 cells, 24 hr TGFβ2: n = 10 cells) shows generally increased current in TGFβ2-treated cells. Data show mean ± SEM ( D, E ). Violin plots of individual cell strains shown in A . Thick dashed line indicates mean, while light dashed line indicates quartiles. ** p < 0.01, *** p < 0.001.

Article Snippet: Lentiviral stock for TGFβ2 (C226,228S) was purchased from VectorBuilder Inc (Chicago, IL) (VB170816-1094fnw, pLV[Exp]-CMV> {hTGFB2[ NM_003238.3 ](C226,228S)}) ( ).

Techniques: Control

( A ) Representative longitudinal 24-well plate scans of collagen type I hydrogels seeded with primary TM (pTM) subjected to the different treatments (dashed lines outline size of contracted constructs). ( B ) Longitudinal quantification of hydrogel construct size compared to the control group at the 0 min time point. ( C ) Detailed comparisons between groups at each experimental time point. n = 6 hydrogels/group. One-way ANOVA with Tukey’s multiple comparisons test, data in ( B, C ) show individual data points over mean ± SD. One pTM strain shown: TGFβ2-induced contractility induction, HC-06-mediated rescue of hypercontractility, and GSK101-induced transient (15 min) contraction were consistent across (3/3) pTM strains tested . **p < 0.01, ***p < 0.001, ****p < 0.0001.

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A ) Representative longitudinal 24-well plate scans of collagen type I hydrogels seeded with primary TM (pTM) subjected to the different treatments (dashed lines outline size of contracted constructs). ( B ) Longitudinal quantification of hydrogel construct size compared to the control group at the 0 min time point. ( C ) Detailed comparisons between groups at each experimental time point. n = 6 hydrogels/group. One-way ANOVA with Tukey’s multiple comparisons test, data in ( B, C ) show individual data points over mean ± SD. One pTM strain shown: TGFβ2-induced contractility induction, HC-06-mediated rescue of hypercontractility, and GSK101-induced transient (15 min) contraction were consistent across (3/3) pTM strains tested . **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Lentiviral stock for TGFβ2 (C226,228S) was purchased from VectorBuilder Inc (Chicago, IL) (VB170816-1094fnw, pLV[Exp]-CMV> {hTGFB2[ NM_003238.3 ](C226,228S)}) ( ).

Techniques: Construct, Control

( A ) Intravitreal injection of LV-TGFβ2 (Week 1), but not LV-Control, elevates intraocular pressure (IOP) in WT mice ( N = 5 eyes/group) as early as 1-week post-injection. Injection of TRPV4 antagonist HC-06, but not PBS, produced multiday IOP reduction in LV-TGFβ2-treated eyes. HC-06 and PBS injections did not affect IOP in LV-Control injected eyes. Two-way ANOVA with Bonferroni post hoc analysis ( B ) Direct comparison of the results of PBS and HC-06 injections in the eyes shown in A . Two-way ANOVA with Bonferroni post hoc analysis. ( C ) Intravitreal injection of LV-TGFβ2 in Trpv4 −/− mice ( N = 6 eyes/group) resulted in only mild OHT; plotted against WT eyes at matching timepoints (3 WT cohorts including the 5 WT eyes shown in A, B, N = 8–15 eyes/group). ( D ) Statistical comparison of the IOP values shown in C . The IOP in LV-TGFβ2 WT eyes was significantly elevated compared to the LV-TGFβ2 Trpv4 −/− eyes from 2 weeks post-injection. LV-Control injected eyes in WT or Trpv4 −/− eyes remain close to the baseline value and are not significantly different. Two-way ANOVA with Bonferroni post hoc analysis. ( A, C ) shows mean ± SEM. Data in ( B, D ) show individual data points over mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 5—source data 1. Source data for Lv-Control IOP and Lv-TFFb2 cohorts treated with HC-06. Figure 5—source data 2. Source data for IOP data from WT and Trpv4 KO eyes treated with TGFβ2.

