Journal: Scientific reports

Article Title: High-volume hemofiltration does not protect human kidney endothelial and tubular epithelial cells from septic plasma-induced injury.

doi: 10.1038/s41598-024-69202-z

Figure Lengend Snippet: Figure 7. Evaluation of TEC polarity after incubation with plasma collected at different HVHF/SVHF time points. (A, B) Evaluation of cellular polarity by analysis of trans-epithelial electrical resistance (TEER in A) and FITC-albumin reabsorption (B) of TEC incubated with plasma collected at different time points after HVHF (black columns) or SVHF (white columns) start. In respect to Vehicle alone, d1h0 plasma induced a significant decrease of TEER and albumin reabsorption (*p < 0.05 d1h0 vs. Vehicle). Plasma samples collected at d1h6 and d1h12 increased TEC polarity (°p < 0.05 d1h6 or d1h12 vs. d1h0). By contrast, TEC monolayers incubated with d3h0 plasma showed a new significant decrease of both TEER and albumin reabsorption (#p < 0.05 d3h0 vs. d1h6 or d1h12). Similar results were observed for HVHF and SVHF. All experiments were performed in triplicate. (C, D) Representative micrographs (C) and relative fluorescence intensity quantification (D) of Megalin (green staining), NKCC1 (green staining) and SGLT-2 (red staining) expression in TEC incubated with plasma collected at different time points after HVHF start. In respect to Vehicle alone, d1h0 plasma induced a significant decrease of Megalin, NKCC1 and SGLT-2 expression (*p < 0.05 d1h0 vs. Vehicle). Plasma samples collected at d1h6 and d1h12 increased the staining for all the proteins on TEC surface (°p < 0.05 d1h6 or d1h12 vs. d1h0). By contrast, TEC monolayers incubated with d3h0 plasma showed a new significant decrease of Megalin, NKCC1 and SGLT-2 expression (#p < 0.05 d3h0 vs. d1h6 or d1h12). Similar results were observed using SVHF plasma. All experiments were performed in triplicate. In fluorescence micrographs, nuclei were counterstained in blue by Hoechst.

Article Snippet: After appropriate stimuli, cultured TEC were fixed in ethanol/acetic acid 2:1 and stained with antibodies directed to human Fas (Upstate Biotechnology, Lake Placid, NY, USA), megalin, NKCC1 or SGLT-2 (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Incubation, Clinical Proteomics, Fluorescence, Staining, Expressing

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: