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botryodiplodia theobromae (ATCC)

Name: botryodiplodia theobromae
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ATCC botryodiplodia theobromae
Botryodiplodia Theobromae, supplied by ATCC, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Expression of LRRC55 in glomerular tissues of FSGS, DN, and MN patients. (A) Volcano plot of differentially expressed genes in glomerular tissues of FSGS patients and normal controls ( n = 5). (B) Isolation of glomerular tissues by laser capture microdissection. (C) The level of LRRC55 mRNA in glomerular tissues of patients with FSGS, DN, and MN ( n = 15, validation cohort). (D) IHC analysis of LRRC55 in glomerular tissues of patients with FSGS, DN, and MN. Black arrows indicate positive staining of LRRC55. (E) Quantification of LRRC55 expression levels in glomerular tissues ( n = 6). (F) Coexpression staining of LRRC55 and synaptopodin (SYNPO) in glomerular tissues of patients with FSGS, DN, and MN. White arrows show regions of superimposition of LRRC55 and SYNPO. Data shown are representative of one (A) or three (B–F) experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for C and E. ***, P < 0.001. Scale bar = 20 µm. FDR, false discovery rate.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Expression of LRRC55 in glomerular tissues of FSGS, DN, and MN patients. (A) Volcano plot of differentially expressed genes in glomerular tissues of FSGS patients and normal controls ( n = 5). (B) Isolation of glomerular tissues by laser capture microdissection. (C) The level of LRRC55 mRNA in glomerular tissues of patients with FSGS, DN, and MN ( n = 15, validation cohort). (D) IHC analysis of LRRC55 in glomerular tissues of patients with FSGS, DN, and MN. Black arrows indicate positive staining of LRRC55. (E) Quantification of LRRC55 expression levels in glomerular tissues ( n = 6). (F) Coexpression staining of LRRC55 and synaptopodin (SYNPO) in glomerular tissues of patients with FSGS, DN, and MN. White arrows show regions of superimposition of LRRC55 and SYNPO. Data shown are representative of one (A) or three (B–F) experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for C and E. ***, P < 0.001. Scale bar = 20 µm. FDR, false discovery rate.

Article Snippet: The transfection of the pREP-NFATc3 plasmid (11790; Addgene), LRRC55 siRNA (sc-96743, 15 nM), TRPC6 siRNA (sc-42672), C/EBPβ siRNA (sc-29229), IRF-2 siRNA (sc-35708), NFATc3 siRNA (sc-29413), STAT4 siRNA (sc-36568), PAX5 siRNA (sc-43996), and ERα siRNA (sc-29305) was conducted with Lipofectamine 2000 (11668–019; Invitrogen).

Techniques: Expressing, Isolation, Laser Capture Microdissection, Staining

The level of LRRC55 in glomerular tissues of patients with interstitial nephritis or MCD. (A) RT-PCR analysis of LRRC55 in glomerular tissues of patients with interstitial nephritis or MCD ( n = 5). (B) IHC analysis of LRRC55 expression in renal tissues of patients with interstitial nephritis or MCD ( n = 5). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for A. ns, not significant. Scale bar = 20 µm.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: The level of LRRC55 in glomerular tissues of patients with interstitial nephritis or MCD. (A) RT-PCR analysis of LRRC55 in glomerular tissues of patients with interstitial nephritis or MCD ( n = 5). (B) IHC analysis of LRRC55 expression in renal tissues of patients with interstitial nephritis or MCD ( n = 5). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for A. ns, not significant. Scale bar = 20 µm.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Knockout of Lrrc55 ameliorates podocyte injury in Ang II–induced mice. (A and B) RT-PCR and Western blot analysis of LRRC55 in podocytes of mice treated with Ang II ( n = 6). Mice glomeruli were isolated by using the magnetic bead–based isolation technique, single-cell suspensions were obtained with enzymatic disaggregation, and then podocytes were enriched by using anti-podocalyxin beads for RT-PCR and Western blot analysis. (C) IHC analysis of LRRC55 in glomerular tissues of mice treated with Ang II ( n = 6). Black arrow indicates positive staining for LRRC55. (D) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from mice treated with Ang II ( n = 6). To analyze the whole-cell recording of the BK current in situ, the isolated glomeruli were attached to poly-L-lysine–coated coverslips, and then the coverslips were placed into a patch-clamp chamber and perfused with bath solution to conduct a patch-clamp experiment. (E) Urinary albumin excretion of mice treated with Ang II ( n = 6). (F) Periodic acid–Schiff staining and electron microscopy analysis of renal sections. (G) Mean width of podocyte foot processes ( n = 6). (H) The number of apoptotic podocytes in glomerular tissues of mice treated with Ang II ( n = 6). (I) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of mice treated with Ang II. Data shown are representative of three experiments. For statistical analysis, a two-tailed Student’s t test was used for A, and one-way ANOVA with Tukey’s post hoc test was used for D, E, G, and H. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Knockout of Lrrc55 ameliorates podocyte injury in Ang II–induced mice. (A and B) RT-PCR and Western blot analysis of LRRC55 in podocytes of mice treated with Ang II ( n = 6). Mice glomeruli were isolated by using the magnetic bead–based isolation technique, single-cell suspensions were obtained with enzymatic disaggregation, and then podocytes were enriched by using anti-podocalyxin beads for RT-PCR and Western blot analysis. (C) IHC analysis of LRRC55 in glomerular tissues of mice treated with Ang II ( n = 6). Black arrow indicates positive staining for LRRC55. (D) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from mice treated with Ang II ( n = 6). To analyze the whole-cell recording of the BK current in situ, the isolated glomeruli were attached to poly-L-lysine–coated coverslips, and then the coverslips were placed into a patch-clamp chamber and perfused with bath solution to conduct a patch-clamp experiment. (E) Urinary albumin excretion of mice treated with Ang II ( n = 6). (F) Periodic acid–Schiff staining and electron microscopy analysis of renal sections. (G) Mean width of podocyte foot processes ( n = 6). (H) The number of apoptotic podocytes in glomerular tissues of mice treated with Ang II ( n = 6). (I) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of mice treated with Ang II. Data shown are representative of three experiments. For statistical analysis, a two-tailed Student’s t test was used for A, and one-way ANOVA with Tukey’s post hoc test was used for D, E, G, and H. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated.

Techniques: Knock-Out, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, Staining, In Situ, Patch Clamp, Electron Microscopy, Two Tailed Test

Ang II activates the BK channel by upregulating LRRC55 and Ca 2+ influx in podocytes. (A) The level of LRRC55 mRNA in podocytes treated with different doses of Ang II for 24 h ( n = 5). (B) Western blot analysis of LRRC55 in podocytes treated with different doses of Ang II for 24 h ( n = 3). (C) The level of LRRC55 mRNA in podocytes treated with Ang II (1 µM) for different durations ( n = 5). (D) Western blot analysis of LRRC55 in podocytes treated with Ang II (1 µM) for different durations ( n = 3). (E) Levels of KCNMA1 , LRRC26 , LRRC38 , and LRRC52 mRNA in podocytes treated with Ang II (1 µM) for 24 h ( n = 5). (F) Representative traces of the whole-cell BK current in podocytes treated with Ang II (1 µM) and LRRC55 siRNA (15 nM) for 24 h. (G) The BK current measured at +80 mV in podocytes treated with Ang II and LRRC55 siRNA for 24 h ( n = 6). (H) The BK current measured at +80 mV in podocytes transfected with the LRRC55 expression plasmid for 24 h ( n = 6). (I) Representative transients of intracellular Ca 2+ dynamics in podocytes treated with Ang II (1 µM). To analyze Ca 2+ influx, cells were incubated with the Ca 2+ indicator, Fluo-4 AM, and then fluorescence images were captured every 5 s. Changes in intracellular Ca 2+ levels were estimated from the fluorescence images as previously described . (J) The level of intracellular Ca 2+ in podocytes treated with Ang II (1 µM) for 4 min ( n = 6). (K) The effect of TRPC6 siRNA and LRRC55 siRNA on the BK current in podocytes treated with Ang II (1 µM) for 24 h ( n = 6). (L) Effect of LRRC55 overexpression and TRPC6 siRNA on the BK current in podocytes treated with Ang II (1 µM) for 4 min ( n = 6). Data shown are representative of three experiments. For statistical analysis, a two-tailed Student’s t test was used for E, H, and J, and one-way ANOVA with Tukey’s post hoc test was used for A, C, G, K, and L. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Scram., scrambled.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Ang II activates the BK channel by upregulating LRRC55 and Ca 2+ influx in podocytes. (A) The level of LRRC55 mRNA in podocytes treated with different doses of Ang II for 24 h ( n = 5). (B) Western blot analysis of LRRC55 in podocytes treated with different doses of Ang II for 24 h ( n = 3). (C) The level of LRRC55 mRNA in podocytes treated with Ang II (1 µM) for different durations ( n = 5). (D) Western blot analysis of LRRC55 in podocytes treated with Ang II (1 µM) for different durations ( n = 3). (E) Levels of KCNMA1 , LRRC26 , LRRC38 , and LRRC52 mRNA in podocytes treated with Ang II (1 µM) for 24 h ( n = 5). (F) Representative traces of the whole-cell BK current in podocytes treated with Ang II (1 µM) and LRRC55 siRNA (15 nM) for 24 h. (G) The BK current measured at +80 mV in podocytes treated with Ang II and LRRC55 siRNA for 24 h ( n = 6). (H) The BK current measured at +80 mV in podocytes transfected with the LRRC55 expression plasmid for 24 h ( n = 6). (I) Representative transients of intracellular Ca 2+ dynamics in podocytes treated with Ang II (1 µM). To analyze Ca 2+ influx, cells were incubated with the Ca 2+ indicator, Fluo-4 AM, and then fluorescence images were captured every 5 s. Changes in intracellular Ca 2+ levels were estimated from the fluorescence images as previously described . (J) The level of intracellular Ca 2+ in podocytes treated with Ang II (1 µM) for 4 min ( n = 6). (K) The effect of TRPC6 siRNA and LRRC55 siRNA on the BK current in podocytes treated with Ang II (1 µM) for 24 h ( n = 6). (L) Effect of LRRC55 overexpression and TRPC6 siRNA on the BK current in podocytes treated with Ang II (1 µM) for 4 min ( n = 6). Data shown are representative of three experiments. For statistical analysis, a two-tailed Student’s t test was used for E, H, and J, and one-way ANOVA with Tukey’s post hoc test was used for A, C, G, K, and L. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Scram., scrambled.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Incubation, Fluorescence, Over Expression, Two Tailed Test

Western blot analysis of LRRC55 or TRPC6 in podocytes. (A) Level of LRRC55 in podocytes transfected with LRRC55 siRNA for 24 h ( n = 3). (B) Level of LRRC55 in podocytes transfected with LRRC55 expression plasmid for 24 h ( n = 3). (C) Level of TRPC6 in podocytes transfected with TRPC6 siRNA for 24 h ( n = 3). Results are representative of three experiments. Scram., scrambled.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Western blot analysis of LRRC55 or TRPC6 in podocytes. (A) Level of LRRC55 in podocytes transfected with LRRC55 siRNA for 24 h ( n = 3). (B) Level of LRRC55 in podocytes transfected with LRRC55 expression plasmid for 24 h ( n = 3). (C) Level of TRPC6 in podocytes transfected with TRPC6 siRNA for 24 h ( n = 3). Results are representative of three experiments. Scram., scrambled.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation

Activation of the BK channel exacerbates TRPC6-mediated Ca 2+ influx in podocytes. (A) I/V relationship of TRPC6 current in podocytes treated with NS1619. (B) TRPC6 current measured at +80 mV in podocytes treated with NS1619 ( n = 6). (C) Level of intracellular Ca 2+ in podocytes treated with NS1619 ( n = 6). (D) TRPC6 current measured at +80 mV in podocytes treated with Ang II and IBTX ( n = 6). (E) Level of intracellular Ca 2+ in podocytes treated with Ang II and IBTX ( n = 6). (F) Effect of LRRC55 siRNA on TRPC6 current in podocytes treated with Ang II for 24 h ( n = 6). (G) Effect of LRRC55 siRNA on intracellular Ca 2+ in podocytes treated with Ang II for 24 h ( n = 6). Data shown are representative of three experiments. For statistical analysis, a two-tailed Student’s t test was used for B and C, and one-way ANOVA with Tukey’s post hoc test was used for D–G. ***, P < 0.001. Scram., scrambled.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Activation of the BK channel exacerbates TRPC6-mediated Ca 2+ influx in podocytes. (A) I/V relationship of TRPC6 current in podocytes treated with NS1619. (B) TRPC6 current measured at +80 mV in podocytes treated with NS1619 ( n = 6). (C) Level of intracellular Ca 2+ in podocytes treated with NS1619 ( n = 6). (D) TRPC6 current measured at +80 mV in podocytes treated with Ang II and IBTX ( n = 6). (E) Level of intracellular Ca 2+ in podocytes treated with Ang II and IBTX ( n = 6). (F) Effect of LRRC55 siRNA on TRPC6 current in podocytes treated with Ang II for 24 h ( n = 6). (G) Effect of LRRC55 siRNA on intracellular Ca 2+ in podocytes treated with Ang II for 24 h ( n = 6). Data shown are representative of three experiments. For statistical analysis, a two-tailed Student’s t test was used for B and C, and one-way ANOVA with Tukey’s post hoc test was used for D–G. ***, P < 0.001. Scram., scrambled.

Techniques: Activation Assay, Two Tailed Test

Decreased intracellular potassium induces cell apoptosis in podocytes. (A) Level of intracellular potassium in podocytes treated with Ang II (1 µM), LRRC55 siRNA, or NS1619 (30 µM) for 24 h ( n = 5). (B) Immunoprecipitation (IP) analysis of the binding between Apaf-1 and cytochrome c (Cyt c) in cytoplasmic extracts incubated with dATP (1 mM), cytochrome c (10 µg/ml), and decreasing concentrations of KCl ( n = 3). (C) Changes in caspase-3 activity in cytoplasmic extracts incubated with dATP, cytochrome c, and decreasing concentrations of KCl ( n = 5). (D) Gel analysis of DNA fragmentation in the mixture of cytoplasmic extracts, nuclei, dATP, cytochrome c, and decreasing concentrations of KCl ( n = 3). (E) IP analysis of the binding between Apaf-1 and cytochrome c in podocytes treated with Ang II, LRRC55 siRNA, or NS1619 for 24 h ( n = 3). (F) Western blot analysis of cleaved caspase-3 (casp-3) in podocytes treated with Ang II, LRRC55 siRNA, or NS1619 for 24 h ( n = 3). (G) Gel analysis of DNA fragmentation in podocytes treated with Ang II, LRRC55 siRNA, or NS1619 for 24 h ( n = 3). (H and I) Flow cytometric analysis of apoptotic cells among podocytes ( n = 5). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for A, C, and I. ***, P < 0.001. AFC, 7-amino-4-trifluoromethylcoumarin; IB, immunoblotting; Scram., scrambled.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Decreased intracellular potassium induces cell apoptosis in podocytes. (A) Level of intracellular potassium in podocytes treated with Ang II (1 µM), LRRC55 siRNA, or NS1619 (30 µM) for 24 h ( n = 5). (B) Immunoprecipitation (IP) analysis of the binding between Apaf-1 and cytochrome c (Cyt c) in cytoplasmic extracts incubated with dATP (1 mM), cytochrome c (10 µg/ml), and decreasing concentrations of KCl ( n = 3). (C) Changes in caspase-3 activity in cytoplasmic extracts incubated with dATP, cytochrome c, and decreasing concentrations of KCl ( n = 5). (D) Gel analysis of DNA fragmentation in the mixture of cytoplasmic extracts, nuclei, dATP, cytochrome c, and decreasing concentrations of KCl ( n = 3). (E) IP analysis of the binding between Apaf-1 and cytochrome c in podocytes treated with Ang II, LRRC55 siRNA, or NS1619 for 24 h ( n = 3). (F) Western blot analysis of cleaved caspase-3 (casp-3) in podocytes treated with Ang II, LRRC55 siRNA, or NS1619 for 24 h ( n = 3). (G) Gel analysis of DNA fragmentation in podocytes treated with Ang II, LRRC55 siRNA, or NS1619 for 24 h ( n = 3). (H and I) Flow cytometric analysis of apoptotic cells among podocytes ( n = 5). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for A, C, and I. ***, P < 0.001. AFC, 7-amino-4-trifluoromethylcoumarin; IB, immunoblotting; Scram., scrambled.

Techniques: Immunoprecipitation, Binding Assay, Incubation, Activity Assay, Western Blot

NFATc3 binds to the LRRC55 promoter and induces LRRC55 overexpression. (A) Screening of TFs binding to the LRRC55 promoter using a promoter-binding TF profiling plate array ( n = 3). Podocytes were treated with Ang II for 24 h to activate TFs. Nuclear extracts were prepared and incubated with TF binding oligo probe mix with or without an LRRC55 promoter DNA fragment. After spin separation of complexes from unbound free biotin-labeled oligos, TF-bound probes were eluted from the column and used for hybridization and analysis. (B) The level of LRRC55 mRNA in podocytes treated with Ang II, C/EBPβ siRNA, IRF-2 siRNA, NFATc3 siRNA, STAT4 siRNA, PAX5 siRNA, and ERα siRNA ( n = 3). (C) Western blot analysis of nuclear NFATc3 in podocytes treated with Ang II (1 µM) for 24 h ( n = 3). (D) Immunofluorescence staining of nuclear NFATc3 in podocytes treated with Ang II (1 µM) for 24 h ( n = 3). (E) Schematic of the NFATc3 binding sites in the upstream sequence of the LRRC55 promoter. (F) ChIP analysis of the binding between NFATc3 and the LRRC55 promoter in podocytes treated with Ang II (1 µM) for 24 h ( n = 3). (G) Schematic of the WT and mutated (MUT) LRRC55 promoter-luciferase reporter plasmids. (H) Normalized luciferase activity of reporter constructs in podocytes cotransfected with NFATc3 plasmid for 24 h ( n = 5). (I) RT-PCR analysis of LRRC55 in podocytes transfected with NFATc3 plasmid for 24 h ( n = 5). (J) Western blot analysis of LRRC55 in podocytes transfected with NFATc3 plasmid ( n = 3). (K) RT-PCR analysis of LRRC55 in podocytes treated with Ang II (1 µM) and NFATc3 siRNA for 24 h ( n = 5). (L) Western blot analysis of LRRC55 in podocytes treated with Ang II (1 µM) and NFATc3 siRNA for 24 h ( n = 3). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for B and K, and a two-tailed Student’s t test was used for H and I. ***, P < 0.001. Scale bar = 20 µm. Scram., scrambled.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: NFATc3 binds to the LRRC55 promoter and induces LRRC55 overexpression. (A) Screening of TFs binding to the LRRC55 promoter using a promoter-binding TF profiling plate array ( n = 3). Podocytes were treated with Ang II for 24 h to activate TFs. Nuclear extracts were prepared and incubated with TF binding oligo probe mix with or without an LRRC55 promoter DNA fragment. After spin separation of complexes from unbound free biotin-labeled oligos, TF-bound probes were eluted from the column and used for hybridization and analysis. (B) The level of LRRC55 mRNA in podocytes treated with Ang II, C/EBPβ siRNA, IRF-2 siRNA, NFATc3 siRNA, STAT4 siRNA, PAX5 siRNA, and ERα siRNA ( n = 3). (C) Western blot analysis of nuclear NFATc3 in podocytes treated with Ang II (1 µM) for 24 h ( n = 3). (D) Immunofluorescence staining of nuclear NFATc3 in podocytes treated with Ang II (1 µM) for 24 h ( n = 3). (E) Schematic of the NFATc3 binding sites in the upstream sequence of the LRRC55 promoter. (F) ChIP analysis of the binding between NFATc3 and the LRRC55 promoter in podocytes treated with Ang II (1 µM) for 24 h ( n = 3). (G) Schematic of the WT and mutated (MUT) LRRC55 promoter-luciferase reporter plasmids. (H) Normalized luciferase activity of reporter constructs in podocytes cotransfected with NFATc3 plasmid for 24 h ( n = 5). (I) RT-PCR analysis of LRRC55 in podocytes transfected with NFATc3 plasmid for 24 h ( n = 5). (J) Western blot analysis of LRRC55 in podocytes transfected with NFATc3 plasmid ( n = 3). (K) RT-PCR analysis of LRRC55 in podocytes treated with Ang II (1 µM) and NFATc3 siRNA for 24 h ( n = 5). (L) Western blot analysis of LRRC55 in podocytes treated with Ang II (1 µM) and NFATc3 siRNA for 24 h ( n = 3). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for B and K, and a two-tailed Student’s t test was used for H and I. ***, P < 0.001. Scale bar = 20 µm. Scram., scrambled.

Techniques: Over Expression, Binding Assay, Incubation, Labeling, Hybridization, Western Blot, Immunofluorescence, Staining, Sequencing, Luciferase, Activity Assay, Construct, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Transfection, Two Tailed Test

Effect of NFATc3 inhibition on podocyte injury in Ang II–treated mice. (A) Nuclear accumulation of NFATc3 in podocytes of mice treated with Ang II and 11R-VIVIT. White arrow indicates nuclear accumulation of NFATc3 in the podocytes. (B) The level of Lrrc55 mRNA in the podocytes of mice ( n = 6). (C) The level of LRRC55 protein in the podocytes of mice ( n = 6). (D) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from mice treated with Ang II and 11R-VIVIT ( n = 6). (E) The level of intracellular potassium in podocytes of freshly isolated glomeruli from mice ( n = 6). (F) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of mice treated with Ang II and 11R-VIVIT ( n = 6). (G) Apoptotic podocytes in glomerular tissues of mice treated with Ang II and 11R-VIVIT ( n = 6). (H) Urinary albumin excretion in mice treated with Ang II and 11R-VIVIT ( n = 6). (I) Periodic acid–Schiff staining and electron microscopy analysis of renal sections. (J) Mean width of podocyte foot processes in mice treated with Ang II and 11R-VIVIT ( n = 6). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for B, D, E, G, H, and J. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Effect of NFATc3 inhibition on podocyte injury in Ang II–treated mice. (A) Nuclear accumulation of NFATc3 in podocytes of mice treated with Ang II and 11R-VIVIT. White arrow indicates nuclear accumulation of NFATc3 in the podocytes. (B) The level of Lrrc55 mRNA in the podocytes of mice ( n = 6). (C) The level of LRRC55 protein in the podocytes of mice ( n = 6). (D) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from mice treated with Ang II and 11R-VIVIT ( n = 6). (E) The level of intracellular potassium in podocytes of freshly isolated glomeruli from mice ( n = 6). (F) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of mice treated with Ang II and 11R-VIVIT ( n = 6). (G) Apoptotic podocytes in glomerular tissues of mice treated with Ang II and 11R-VIVIT ( n = 6). (H) Urinary albumin excretion in mice treated with Ang II and 11R-VIVIT ( n = 6). (I) Periodic acid–Schiff staining and electron microscopy analysis of renal sections. (J) Mean width of podocyte foot processes in mice treated with Ang II and 11R-VIVIT ( n = 6). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for B, D, E, G, H, and J. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated.

Techniques: Inhibition, Isolation, Western Blot, Staining, Electron Microscopy

Effects of losartan on LRRC55 expression and podocyte injury in ADR-treated mice. (A) The level of Lrrc55 mRNA in podocytes of ADR-treated mice treated with losartan or LRRC55 siRNA ( n = 6). (B) Western blot analysis of LRRC55 expression in podocytes of ADR-treated mice ( n = 6). (C) IHC analysis of LRRC55 expression in the renal tissues of ADR-treated mice ( n = 6). Black arrows indicate positive staining of LRRC55. (D) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from ADR-treated mice ( n = 6). (E) Intracellular potassium levels in podocytes of freshly isolated glomeruli from ADR-treated mice ( n = 6). (F) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of ADR-treated mice ( n = 6). (G) The number of apoptotic podocytes in glomerular tissues of ADR-treated mice ( n = 6). (H) Urinary albumin excretion of ADR-treated mice ( n = 6). (I) Periodic acid–Schiff staining and electron microscopy analysis of renal sections. (J) Mean width of podocyte foot processes ( n = 6). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for A, D, E, G, H, and J. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated. Scram., scrambled.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Effects of losartan on LRRC55 expression and podocyte injury in ADR-treated mice. (A) The level of Lrrc55 mRNA in podocytes of ADR-treated mice treated with losartan or LRRC55 siRNA ( n = 6). (B) Western blot analysis of LRRC55 expression in podocytes of ADR-treated mice ( n = 6). (C) IHC analysis of LRRC55 expression in the renal tissues of ADR-treated mice ( n = 6). Black arrows indicate positive staining of LRRC55. (D) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from ADR-treated mice ( n = 6). (E) Intracellular potassium levels in podocytes of freshly isolated glomeruli from ADR-treated mice ( n = 6). (F) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of ADR-treated mice ( n = 6). (G) The number of apoptotic podocytes in glomerular tissues of ADR-treated mice ( n = 6). (H) Urinary albumin excretion of ADR-treated mice ( n = 6). (I) Periodic acid–Schiff staining and electron microscopy analysis of renal sections. (J) Mean width of podocyte foot processes ( n = 6). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for A, D, E, G, H, and J. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated. Scram., scrambled.

Techniques: Expressing, Western Blot, Staining, Isolation, Electron Microscopy

Effects of losartan on LRRC55 expression and podocyte injury in experimental DN. (A) The level of Lrrc55 mRNA in podocytes of diabetic mice treated with losartan or LRRC55 siRNA ( n = 6). (B) Western blot analysis of LRRC55 expression in podocytes of diabetic mice ( n = 6). (C) IHC analysis of LRRC55 expression in renal tissues ( n = 6). Black arrows indicate positive staining for LRRC55. (D) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from diabetic mice treated with losartan or LRRC55 siRNA ( n = 6). (E) Intracellular potassium levels in podocytes of freshly isolated glomeruli from diabetic mice treated with losartan or LRRC55 siRNA ( n = 6). (F) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of diabetic mice ( n = 6). (G) The number of apoptotic podocytes in glomerular tissues of diabetic mice ( n = 6). (H) Urinary albumin excretion of diabetic mice ( n = 6). (I) Periodic acid–Schiff staining and electron microscopy analysis of renal sections. (J) Mean width of podocyte foot processes ( n = 6). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for A, D, E, G, H, and J. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated. Scram., scrambled.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Effects of losartan on LRRC55 expression and podocyte injury in experimental DN. (A) The level of Lrrc55 mRNA in podocytes of diabetic mice treated with losartan or LRRC55 siRNA ( n = 6). (B) Western blot analysis of LRRC55 expression in podocytes of diabetic mice ( n = 6). (C) IHC analysis of LRRC55 expression in renal tissues ( n = 6). Black arrows indicate positive staining for LRRC55. (D) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from diabetic mice treated with losartan or LRRC55 siRNA ( n = 6). (E) Intracellular potassium levels in podocytes of freshly isolated glomeruli from diabetic mice treated with losartan or LRRC55 siRNA ( n = 6). (F) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of diabetic mice ( n = 6). (G) The number of apoptotic podocytes in glomerular tissues of diabetic mice ( n = 6). (H) Urinary albumin excretion of diabetic mice ( n = 6). (I) Periodic acid–Schiff staining and electron microscopy analysis of renal sections. (J) Mean width of podocyte foot processes ( n = 6). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for A, D, E, G, H, and J. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated. Scram., scrambled.

Techniques: Expressing, Western Blot, Staining, Isolation, Electron Microscopy

Effects of losartan on LRRC55 expression and podocyte injury in experimental MN. (A) The level of Lrrc55 mRNA in podocytes of PHN rats treated with losartan or LRRC55 siRNA ( n = 6). (B) Western blot analysis of LRRC55 expression in podocytes of PHN rats ( n = 6). (C) IHC analysis of LRRC55 expression in renal tissues ( n = 6). Black arrows indicate positive staining of LRRC55. (D) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from PHN rats treated with losartan or LRRC55 siRNA ( n = 6). (E) Intracellular potassium level in podocytes of freshly isolated glomeruli from PHN rats treated with losartan or LRRC55 siRNA ( n = 6). (F) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of PHN rats ( n = 6). (G) The number of apoptotic podocytes in glomerular tissues of PHN rats ( n = 6). (H) Urinary albumin excretion of PHN rats ( n = 6). (I) Electron microscopy analysis of renal sections. (J) Mean width of podocyte foot processes ( n = 6). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for A, D, E, G, H, and J. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated. Scram., scrambled.

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Effects of losartan on LRRC55 expression and podocyte injury in experimental MN. (A) The level of Lrrc55 mRNA in podocytes of PHN rats treated with losartan or LRRC55 siRNA ( n = 6). (B) Western blot analysis of LRRC55 expression in podocytes of PHN rats ( n = 6). (C) IHC analysis of LRRC55 expression in renal tissues ( n = 6). Black arrows indicate positive staining of LRRC55. (D) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from PHN rats treated with losartan or LRRC55 siRNA ( n = 6). (E) Intracellular potassium level in podocytes of freshly isolated glomeruli from PHN rats treated with losartan or LRRC55 siRNA ( n = 6). (F) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of PHN rats ( n = 6). (G) The number of apoptotic podocytes in glomerular tissues of PHN rats ( n = 6). (H) Urinary albumin excretion of PHN rats ( n = 6). (I) Electron microscopy analysis of renal sections. (J) Mean width of podocyte foot processes ( n = 6). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for A, D, E, G, H, and J. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated. Scram., scrambled.

Techniques: Expressing, Western Blot, Staining, Isolation, Electron Microscopy

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