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A ) Intravitreal injection of LV-TGFβ2 (Week 1), but not LV-Control, elevates intraocular pressure (IOP) in WT mice ( N = 5 eyes/group) as early as 1-week post-injection. Injection of TRPV4 antagonist HC-06, but not PBS, produced multiday IOP reduction in LV-TGFβ2-treated eyes. HC-06 and PBS injections did not affect IOP in LV-Control injected eyes. Two-way ANOVA with Bonferroni post hoc analysis ( B ) Direct comparison of the results of PBS and HC-06 injections in the eyes shown in A . Two-way ANOVA with Bonferroni post hoc analysis. ( C ) Intravitreal injection of LV-TGFβ2 in Trpv4 −/− mice ( N = 6 eyes/group) resulted in only mild OHT; plotted against WT eyes at matching timepoints (3 WT cohorts including the 5 WT eyes shown in A, B, N = 8–15 eyes/group). ( D ) Statistical comparison of the IOP values shown in C . The IOP in LV-TGFβ2 WT eyes was significantly elevated compared to the LV-TGFβ2 Trpv4 −/− eyes from 2 weeks post-injection. LV-Control injected eyes in WT or Trpv4 −/− eyes remain close to the baseline value and are not significantly different. Two-way ANOVA with Bonferroni post hoc analysis. ( A, C ) shows mean ± SEM. Data in ( B, D ) show individual data points over mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 5—source data 1. Source data for Lv-Control IOP and Lv-TFFb2 cohorts treated with HC-06. Figure 5—source data 2. Source data for IOP data from WT and Trpv4 KO eyes treated with TGFβ2.

Article Snippet: Lentiviral stock for TGFβ2 (C226,228S) was purchased from VectorBuilder Inc (Chicago, IL) (VB170816-1094fnw, pLV[Exp]-CMV> {hTGFB2[ NM_003238.3 ](C226,228S)}) ( ).

Techniques: Injection, Control, Produced, Comparison

Intraocular pressure (IOP) in LV-TGFβ2-injected eyes was significantly elevated compared to both LV-Ctrl injected WT and Trpv4 −/− eyes, as well as LV-TGFβ2-injected Trpv4 −/− eyes ( N = 6 eyes/condition).

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: Intraocular pressure (IOP) in LV-TGFβ2-injected eyes was significantly elevated compared to both LV-Ctrl injected WT and Trpv4 −/− eyes, as well as LV-TGFβ2-injected Trpv4 −/− eyes ( N = 6 eyes/condition).

Article Snippet: Lentiviral stock for TGFβ2 (C226,228S) was purchased from VectorBuilder Inc (Chicago, IL) (VB170816-1094fnw, pLV[Exp]-CMV> {hTGFB2[ NM_003238.3 ](C226,228S)}) ( ).

Techniques: Injection

( A, B ) Post-LV injection daytime (12–2:00 P.M) and nocturnal (9–10:00 P.M.) IOP compared in WT mice ( N = 4–6 eyes/group) before drug treatment. LV-TGFβ2 eyes were elevated at daytime, but nocturnal ocular hypertension (OHT) was not significantly different between LV-Ctrl and LV-TGFβ2 eyes in two separate cohorts of mice measured under isoflurane anesthesia ( A ) or while awake ( B ). ( C, D ) Anesthesia had no significant effect on measured nocturnal IOP. One-way ANOVA with Tukey’s multiple comparisons test. ( E, F ) PBS-injected eyes did not exhibit changes in daytime or nighttime intraocular pressure; however, HC-06 injection reduced TGFβ2-induced IOP elevations during the day and LV-Ctrl and LV-TGFβ2 nocturnal IOPs ( N = 4 eyes/group); two-way ANOVA with Bonferroni post hoc analysis. Figures show data points over mean ± SEM, *p < 0.05 , ** p < 0.01, ****p < 0.0001. Figure 6—source data 1. Source data for nocturnal and diurnal IOP cohorts from control mice and animals treated with LV-TGFβ2.

