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

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A : Mice were subjected to either intraperitoneal (ip.) or intracortical (cor.) LPS injections, while NKCC1 was blocked by ip. bumetanide (Bum) administration. Central LPS injection triggers high cytokine (G-CSF, IL-1α, IL-1β) and chemokine (KC) responses in the brain compared to ip. LPS injection, which is blocked by ip. Bum administration. B : Central NKCC1 inhibition by intracortical Bum administration significantly increases GCSF and IL-1β levels. See also Supplementary Figure 1 for effects of systemic vs. central blockade of NKCC1 on LPS-induced cytokine responses in the periphery. C : Flow cytometric dot plots show that cortical administration of Bum does not affect the number of microglia (CD45 int /P5 gate), and recruitment of leukocytes (CD45 high /P4 gate), including monocytes (CD11b + , Ly6C high /P9 gate), and granulocytes (CD11b + , Ly6G high /P7 gate) upon central LPS injection. D : The main source of IL-1α and IL-1β in the brain are microglia cells. Confocal images of Cx3CR1 +/GFP brain slices show IL-1α-CD45-P2Y12R (above, red arrowheads) and IL-1β-CD45-P2Y12R (below, red arrowheads) labelled cells after cortical LPS injection-induced inflammation. All data are expressed as mean±SEM. E : NKCC1 (encoded by Slc12a2 ) and P2Y12R gene expression is downregulated in microglia isolated from adult mice 24 hours after cisterna magna LPS application. A : One-way ANOVA followed by Tukey’s multiple comparison test; * p <0.05; N=6/group; B : Unpaired t-test; * p <0.05; N=9/group; Data were pooled from two independent studies. C : One-way ANOVA followed by Tukey’s multiple comparison test; * p <0.05; N=4/group. D : Scale: 25 μm; E : Unpaired t-test; ** p <0.01, *** p <0.001; N (WT)=6, N (KO)=5; Abbreviations: veh.: vehicle; ip: intraperitoneal; cor.: cortical; Bum: bumetanide; ns: not significant

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Injection, Inhibition, Expressing, Isolation

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A: NKCC1 mRNA expression levels in newborn and adult microglia isolated from C57BL/6J mice compared to neural progenitors derived from E17 embryonic hippocampi. Note, that NKCC1 mRNA levels decrease dramatically during in vitro maintenance (DIV10). B: We generated a novel microglia-specific conditional NKCC1 KO transgenic mouse line by crossing NKCC1 fl/fl (exon 8 of the Slc12a2 gene was flanked with lox P sites) and Cx3CR1-Cre ERT2 mice. C: NKCC1 mRNA levels in isolated NKCC1 KO microglia was markedly reduced in comparison to wild-type cells. D-E: NKCC1 protein expression in a large number of randomly sampled NKCC1 KO microglia cells is markedly reduced compared to WT cells. Inserts show plasma membrane localization of NKCC1. F-G: Automated morphological analysis and maximum intensity projections of confocal images. Inserts show cells marked with white asterisks in 3D. Arrowheads indicate altered branch structure of NKCC1 KO microglia. Automated morphological analysis shows that features of NKCC1 deficient microglia significantly differ from WT microglia. A: One-way ANOVA, followed by Holm-Sidak’s post hoc test. N=3/group. **: p <0.01; n.s.: not significant C: Mann-Whitney test, N=3/group. **: p <0.01 D: Mann-Whitney test, N (WT)=142 cells from 2 mice, N (KO)=83 cells from 1 mouse. ****: p <0.0001 E: Scale: 2 μm F-G: Scale: 10 μm; Mann-Whitney test, N (WT)=78 cells from 3 mice, N (KO)=136 cells from 5 mice. **: p <0.01, ***: p <0.001, ****: p <0.0001 Abbreviations: DIV: days in vitro; n.s.: not significant; TMX: tamoxifen

