Journal: Nature cell biology

Article Title: FBP17 and CIP4 recruit SHIP2 and Lamellipodin to prime the plasma membrane for Fast Endophilin-Mediated Endocytosis

doi: 10.1038/s41556-018-0146-8

Figure Lengend Snippet: a, Colocalization of named EGFP-tagged BAR proteins on endogenous Endophilin spots in BSC1 cells. Histograms show the mean ± SEM from three independent biological experiments (n>150 puncta per construct). b, Colocalization of named endogenous BAR proteins on Endophilin spots at the leading edge of resting BSC1 cells. Histograms show the mean ± SEM from three independent biological experiments (n=150 puncta per condition). c, Intracellular accumulation of β1 adrenergic receptor (β1-AR) in Endophilin triple knocked-down (TKD), AP2 knocked-down (KD), Amphiphysin and Bin1 double knocked-down (DKD) or FBP17 and CIP4 DKD cells, treated with 10μM dobutamine for 30min. Counterstaining of the targeted proteins (red) validated the knock-downs in the cells imaged. Histograms show the mean ± SEM from three independent biological experiments (n=30 cells per condition), normalized to control cells. d, β1-AR uptake into FEME carriers (cytoplasmic Endophilin-positive assemblies) following 4min addition of 10μM dobutamine in cells depleted for FBP17 and CIP4 (F+C DKD) or not. Histograms show the mean ± SEM from three independent biological experiments (n=30 cells per condition). e, Pull-down using GST or GST-SH3 domains of Endophilin A2, FBP17, CIP4 and TOCA-1 and cell extracts expressing EGFP-tagged third intracellular loops (TIL) of β1-, β2-, β3-, α2a- or α2b-adrenergic receptors. Inputs correspond to 1 to 5% of the cell extracts. Unprocessed original scans are provided in Supplementary Fig. 6. All images are representative of at least 10 captures taken from three independent biological experiments for each condition. All experiments were repeated independently at least three times with similar results. Statistical analysis was performed by one-way ANOVA (a) or two-way ANOVA (b,c); NS, non significant P>0.99. Statistical source data are provided in Supplementary Table 2. Scale bars, 10 (c) and 5μm (b,d).

Article Snippet: Cell culture BSC1 (ECACC 85011422) and HEK293 cells (ATCC CRL-1573) were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS, Gold PAA), 1mM GlutaMAX-I (Gibco).

Techniques: Construct, Control, Expressing

Journal: Nature cell biology

Article Title: FBP17 and CIP4 recruit SHIP2 and Lamellipodin to prime the plasma membrane for Fast Endophilin-Mediated Endocytosis

doi: 10.1038/s41556-018-0146-8

Figure Lengend Snippet: a, colocalization of endogenous CIP4 and Endophilin in resting BSC1 cells. b, Colocalization of CIP4 on Endophilin spots at the plasma membrane but not on FEME carriers (10μM isoproterenol for 4min). c, Recruitment of endogenous CIP4 and Endophilin in resting cells depleted or not for FBP17 and CIP4 (F+C DKD) or Endophilin (Endo TKD). Histograms show the mean ± SEM from three independent biological experiments (n=150 cells per condition), normalized to control. d, Endogenous Endophilin cell surface spots and FEME carriers in cells overexpressing CIP4-EGFP (CIP4 OEx). Histograms show the mean ± SEM from three independent biological experiments (n>30 cells per condition), normalized to control. e, Proximity Ligation Assays between endogenous β1-AR and Endophilin in CIP4 OEx or control cells. Cells were pre-treated with 20nM GDC-0941 (PI3Ki) for 5min, before stimulation with 10μM dobutamine for 4min, as indicated. Histograms show the mean ± SEM from three independent biological experiments (n>30 cells per condition). f, β1-AR uptake into FEME carriers in cells overexpressing CIP4-Myc and treated as in e. Histograms show the mean ± SEM from three independent biological experiments (n=30 cells per condition). g, Kymographs and till views from leading edge or ventral surface of resting cells expressing low levels of FBP17-EGFP and EndophilinA2-RFP and imaged at 0.5Hz (top) and 2Hz (middle). Right, percentage of maximum signals (means ± SEM, n=50 spots from three independent biological experiments). h, Kymographs from cells expressing low levels of β1-AR-EGFP or EndophilinA2-RFP and OEx or not CIP4-EGFP, treated with dobutamine and GDC-0941 as indicated and imaged at 2Hz. Kymographs are representative of 5 captures from three independent biological experiments. Histograms show the mean ± SEM from three independent biological experiments (n=5 cells per condition). All images are representative of at least 10 captures taken from three independent biological experiments for each condition. All experiments were repeated independently at least three times with similar results. Statistical analysis was performed by one-way ANOVA (e,f) or two-way ANOVA (c,d,h); NS, non significant P>0.99. Statistical source data are provided in Supplementary Table 2. Scale bars, 20 (a) and 5μm (b,c,d,f).

Article Snippet: Cell culture BSC1 (ECACC 85011422) and HEK293 cells (ATCC CRL-1573) were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS, Gold PAA), 1mM GlutaMAX-I (Gibco).

Techniques: Clinical Proteomics, Membrane, Control, Ligation, Expressing

Journal: Nature cell biology

Article Title: FBP17 and CIP4 recruit SHIP2 and Lamellipodin to prime the plasma membrane for Fast Endophilin-Mediated Endocytosis

doi: 10.1038/s41556-018-0146-8

Figure Lengend Snippet: a, Pull-down experiments with GST-tagged SH3 domains of the indicated proteins and EGFP-tagged SHIP2, Lpd or N-WASP (positive control). Inputs correspond to 1 to 5% of the cell extracts. Unprocessed original scans are provided in Supplementary Fig. 6. b-e, Recruitment of endogenous CIP4, SHIP2, Lpd or Endophilin in resting BSC1 cells depleted or not for FBP17 and CIP4 (F+C DKD), SHIP (SHIP1+2 DKD) or Lpd, as indicated. Histograms show the mean ± SEM from three independent biological experiments (n=30 cells per condition), normalized to control. f, Recruitment endogenous SHIP2 or Lpd at the leading edge of resting cells overexpressing the indicated constructs. Histograms show the mean ± SEM from three independent biological experiments (n=30 cells per condition), normalized to control. g, Kymograph from a cell expressing low levels of CIP4-EGFP and EndophilinA2-RFP, treated with AS19499490 (SHIP2i) as indicated and imaged at 2Hz. The kymograph is representative of 9 captures from three independent biological experiments. h, Proximity Ligation Assays between endogenous β1-AR and Endophilin in cells pre-treated or not with 10μM AS19499490 (SHIP2i) for 5min before stimulation with 10μM dobutamine for 4min, as indicated. i, β1-AR uptake into FEME carriers (cytoplasmic Endophilin-positive assemblies) in cells treated as in h. j, Histograms show the mean ± SEM (left and middle, n=30 cells per condition, right, n=5 cells per condition) of cells treated as in g-i, respectively. All images are representative of at least 10 captures taken from three independent biological experiments. All experiments were repeated independently at least three times with similar results. Statistical analysis was performed by one-way ANOVA (j, middle and right) or two-way ANOVA (e,f,j left); NS, non significant P>0.99. Statistical source data are provided in Supplementary Table 2. Scale bars, 20 (h) and 5μm (b,c,d,f,i).

Article Snippet: Cell culture BSC1 (ECACC 85011422) and HEK293 cells (ATCC CRL-1573) were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS, Gold PAA), 1mM GlutaMAX-I (Gibco).

Techniques: Positive Control, Control, Construct, Expressing, Ligation

Journal: Nature cell biology

Article Title: FBP17 and CIP4 recruit SHIP2 and Lamellipodin to prime the plasma membrane for Fast Endophilin-Mediated Endocytosis

doi: 10.1038/s41556-018-0146-8

Figure Lengend Snippet: a-c, Recruitment of endogenous CIP4, SHIP2, or Endophilin in resting BSC1 cells depleted or not for Endophilin (Endo TKD), and treated with 10μM ML141 (Cdc42i 1) for 10min with as indicated. Images are representative of 10 captures from three independent biological experiments for each condition. d, Recruitment of endogenous CIP4, Lpd or Endophilin in resting cells overexpressing EGFP-tagged dominant negative (Cdc42-DN, T17N mutant) or constitutively active (Cdc42-CA, Q61L mutant) versions of Cdc42, as indicated. Focal planes were located at the bottom membrane or in the middle of the cells, as indicated. Images are representative of 6 captures from three independent biological experiments for each condition. e, Histograms show the mean ± SEM from three independent biological experiments (n=30 cells per condition), treated as in a-d and normalized to the respective controls. Secramine (Cdc42i 2) was used at 10μM for the indicated time). f, scheme of the EGFP-tagged full-length or truncated versions of CIP4 used. g, h, Recruitment of EGFP-tagged full-length or truncated versions of CIP4 in cells depleted for endogenous FBP17, CIP4 and TOCA1 (TKD) and treated or not with 10μM ML141 for 10min (+Cdc42i), as indicated. Cells were immunostained for endogenous CIP4 (red) to control for the depletion in the cells imaged. Images are representative of at least 6 captures taken from three independent biological experiments for each condition. Histograms show the mean ± SEM (n>6 cells per condition). All experiments were repeated at least three times with similar results. Statistical analysis was performed by one-way ANOVA (g) or two-way ANOVA (e). Statistical source data are provided in Supplementary Table 2. Scale bars, 5μm.

Article Snippet: Cell culture BSC1 (ECACC 85011422) and HEK293 cells (ATCC CRL-1573) were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS, Gold PAA), 1mM GlutaMAX-I (Gibco).

Techniques: Dominant Negative Mutation, Mutagenesis, Membrane, Control

Journal: Nature cell biology

Article Title: FBP17 and CIP4 recruit SHIP2 and Lamellipodin to prime the plasma membrane for Fast Endophilin-Mediated Endocytosis

doi: 10.1038/s41556-018-0146-8

Figure Lengend Snippet: a, Recruitment of endogenous Endophilin in resting BSC1 cells overexpressing wild-type EGFP-tagged RICH1 or R288A mutant (GAP*), as indicated. Images are representative of 10 captures from three independent biological experiments. b, Recruitment of endogenous Endophilin and RICH1 in resting cells depleted or not for Endophilin (Endo TKD) or RICH1 and SH3BP1 (R+S DKD). Images are representative of 10 captures from three independent biological experiments. c, Histograms show the mean ± SEM from three independent biological experiments (n=30 cells per condition) treated as indicated and normalized to the respective controls. d, Kymograph from a R+S DKD cell expressing low levels of CIP4-EGFP and EndophilinA2-RFP and stimulated with dobutamine at the indicated times. The kymograph is representative of 5 captures from three independent biological experiments. e, Proximity Ligation Assays between endogenous β1-AR and Endophilin in cells R+S DKD depleted pre-treated or not with 20nM GDC-0941 (PI3Ki) for 5min before stimulation with 10μM dobutamine for 4min, as indicated. f, β1-AR uptake into FEME carriers (cytoplasmic Endophilin-positive assemblies) in cells treated as in e. g, Histograms show the mean ± SEM (left and middle, n=30 cells per condition, right, n=3 cells per condition) of cells treated as in d-f, respectively. (h) Model summarizing the priming cycle of FEME in resting cells: Step 1, active GTP-loaded Cdc42 recruits FBP17 and CIP4 through their REM domains. Step 2, FBP17 and CIP4 cluster 5’-phosphatases SHIP1 and 2 as well as Lpd through their SH3 domains. Lpd is further stabilized by Pi(3,4)P2 locally produced by SHIP1/2, Step 3, Endophilin is recruited and concentrated by Lpd. From there, pre-enriched Endophilin mediates prompt FEME carrier formation upon cargo activation. In absence of cargo activation, the FEME priming complex disassembled (Step 4), upon local Cdc42 deactivation by the GAPs RICH1 and SH3BP1. All experiments were repeated at least three times with similar results. Statistical analysis was performed by one-way ANOVA (c, g middle and right) or two-way ANOVA (g left); NS, non significant P>0.99. Statistical source data are provided in Supplementary Table 2. Scale bars, 20 (e) and 5μm (a,b,f).

Article Snippet: Cell culture BSC1 (ECACC 85011422) and HEK293 cells (ATCC CRL-1573) were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS, Gold PAA), 1mM GlutaMAX-I (Gibco).

Techniques: Mutagenesis, Expressing, Ligation, Produced, Activation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: The β3‐AR agonist BRL37344 ameliorates the main symptoms of X‐linked nephrogenic diabetes insipidus in the mouse model of the disease

doi: 10.1111/jcmm.18301

Figure Lengend Snippet: Multiple repeated BRL37344 1 mg/kg i.p. injections promoted NKCC2 activation in the thick ascending limb of X‐NDI mice. (A) At the end of the 6 i.p injections/24 h experiment, a quantitative reverse transcription polymerase chain reaction was performed on kidneys from X‐NDI treated with saline (CTR, n = 4) or with BRL37344 (BRL, n = 8). Relative quantification of gene expression (RQ) was performed setting the amount of NKCC2 mRNA in CTR as 1. No differences were seen in NKCC2 transcription between the two groups. The experiment was repeated three times and comparable results were obtained. In the scatter plot data were given as mean ± SEM and each dot represents the average of data from three experiments for each mouse. (B) Western blotting with anti‐NKCC2 and antiphosphorylated NKCC2 antibodies was carried out using homogenates prepared from whole kidneys of CTR ( n = 3) and BRL ( n = 9) mice. Representative lanes were reported in the figure. The expression levels of each protein were normalized to total protein content using Stain‐free™ gels technologies. Densitometric analysis showed a two‐fold increase in pNKCC2 (active form), normalized to total NKCC2, in BRL compared to CTR mice. No differences were seen in NKCC2 abundance. The experiment was repeated three times and comparable results were obtained. In the scatter plot, data were given as mean ± SEM and each dot represents the average of data from three experiments for each mouse. **** p < 0.0001 with two‐tailed unpaired Student's t‐test (C) Kidneys from CTR and BRL mice were subjected to immunofluorescence localization of NKCC2 (in green) and pNKCC2 (in green) and counterstained with Evans blue (red) (CTR = 3, BRL = 5). The number of NKCC2‐positive TAL cells and the localization of NKCC2 were similar in two groups but the fluorescence intensity of pNKCC2 was increased in BRL mice. Representative images were shown. The experiment was repeated three times and comparable results were obtained. (bar = 25 μm).

Article Snippet: For quantification of NKCC2, NCC and AQP2 mRNA, real‐time PCR was performed in triplicate using the Applied Biosystem StepONE Real‐time PCR system and the following TaqMan GenExpression Assay (Thermo Fisher Scientific): Mm00490213_m1 for mouse NCC, Mm01275821_m1 for mouse NKCC2 and Mm00437575_m1 for mouse AQP2.

Techniques: Activation Assay, Reverse Transcription, Polymerase Chain Reaction, Saline, Quantitative Proteomics, Gene Expression, Western Blot, Expressing, Staining, Two Tailed Test, Immunofluorescence, Fluorescence

Journal: Chem

Article Title: Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders

doi: 10.1016/j.chempr.2020.06.017

Figure Lengend Snippet: In Vitro Selection of the Selective NKCC1 Inhibitor ARN23746 as a Lead Compound (A) Schematic representation of the intervention point in bumetanide’s structure for synthesizing novel bumetanide analogs. (B) Quantification of the inhibitory activity of bumetanide (Bume) and bumetanide analogs (10, 100 μM) in NKCC1-(left) or NKCC2-(right) transfected HEK293 cells in the Cl − influx assay. Data are presented as a percentage of the respective control DMSO. Data represent mean ± standard error of the mean (SEM) from 3–4 independent experiments (Kruskal-Wallis one way ANOVA on Ranks, NKCC1 10 μM: H = 84.898, DF = 6, p < 0.001; NKCC1 100 μM: H = 86.799, DF=6, p < 0.001; NKCC2 10 μM: H = 40.700, DF = 6, p < 0.001; NKCC2 100 μM: H = 70.569, DF = 6, p < 0.001, Dunn’s post hoc test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (C) Representation of the ligand-based computational strategy to discover novel molecular scaffolds that inhibit NKCC1. The obtained bumetanide pharmacophore ( 1 ) consists of three H-bond acceptor (HBA) features (red spheres), three H-bond donor (HBD) interactions (blue spheres), one lipophilic feature (green sphere), and one stacking feature (brown sphere) anchored around the central aromatic core. Ligand disposition was then implemented by superimposing other known unspecific NKCC1 inhibitors ( 2 ), revealing shared features and different dihedral dispositions of substituent around the central aromatic core. This model was used as a search filter for the virtual screening ( 3 ) of our internal chemical collection (∼15,000 compounds). Results generated from in vitro testing of the 165 initial hits ( 4 ) were then used to retrain the model ( 5 ) and perform a second more specific screening of our chemical library and commercial chemical libraries (∼135,000 compounds). This iterative computational cycle led to hit compounds ( 6 ) ARN22393 and ARN22394. (D) Quantification of the inhibitory activity of the indicated compounds (10, 100 μM) in NKCC1-transfected HEK293 cells (Cl − influx assay). Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from 3–4 independent experiments (Kruskal-Wallis one way ANOVA on Ranks, 10 μM: H = 37.119, DF = 3, p < 0.001; 100 μM: H = 33.724, DF = 3, p < 0.001, Dunn’s post hoc test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (E) Chemical structures of NKCC1 inhibitors with novel scaffold. (F) Left, example traces obtained in the Cl − influx assay on NKCC1-transfected HEK293 cells for each compound (100 μM). The arrow indicates the addition of NaCl (74 mM) to initiate the NKCC1-mediated Cl − influx. Right, quantification of the NKCC1 inhibitory activity of the indicated compounds (10, 100 μM) in experiments such as those on the right. Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from 3–4 independent experiments (10 μM: one way ANOVA, F (4,84) = 33.048, p < 0.001, Dunnett's post hoc test, ∗p < 0.05, ∗∗∗p < 0.001; 100 μM: Kruskal-Wallis one way ANOVA on Ranks, H = 50.796, DF = 4, p < 0.001, Dunn’s post hoc test, ∗∗∗p < 0.001. (G) Left, example traces obtained in the Ca 2+ influx assay on 3DIV hippocampal mouse neurons for each tested compound (100 μM). The arrows indicates the addition of GABA (100 μM) and KCl (90 mM). Right, quantification of the effect of the indicated compounds (10, 100 μM) in the Ca 2+ influx assay on 3DIV neurons. Data are presented as a percentage of the control DMSO. Data represent mean ± SEM from three independent experiments. 10 μM: one-way ANOVA, F (5,161) = 77.184, p < 0.001, Dunnett's post hoc test ∗∗p < 0.01, ∗∗∗p < 0.001; 100 μM: Kruskal-Wallis One ANOVA on Ranks, H = 134.681, DF = 5, p < 0.001, Dunn’s post hoc test, ∗∗∗p < 0.001). (H) Amplitude change average and single cell data points of GABA-evoked currents obtained by voltage-clamp recordings of 12–20 DIV hippocampal mouse neurons before (gray example recordings in the inset above) and after (black example traces) bath application of the indicated drugs (10 μM). Data are presented as mean ± SEM (Paired t test, ∗p < 0.05, ∗∗p < 0.01). Scale bars: 250 pA, 250 ms. See also A–S1D; and .

Article Snippet: To assess NKCC1 and NKCC2 activity (Cl − influx assay), 3 million HEK cells were plated in a 10 cm cell-culture dish and transfected with a transfection mixture comprising 5 mL of DMEM, 4 mL Opti-MEM, 8 μg of DNA plasmid coding for NKCC1 (PRK-NKCC1 obtained from Medical Research Council and the University of Dundee), NKCC2 (OriGene plasmid #RC216145) subcloned in PRK5 plasmid, or mock control (empty vector), together with 8 μg of a plasmid coding for the Cl − -sensitive variant of the mbYFPQS (Addgene plasmid #80742), and 32 μL of Lipofectamin 2000.

Techniques: In Vitro, Selection, Activity Assay, Transfection, Generated

Journal: Chem

Article Title: Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders

doi: 10.1016/j.chempr.2020.06.017

Figure Lengend Snippet: ARN23746 Restores [Cl − ] i in DS Neurons, Does Not Inhibit NKCC2 in HEK Cells and Shows Improved Brain Penetration In Vivo in Comparison to Bumetanide (A) Left, representative pseudo-color images (colored scale below) of the [Cl − ] i measured with the MQAE Cl − -sensitive dye, in WT and Ts65Dn hippocampal neurons at 15 DIV, after treatment with control DMSO (0.1%) and the indicated compounds (10 μM). Scale bar: 70 μm. Right, quantification of the indicated compounds (10 μM) in modulating [Cl − ] i in experiments such as those on the left. Data represent mean ± SEM from three independent experiments (two-way ANOVA, F interaction (2,30) = 3.815, p = 0.033, Tukey’s post hoc test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (B) Left, example traces obtained in the Cl − influx assay on NKCC2-transfected HEK293 cells for each compound (10 μM). The arrow indicates the addition of NaCl (74 mM) to initiate the NKCC2-mediated Cl − influx. Right, quantification of the NKCC2 inhibitory activity in experiments such as those on the right. Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from four independent experiments (Kruskal-Wallis one way ANOVA on Ranks, H = 16,962, DF = 2, p < 0.001, Dunn’s post hoc test, ∗∗∗p < 0.001). (C) Left, example traces obtained in the Tl influx assay on KCC2-transfected HEK293 cells for each compound (10 μM). The arrows indicate the additions of TlSO 4 and NaCl. Right, quantification of the KCC2 inhibitory activity in experiments such as those on the left. Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from 4 independent experiments (one way ANOVA, F (2,51) = 10.676, p < 0.001, Dunnett's post hoc test ∗∗∗p < 0.001). (D) Quantification of the level of bumetanide and ARN23746 in the brain at diverse time points (5, 15, 30, 60, 120 min) after injection in C57BL/6N mice (BioLASCO Taiwan). (Two-way ANOVA, F treatment (1,50) = 6.510, p = 0.014, Tukey’s post hoc test, ∗p < 0.05.). (E) Quantification of the ratio between brain and plasma concentration of bumetanide and ARN2346 5 min after the injection (two-tailed t test, t = 7.915, p < 0.001). See also E–S1G.

Article Snippet: To assess NKCC1 and NKCC2 activity (Cl − influx assay), 3 million HEK cells were plated in a 10 cm cell-culture dish and transfected with a transfection mixture comprising 5 mL of DMEM, 4 mL Opti-MEM, 8 μg of DNA plasmid coding for NKCC1 (PRK-NKCC1 obtained from Medical Research Council and the University of Dundee), NKCC2 (OriGene plasmid #RC216145) subcloned in PRK5 plasmid, or mock control (empty vector), together with 8 μg of a plasmid coding for the Cl − -sensitive variant of the mbYFPQS (Addgene plasmid #80742), and 32 μL of Lipofectamin 2000.

Techniques: In Vivo, Transfection, Activity Assay, Injection, Concentration Assay, Two Tailed Test

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Toolbox to Characterize Human Induced Pluripotent Stem Cell–Derived Kidney Cell Types and Organoids

doi: 10.1681/ASN.2019030303

Figure Lengend Snippet: iPSC reporter line details and localizations

Article Snippet: Live imaging of MAFB mTagBFP2 :GATA3 mCherry organoids was performed on the Dragonfly Spinning Disk confocal within a humidified incubation chamber set at 37°C and 5% O 2 . table ft1 table-wrap mode="anchored" t5 Table 4. caption a7 Specificity Host Species Dilution Range Manufacturer and Identifier NANOG Mouse 1:50 DSHB (PCRP-NANOGP1–2D8) CITED1 Rabbit 1:300 Thermo Fisher Scientific (RB-9219-P1) GATA3 Goat 1:300 R&D Systems (AF2605) GFP Chicken 1:300 Sapphire Biosciences (Ab13970) ECADHERIN Mouse 1:300 BD Biosciences (610181) EpCAM (Alexa488 conjugate) Mouse 1:300 BioLegend (324210) LAMININ Rabbit 1:400–1:500 Sigma-Aldrich (L9393) mCherry (RFP) Rabbit 1:300–1:400 MBL Medical & Biologic Laboratories Co. Ltd. (PM005) MECA-32 Rat 1:100 Novus Biologic (NB100–77668) mTagBFP2 Rabbit 1:500 Evrogen (AB233) NEPHRIN Sheep 1:300 R&D Systems (AF4269) PECAM-1 Goat 1:100 Santa Cruz Biotechnology (SC-1506) Proximal tubule brush border membrane Lotus tetragonolobus lectin (LTL) 1:300–1:500 Vector Laboratories (B-1325) SIX2 Rabbit 1:300 Proteintech Group (11562–1-AP) SLC12A1 Rabbit 1:300 Proteintech (18970–1-AP) Open in a separate window Antibodies and lectins used for immunofluorescence Organoid Transplantation Transplantation of D7+12 (7 days of monolayer differentiation followed by 12 days of organoid culture) LRP2 mTagBFP2 organoids was performed with approved animal ethics protocols (MCRI AEC A873 and A888) using immunocompromised 10-week-old female NSG mice (Jackson Laboratories) and D7+12 organoids as described previously for subcapsular transplants.

Techniques: Expressing, Activation Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Toolbox to Characterize Human Induced Pluripotent Stem Cell–Derived Kidney Cell Types and Organoids

doi: 10.1681/ASN.2019030303

Figure Lengend Snippet: Antibodies and lectins used for immunofluorescence

Article Snippet: Live imaging of MAFB mTagBFP2 :GATA3 mCherry organoids was performed on the Dragonfly Spinning Disk confocal within a humidified incubation chamber set at 37°C and 5% O 2 . table ft1 table-wrap mode="anchored" t5 Table 4. caption a7 Specificity Host Species Dilution Range Manufacturer and Identifier NANOG Mouse 1:50 DSHB (PCRP-NANOGP1–2D8) CITED1 Rabbit 1:300 Thermo Fisher Scientific (RB-9219-P1) GATA3 Goat 1:300 R&D Systems (AF2605) GFP Chicken 1:300 Sapphire Biosciences (Ab13970) ECADHERIN Mouse 1:300 BD Biosciences (610181) EpCAM (Alexa488 conjugate) Mouse 1:300 BioLegend (324210) LAMININ Rabbit 1:400–1:500 Sigma-Aldrich (L9393) mCherry (RFP) Rabbit 1:300–1:400 MBL Medical & Biologic Laboratories Co. Ltd. (PM005) MECA-32 Rat 1:100 Novus Biologic (NB100–77668) mTagBFP2 Rabbit 1:500 Evrogen (AB233) NEPHRIN Sheep 1:300 R&D Systems (AF4269) PECAM-1 Goat 1:100 Santa Cruz Biotechnology (SC-1506) Proximal tubule brush border membrane Lotus tetragonolobus lectin (LTL) 1:300–1:500 Vector Laboratories (B-1325) SIX2 Rabbit 1:300 Proteintech Group (11562–1-AP) SLC12A1 Rabbit 1:300 Proteintech (18970–1-AP) Open in a separate window Antibodies and lectins used for immunofluorescence Organoid Transplantation Transplantation of D7+12 (7 days of monolayer differentiation followed by 12 days of organoid culture) LRP2 mTagBFP2 organoids was performed with approved animal ethics protocols (MCRI AEC A873 and A888) using immunocompromised 10-week-old female NSG mice (Jackson Laboratories) and D7+12 organoids as described previously for subcapsular transplants.

Techniques: Membrane, Plasmid Preparation

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Toolbox to Characterize Human Induced Pluripotent Stem Cell–Derived Kidney Cell Types and Organoids

doi: 10.1681/ASN.2019030303

Figure Lengend Snippet: Validation and characterization of iPSC reporter lines for proximal/distal nephron and ureteric epithelium. (A) Characterization of proximal nephron iPSC lines, LRP2mTagBFP2 and HNF4αYFP. Immunofluorescence of D7+12 organoids (Ai) confirmed expression of markers for proximal tubules (LTL+; blue), distal tubules, (ECADHERIN [ECAD]+/LTL−; green), ureteric epithelium/connecting segment (ECAD+/GATA3+; green and red), and podocytes of the glomeruli (NPHS1+; gray). YFP and mTagBFP2 reporter fluorescence was detectable both by live confocal imaging (Aii) and flow cytometry (Aiii) of LRP2mTagBFP2 and HNF4αYFP organoids. FACS-isolated mTagBFP2+ and YFP+ cells (Aiv; blue and yellow bars) from LRP2mTagBFP2 and HNF4αYFP organoids showed enriched endogenous expression of their targeted genes, LRP2 and HNF4α, compared with the reporter-negative control populations (gray bars) via quantitative RT-PCR (qRT-PCR). Sorted reporter-positive populations also showed enrichment for additional proximal tubule markers such as CUBN. Error bars depict SEM and significance was determined using a t test on normalized (∆Ct) values (*P≤0.05; ***P≤0.001; ****P<0.001). Transplanted D7+9 LRP2mTagBFP2 organoids (Avii) retrieved from subcapsular (left panel) and omental (right panels) transplants 21 days after surgery (D7+9+21) showed endogenous mTagBFP2 expression and human and mouse derived vasculature surrounding mTagBFP2+ tubules (cyan) via immunofluorescence (vasculature marker; PECAM-1 [red], mouse-specific cellular antigen; MECA-32 [green]). (B) Characterization of distal nephron/ureteric epithelium iPSC lines, GATA3mCherry and MAFBmTagBFP2:GATA3mCherry. Immunofluorescence of representative D7+12 MAFBmTagBFP2:GATA3mCherry organoids (Bi) demonstrates expression of nephron segment-specific markers for proximal tubules (LTL+; top panel [blue] and HNF4α; bottom panel [green]), distal tubules, (ECAD+/LTL−; top panel [green/blue]), thick ascending limb of the loop of Henle (SLC12A1+; bottom panel [red]), ureteric epithelium/connecting segment (ECAD+/GATA3+; top panel [green/red] and bottom panel [GATA3 only, gray]), and podocytes of the glomeruli (NPHS1+; top panel [gray]). Endogenous mCherry (red) and mTagBFP2 (blue) reporter expression GATA3mCherry and MAFBmTagBFP2:GATA3mCherry organoids (Bii) was detected via live confocal imaging and flow cytometry. Immunofluorescence of GATA3mCherry organoids (Biii) confirmed colocalization of mCherry (red) with GATA3 (green) protein and qRT-PCR of FACS-isolated mCherry+ cells from these organoids showed enriched GATA3 gene expression (red bar) compared with the mCherry−/EpCam+ epithelial control cell population (gray bar). Error bars represent SEM and significance was determined using a t test on normalized (∆Ct) values (****P<0.001). (C) Representative still images from confocal time-lapse imaging of a D7+9 MAFBmTagBFP2:GATA3mCherry organoid across a 35-hour period demonstrates endogenous mCherry (red) and mTagBFP2 (blue) expression. Scale bars in (A and B) represent 50 µm. Scale bar in (C) represents 500 µm.

Article Snippet: Live imaging of MAFB mTagBFP2 :GATA3 mCherry organoids was performed on the Dragonfly Spinning Disk confocal within a humidified incubation chamber set at 37°C and 5% O 2 . table ft1 table-wrap mode="anchored" t5 Table 4. caption a7 Specificity Host Species Dilution Range Manufacturer and Identifier NANOG Mouse 1:50 DSHB (PCRP-NANOGP1–2D8) CITED1 Rabbit 1:300 Thermo Fisher Scientific (RB-9219-P1) GATA3 Goat 1:300 R&D Systems (AF2605) GFP Chicken 1:300 Sapphire Biosciences (Ab13970) ECADHERIN Mouse 1:300 BD Biosciences (610181) EpCAM (Alexa488 conjugate) Mouse 1:300 BioLegend (324210) LAMININ Rabbit 1:400–1:500 Sigma-Aldrich (L9393) mCherry (RFP) Rabbit 1:300–1:400 MBL Medical & Biologic Laboratories Co. Ltd. (PM005) MECA-32 Rat 1:100 Novus Biologic (NB100–77668) mTagBFP2 Rabbit 1:500 Evrogen (AB233) NEPHRIN Sheep 1:300 R&D Systems (AF4269) PECAM-1 Goat 1:100 Santa Cruz Biotechnology (SC-1506) Proximal tubule brush border membrane Lotus tetragonolobus lectin (LTL) 1:300–1:500 Vector Laboratories (B-1325) SIX2 Rabbit 1:300 Proteintech Group (11562–1-AP) SLC12A1 Rabbit 1:300 Proteintech (18970–1-AP) Open in a separate window Antibodies and lectins used for immunofluorescence Organoid Transplantation Transplantation of D7+12 (7 days of monolayer differentiation followed by 12 days of organoid culture) LRP2 mTagBFP2 organoids was performed with approved animal ethics protocols (MCRI AEC A873 and A888) using immunocompromised 10-week-old female NSG mice (Jackson Laboratories) and D7+12 organoids as described previously for subcapsular transplants.

Techniques: Biomarker Discovery, Immunofluorescence, Expressing, Fluorescence, Imaging, Flow Cytometry, Isolation, Negative Control, Quantitative RT-PCR, Derivative Assay, Marker, Gene Expression, Control

Journal: Nature Communications

Article Title: Integrative single-cell characterization of a frugivorous and an insectivorous bat kidney and pancreas

doi: 10.1038/s41467-023-44186-y

Figure Lengend Snippet: A UMAPs of type B intercalated cell marker gene SLC26A4 expression in each species (Jamaican fruit (JF) and big brown (BB) bat). B (Left) Representative images of SLC26A4 immunofluorescence (red) in bat kidneys. Nuclei are stained with DAPI (blue). (Right) Quantification of SLC26A4 immunofluorescence normalized to nuclei in bat kidneys. Results represent arbitrary units of fluorescence (AU) mean ± standard error of the mean (SEM) derived from 3 insectivorous and 3 frugivorous bats ( n = 3/phenotype, n = 10 images/individual [see Methods]). Mixed effects model (two-sided) *** p -value = 0. Exact p -value C (Left) Representative images of SLC12A1 immunofluorescence (red) in bat kidneys. Nuclei are stained with DAPI (blue). (Right) Quantification of SLC12A1 immunofluorescence normalized to nuclei in bat kidneys. Results represent mean (AU) ± standard error of the mean (SEM) derived from 3 insectivorous and 3 frugivorous bats ( n = 3/phenotype, n = 10 images/individual [see Methods]). Mixed effects model (two-sided) *** p -value = 0. D Bar plots showing Kyoto Encyclopedia of Genes and Genomes (KEGG) Human 2021 pathways enriched in fruit bat type B intercalated cells. Q -values were calculated with a one-sided Fisher’s exact test and corrected with the Benjamini-Hochberg method. E Violin plot of ATP6V1G3 expression in type B intercalated cells. F Bar plots showing KEGG Human 2021 pathways enriched in fruit bat type A intercalated cells. P -value calculated with a two-sided Wilcoxon rank-sum test and the Bonferroni correction. G Violin plots of SLC2A9 , RHBG , KCNMA1 , and KLK1 in type A intercalated cells. P -values were calculated with a two-sided Wilcoxon rank-sum test and the Bonferroni correction. H Bar plots showing KEGG Human 2021 pathways enriched in insectivore thick ascending limbs. Q -values were calculated with a one-sided Fisher’s exact test and corrected with the Benjamini-Hochberg method. I Violin plots of SLC9A3 , OXSR1 , STK39 , and KCNJ1 expression in TAL. P -values calculated with two-sided Wilcoxon rank-sum test and the Bonferroni correction. J Volcano plot showing differentially expressed genes between species in proximal tubules cells. P -values calculated with two-sided Wilcoxon rank-sum test and the Bonferroni correction. K Dot plots showing the expression of gluconeogenesis and various SLC5 genes in bat proximal tubules and proximal tubules-like cells. Jamaican fruit bat is depicted as JF and big brown bat as BB in the various panels. Source data are provided as a Source Data file.

Article Snippet: Sections were then incubated overnight at 4 degrees Celsius in a humidity chamber protected from light with one of the following primary antibodies from Thermo Scientific TM at respective dilutions in blocking buffer: SLC12A1 (18970-1-AP; 1:100), AQP2 (PA5-78808; 1:100), SLC26A4 (PA5-115911; 1:100), INS (15848-1-AP; 1:500), GCG (15954-1-AP; 1:500).

Techniques: Marker, Expressing, Immunofluorescence, Staining, Fluorescence, Derivative Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: Effects of extreme potassium stress on blood pressure and renal tubular sodium transport

doi: 10.1152/ajprenal.00527.2019

Figure Lengend Snippet: Dietary K+ maneuvers affect the abundance of transporters and channels along the nephron. Kidney lysates from mice subjected to various dietary K+ maneuvers for 10 days were assessed by Western blot analysis. Equivalent protein loading was determined by BCA Protein Assay and Coomassie staining. Results are shown as means ± SE; n = 6 mice per diet. One-way ANOVA with Sidak’s post hoc analysis, *P ≤ 0.05 and **P ≤ 0.01 compared with control; φsignificant difference between K+ basic and KCl diets. A: we probed for a variety of channels and transporters expressed from the proximal tubule to the collecting duct, as indicated. B: Western blots of kidney homogenates from mice subjected to various K+ diets. All samples were of the kidney cortex unless noted [Na+-K+-2Cl− cotransporter (NKCC2) in the whole kidney]. #Multimerization of Na+-bicarbonate cotransporter 1A (NBCe1A) (39) and pendrin (62). C: graphical summary of the results. The dotted line depicts control values normalized to 1.0. DCT, distal convoluted tubule; γ-ENaC, γ-subunit of the epithelial Na+ channel; IC, intercalated cells; NCC, NaCl cotransporter; NHE3, Na+/H+ exchanger 3; ROMK, renal outer medullary K+ channel; NDCBE, Na+-driven Cl−/bicarbonate exchanger; PC, principal cells; PCT, proximal convoluted tubule; SGLT2, Na+-glucose transporter 2; TAL, thick ascending limb.

Article Snippet: The following antibodies were used for immunoblot analysis: aquaporin 2 (Aqp2; 29 kDa unglycosylated and 35−45 kDa mature, sc-515770, Santa Cruz Biotechnology) ( 46 ), Na + /H + exchanger 3 (NHE3; 84 kDa, SPC-400, Stressmarq) ( 28 ), Na + -bicarbonate cotransporter 1A (NBCe1A; 130 kDa denatured and 280 kDa dimer, AB3212-I, Millipore) ( 39 , 49 ), Na + -glucose transporter 2 (SGLT2; 74 kDa, ab37296, Abcam) ( 44 ), Na + -K + -2Cl − cotransporter (NKCC2; 160 kDa, SPC-401, Stressmarq) ( 6 ), NaCl cotransporter (NCC; 130 kDa, kindly provided by David Ellison) ( 8 ), phosphorylated (p)NCC (Thr 53 ; 130 kDa, P1311-53, Phospho-solutions) ( 40 ), γ-subunit of the epithelial Na + channel (γ-ENaC; 83 kDa cleaved and 95 kDa uncleaved, SPC-405, Stressmarq) ( 6 ), renal outer medullary K + channel (ROMK; 50−65 kDa complex glycosylated, R-80, kindly provided by James Wade) ( 61 ), electroneutral Cl − exchanger (pendrin; 85−110 kDa denatured and 220 kDa dimer, sc16894, Santa Cruz Biotechnology) ( 25 , 62 ), and Na + -driven Cl − /bicarbonate exchanger (NDCBE; 123 kDa, no. 12531-1, ProteinTech) ( 53 ).

Techniques: Western Blot, Bicinchoninic Acid Protein Assay, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Effects of extreme potassium stress on blood pressure and renal tubular sodium transport

doi: 10.1152/ajprenal.00527.2019

Figure Lengend Snippet: Summary of the effects of extreme dietary K+ maneuvers on Na+ transport. The effect of dietary K+ depletion and loading are summarized based on transporter abundance and activity obtained from immunoblot and diuretic experiments. Extreme K+ depletion had a significant effect on all Na+ transporters and channels studied due to the combined effects of volume loss and K+ deficiency. K+ loading had similar stimulatory effects on Na+-K+-2Cl− cotransporter (NKCC2) and epithelial Na+ channel (ENaC) and inhibitory effects on NaCl cotransporter (NCC). The K+ anion (basic vs. Cl−) had opposing effects in the proximal tubule and intercalated cells. CCD, cortical collecting duct; CNT, connecting tubule; cTAL, cortical thick ascending limb; DCT, distal convoluted tubule; IC, intercalated cells; mTAL, medullary thick ascending limb; PC, principal cells; PT, proximal tubule.

Article Snippet: The following antibodies were used for immunoblot analysis: aquaporin 2 (Aqp2; 29 kDa unglycosylated and 35−45 kDa mature, sc-515770, Santa Cruz Biotechnology) ( 46 ), Na + /H + exchanger 3 (NHE3; 84 kDa, SPC-400, Stressmarq) ( 28 ), Na + -bicarbonate cotransporter 1A (NBCe1A; 130 kDa denatured and 280 kDa dimer, AB3212-I, Millipore) ( 39 , 49 ), Na + -glucose transporter 2 (SGLT2; 74 kDa, ab37296, Abcam) ( 44 ), Na + -K + -2Cl − cotransporter (NKCC2; 160 kDa, SPC-401, Stressmarq) ( 6 ), NaCl cotransporter (NCC; 130 kDa, kindly provided by David Ellison) ( 8 ), phosphorylated (p)NCC (Thr 53 ; 130 kDa, P1311-53, Phospho-solutions) ( 40 ), γ-subunit of the epithelial Na + channel (γ-ENaC; 83 kDa cleaved and 95 kDa uncleaved, SPC-405, Stressmarq) ( 6 ), renal outer medullary K + channel (ROMK; 50−65 kDa complex glycosylated, R-80, kindly provided by James Wade) ( 61 ), electroneutral Cl − exchanger (pendrin; 85−110 kDa denatured and 220 kDa dimer, sc16894, Santa Cruz Biotechnology) ( 25 , 62 ), and Na + -driven Cl − /bicarbonate exchanger (NDCBE; 123 kDa, no. 12531-1, ProteinTech) ( 53 ).

Techniques: Activity Assay, Western Blot

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Epidermal growth factor deficiency predisposes to progressive renal disease

doi: 10.1096/fj.202101837R

Figure Lengend Snippet: Western blotting of whole kidney lysates from the following mice: WT mice with normal kidney function (1-, 2- and 11-months-old), 1 months-old Egf−/− mice with normal kidney function, 4 months-old Egf−/− mouse with renal failure and fibrosis (BUN 497; shown in Figures 2 and ​and3),3), 11-months-old Egf−/− mouse with a moderate increase in BUN (56) and no renal fibrosis, 2-months-old Egf−/WT mouse (BUN 29) and 4-months-old Egf−/WTTgfa−/− mouse with normal kidney function (BUN 34). The 4 months-old Egf−/− mouse kidney with renal failure and fibrosis shows diminished protein levels of NCC and Slc3a1 (but not of NKCC2), increased active β-catenin levels and mTOR activation, with an increase in phosphorylation of 4E-BP1 at Thr37/46, p70 S6 kinase and TFEB. In contrast, the kidney of the 11-months-old Egf−/− mouse with a moderate increase in BUN (56) and no renal fibrosis did not show these changes. Black arrowheads indicate protein size (based on protein ladder, indicated in kDa). Values show densitometric values for Western blot bands normalized to β-actin.

Article Snippet: The following primary antibodies were used: antibodies against NKCC2 (38436, Cell Signaling Technology), NCC (Millipore Cat# AB3553, RRID:AB_571116), active β-catenin (non-phospho Ser33/37/Thr41) (Cell Signaling Technology Cat# 8814S, RRID:AB_11127203), phospho 4E-BP1 (Thr37/46) (Cell Signaling Technology Cat# 2855, RRID:AB_560835), p70 S6 kinase (Cell Signaling Technology Cat# 2708, RRID:AB_390722), rabbit anti-TFEB (Bethyl Cat# A303–672A, RRID:AB_11204598), and rabbit anti-Scl3a1 (Proteintech Cat# 16343–1-AP; RRID:AB_2239419).

Techniques: Western Blot, Activation Assay, Phospho-proteomics

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Epidermal growth factor deficiency predisposes to progressive renal disease

doi: 10.1096/fj.202101837R

Figure Lengend Snippet: Immunolabeling of a 4 months-old Egf−/− mouse kidney with renal failure and fibrosis shows dilated DCTs (Pvalb+NCC+) (yellow arrows) and CTs/cortical CDs (Aqp2+) (blue arrow), increased macrophage infiltration (F4/80+), and fibrosis with SMA+ myofibroblasts particularly in a periglomerular distribution (orange arrows), whereas TALs (NKCC2+) are not dilated (red arrow). These changes are not seen in a WT age-matched littermate. PNA (rhodamine-conjugated peanut agglutinin) labels distal nephron epithelial cells, whereas contiguous red labeling is seen in PTs. Scale bars 100 μm.

Article Snippet: The following primary antibodies were used: antibodies against NKCC2 (38436, Cell Signaling Technology), NCC (Millipore Cat# AB3553, RRID:AB_571116), active β-catenin (non-phospho Ser33/37/Thr41) (Cell Signaling Technology Cat# 8814S, RRID:AB_11127203), phospho 4E-BP1 (Thr37/46) (Cell Signaling Technology Cat# 2855, RRID:AB_560835), p70 S6 kinase (Cell Signaling Technology Cat# 2708, RRID:AB_390722), rabbit anti-TFEB (Bethyl Cat# A303–672A, RRID:AB_11204598), and rabbit anti-Scl3a1 (Proteintech Cat# 16343–1-AP; RRID:AB_2239419).

Techniques: Immunolabeling, Labeling