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A, B ) Post-LV injection daytime (12–2:00 P.M) and nocturnal (9–10:00 P.M.) IOP compared in WT mice ( N = 4–6 eyes/group) before drug treatment. LV-TGFβ2 eyes were elevated at daytime, but nocturnal ocular hypertension (OHT) was not significantly different between LV-Ctrl and LV-TGFβ2 eyes in two separate cohorts of mice measured under isoflurane anesthesia ( A ) or while awake ( B ). ( C, D ) Anesthesia had no significant effect on measured nocturnal IOP. One-way ANOVA with Tukey’s multiple comparisons test. ( E, F ) PBS-injected eyes did not exhibit changes in daytime or nighttime intraocular pressure; however, HC-06 injection reduced TGFβ2-induced IOP elevations during the day and LV-Ctrl and LV-TGFβ2 nocturnal IOPs ( N = 4 eyes/group); two-way ANOVA with Bonferroni post hoc analysis. Figures show data points over mean ± SEM, *p < 0.05 , ** p < 0.01, ****p < 0.0001. Figure 6—source data 1. Source data for nocturnal and diurnal IOP cohorts from control mice and animals treated with LV-TGFβ2.

Article Snippet: Lentiviral stock for TGFβ2 (C226,228S) was purchased from VectorBuilder Inc (Chicago, IL) (VB170816-1094fnw, pLV[Exp]-CMV> {hTGFB2[ NM_003238.3 ](C226,228S)}) ( ).

Techniques: Injection, Control

Expanded time series of IOP measured weekly from a second cohort of mouse eyes injected with LV-Ctrl ( n = 6 eyes) or LV-TGFβ2 ( n = 4 eyes). LV-TGFβ2 resulted in elevated diurnal IOP, which gradually approached the IOP seen in nocturnal measurements but did not further elevate IOP above nocturnal values. In this cohort, both diurnal and nocturnal measurements were made in awake animals.

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: Expanded time series of IOP measured weekly from a second cohort of mouse eyes injected with LV-Ctrl ( n = 6 eyes) or LV-TGFβ2 ( n = 4 eyes). LV-TGFβ2 resulted in elevated diurnal IOP, which gradually approached the IOP seen in nocturnal measurements but did not further elevate IOP above nocturnal values. In this cohort, both diurnal and nocturnal measurements were made in awake animals.

Article Snippet: Lentiviral stock for TGFβ2 (C226,228S) was purchased from VectorBuilder Inc (Chicago, IL) (VB170816-1094fnw, pLV[Exp]-CMV> {hTGFB2[ NM_003238.3 ](C226,228S)}) ( ).

Techniques: Injection

Chronic exposure to TGFβ2 induces upregulation of functional TRPV4 channels alongside the autoinhibitory canonical modulator SMAD7. TRPV4-mediated Ca 2+ influx, canonical, and non-canonical TGFβ2 signaling stimulate the Rho/ROCK pathway to augment cytoskeletal contractility and stimulate extracellular matrix (ECM) release. Actomyosin contractility promotes outflow resistance and drives OHT and underpins a vicious feedforward TRPV4-dependent loop that maintains OHT. This figure was created using BioRender.com .

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: Chronic exposure to TGFβ2 induces upregulation of functional TRPV4 channels alongside the autoinhibitory canonical modulator SMAD7. TRPV4-mediated Ca 2+ influx, canonical, and non-canonical TGFβ2 signaling stimulate the Rho/ROCK pathway to augment cytoskeletal contractility and stimulate extracellular matrix (ECM) release. Actomyosin contractility promotes outflow resistance and drives OHT and underpins a vicious feedforward TRPV4-dependent loop that maintains OHT. This figure was created using BioRender.com .

Article Snippet: Lentiviral stock for TGFβ2 (C226,228S) was purchased from VectorBuilder Inc (Chicago, IL) (VB170816-1094fnw, pLV[Exp]-CMV> {hTGFB2[ NM_003238.3 ](C226,228S)}) ( ).

Techniques: Functional Assay

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