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Expressing, Isolation, Derivative Assay, In Vitro, Generated, Transgenic Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A : Baseline NLRP3 and IL-1β mRNA expression is increased in isolated NKCC1 KO microglia compared to WT cells. B: Experimental outline of automated morphological analysis, cytokine array and flow cytometry. C-D : Automated morphological analysis show that activated NKCC1 deficient microglia are slightly smaller than their wild-type counterparts. E : LPS-induced cytokine levels are significantly higher in the cortices of microglial NKCC1 KO mice than in WT. F: Flow cytometric dot plots show that microglial NKCC1 deficiency does not alter the number of CD11b + , CD45 int microglia (P4 gate) or numbers of infiltrating CD11b + , CD45 high leukocytes (P5 gate), CD11b + , Ly6C high monocytes (P9 gate) and CD11b + , Ly6G high granulocytes (P7 gate) in response to intracortical LPS administration. See corresponding data on peripheral cytokine levels and immune cell populations in Supplementary Figure 3 . G: Increased NLRP3 and IL-1β mRNA levels are sustained in NKCC1 KO and WT microglia 24 hours after intracisternal LPS administration . A: Unpaired t-test; * p <0.05, ** p <0.01; N (WT)=6, N (KO)=5 D: Mann-Whitney test, N (WT)=171 cells from 6 mice, N (KO)= 85 cells from 4 mice, ** p <0.01, *** p <0.001 E: Mann-Whitney test, *: p <0.05; N (WT)=7, N (KO)=6 F: unpaired t-test, N (WT)=4, N (KO)=4 G: Unpaired t-test; * p <0.05; N (WT)=5, N (KO)=5; n. s.: not significant.

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Expressing, Isolation, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A : Schematic representation of experiment. Perforated patch-clamp recordings were performed on microglial cells in acute hippocampal slice preparations. Current responses to a train of voltage steps from -100 to 100 mV with 20 mV increments and a duration of 100 ms were measured in voltage-clamp mode (holding potential: -40 mV) both in normotonic, and after 5 minute perfusion with hypotonic ACSF (50% dilution). B: Example traces of recordings from WT (black: normotonic, grey: hypotonic ACSF) and NKCC1 KO (purple: normotonic, violet: hypotonic ACSF) animal. Traces show responses for -100 and +100 mV stimulations in both conditions. C: Average I-V curve responses from WT (black squares with SEM) and NKCC1 KO cells (violet circles with SEM) in normotonic (left) and after 5 minute perfusion of hypotonic ACSF (right) D: Resting membrane potential in normotonic condition of WT vs. NKCC1 KO microglial cells. E: I-V curves calculated by the subtraction of measured values in normotonic conditions from ones in hypotonic medium, resulting in I-V curves representing the currents evoked by cell-swelling due to osmotic change. F: Reversal potentials of the swelling-induced currents measured from WT (grey) or NKCC1 KO (violet) animals (left), with corresponding intracellular Cl - concentrations (right) calculated via the Nernst equation. All parametric data are expressed as mean±SEM. C: Unpaired t-test; N(WT)=8 cells from 7 animal, N(KO)=8 cell from 6 animal; *: p<0.05, **: p<0.01 D: Unpaired t-test; N(WT)=13 microglial cells from 12 mice, N(KO)=12 microglial cells from 11 mice; **: p <0.01 E: Unpaired t-test; *: p <0.05 F: Unpaired t-test; N(WT)=8 cell, N (KO)=8 cell; *: p<0.05.

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Patch Clamp

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A-B : Microglial NKCC1-deficient mice (KO) show larger infarct volume as assessed on cresyl violet-stained brain sections and more severe neurological outcome compared to wild type mice. C: Cytokine levels in the cortex do not differ 8 hours after MCAo in KO mice compared to WT. D-E: Microglial NKCC1 deletion results in higher levels of IL-1α and IL-1β 24 hours after MCAo. B: Unpaired t-test, N (WT)=7, N (KO)=9; *: p <0.05, **: p <0.01 C: Kruskall-Wallis test followed by Dunn’s multiple comparison test; N=6/group. *: p <0.05, **: p <0.01 D: Scale: 50 μm E: Mann-Whitney test, *: p <0.05; N (WT)=7, N (KO)=9. n.s.: not significant

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Staining, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: Atlas of human dental pulp cells at multiple spatial and temporal levels based on single-cell sequencing analysis

doi: 10.3389/fphys.2022.993478

Figure Lengend Snippet: The top 20 genes in odontoblasts.

Article Snippet: The following antibodies were used in our study: CD163 (1:500, Abcam, United States), SLC12A2 (1:100, Proteintech, China), ST8SIA1 (1:100, Proteintech), CD24 (1: 50, Santa Cruz, United States), WISP1 (1:100, Proteintech), CD146/MCAM (1: 250, Abcam), and CD90 (1:200, Abcam).

Techniques:

Journal: Frontiers in Genetics

Article Title: Cell-based analysis of CLIC5A and SLC12A2 variants associated with hearing impairment in two African families

doi: 10.3389/fgene.2022.924904

Figure Lengend Snippet: Mutant (MT) CLIC5A and SLC12A2 proteins are expressed at higher levels in HEK-293 cells, relative to the wild type proteins (WT). (A) Western blot of total proteins from HEK-293 cells transfected with wild-type (WT) or (C) 224T>C; p.(L75P) mutant (MT) CLIC5A plasmids; using anti-Myc antibody (N-terminal). (B) Densitometric analysis of western blot of CLIC5. (C) Western blot of total protein isolated from HEK-293 cells transfected with WT and (C) 2935G>A:p.(E979K) MT SLC12A2 plasmids; using anti-flag antibody (N-terminal). (D) Densitometric analysis of western blots of SLC12A2 for the WT and MT SLC12A2 treated cells using ImageJ. The uncropped western blot pictures are shown in . Schematic diagrams showing (E) CLIC5 p.(L75P) and (F) SLC12A2 p(E979K) variant positions. The protein domains were predicted using the Protein Families Database (Pfam) ( https://doi.org/10.1093/nar/gkaa913 ).

Article Snippet: Mammalian expression constructs containing human CLIC5A (NM_001256023, UniProtKB - Q9NZA1) and SLC12A2 (NM_001046, UniProtKB - P55011) cDNAs were purchased from ORIGENE (Rockville, Maryland).

Techniques: Mutagenesis, Western Blot, Transfection, Isolation, Variant Assay

Journal: Frontiers in Genetics

Article Title: Cell-based analysis of CLIC5A and SLC12A2 variants associated with hearing impairment in two African families

doi: 10.3389/fgene.2022.924904

Figure Lengend Snippet: SLC12A2 WT HEK293 expressing cells display distinct morphology not observed in mutant-expressing cells. Live HEK293 cells expressing WT or MT GFP-tagged SLC12A2 protein and stained with Hoechst were observed using confocal microscopy 72 h post transfection. The red arrows point to axon-like structures present in the WT- SLC12A2 population of cells.

Article Snippet: Mammalian expression constructs containing human CLIC5A (NM_001256023, UniProtKB - Q9NZA1) and SLC12A2 (NM_001046, UniProtKB - P55011) cDNAs were purchased from ORIGENE (Rockville, Maryland).

Techniques: Expressing, Mutagenesis, Staining, Confocal Microscopy, Transfection

Journal: Frontiers in Genetics

Article Title: Cell-based analysis of CLIC5A and SLC12A2 variants associated with hearing impairment in two African families

doi: 10.3389/fgene.2022.924904

Figure Lengend Snippet: Mutant (MT) SLC12A2 expressing cells had relatively less phosphorylated p38 (Pp38) compared to the wild type (WT). (A) Western blot showing Pp38 expression in HEK293 cells transfected with WT and (C) 2935G>A: p.(E979K) MT SLC12A2 plasmids. Total p38 was used for normalization (B) Densitometric analysis of western blots of Pp38 the WT and MT SLC12A2 treated cells using ImageJ. The densitometric analysis was conducted 3 times and the mean measurements were recorded. The uncropped western blot pictures are shown in .

Article Snippet: Mammalian expression constructs containing human CLIC5A (NM_001256023, UniProtKB - Q9NZA1) and SLC12A2 (NM_001046, UniProtKB - P55011) cDNAs were purchased from ORIGENE (Rockville, Maryland).

Techniques: Mutagenesis, Expressing, Western Blot, Transfection

Journal: Frontiers in Genetics

Article Title: Cell-based analysis of CLIC5A and SLC12A2 variants associated with hearing impairment in two African families

doi: 10.3389/fgene.2022.924904

Figure Lengend Snippet: Association of CLIC5 and SLC12A2 variants with hearing impairment in patients.

Article Snippet: Mammalian expression constructs containing human CLIC5A (NM_001256023, UniProtKB - Q9NZA1) and SLC12A2 (NM_001046, UniProtKB - P55011) cDNAs were purchased from ORIGENE (Rockville, Maryland).

Techniques: