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.

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: 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

Journal: PLOS ONE

Article Title: Cleavage of the V-ATPase associated prorenin receptor is mediated by PACE4 and is essential for growth of prostate cancer cells

doi: 10.1371/journal.pone.0288622

Figure Lengend Snippet: ( A ) Western blot analysis showing Prorenin Receptor (PRR), furin, and PACE4 expression, sPRR secretion, and cell lysate and conditioned media total lane protein (CLTLP, CMTLP) in LNCaP cells infected with a control non-target shRNA (NT), Furin shRNA (shfurin), or PACE4 shRNA (shPACE4). ( B ) Western Blot analysis demonstrating the expression of PRR and secretion of sPRR in LNCaP cells infected with an empty pLenti6 vector or with pLenti6-PACE4 to overexpress PACE4. ( C ) Quantification of sPRR levels in pLenti6 and pLenti-PACE4-infected LNCaP cells (*P<0.05, n = 3). ( D ) Western blot showing the reduction in PRR processing resembled as a ratio of HA-tagged M8.9 (M8.9-HA) to HA-tagged full-length PRR (PRR-HA) in cellular extract of LNCaP cells after a 50 μM PACE4 inhibitor LLLRVK-amidinobenzylamide (Amba) (C23) treatment. ( E ) Corresponding quantification of the ratio of M8.9-HA to PRR-HA standardized over total lane protein (TLP) (*P<0.05, n = 3). ( F ) Analysis of PRR peptide cleavage by recombinant PACE4 (rPACE4) or recombinant furin (rfurin) monitored after a 2-hour incubation by high pressure liquid chromatography (HPLC). Mass spectrometry was done to confirm identity of peptide after cleavage. Cleavage site is underlined on the peptide sequence. Western blot analysis of PRR expression and sPRR secretion and quantification of sPRR secretion after DMSO (Vehicle), 50 μM multi-Leucine peptide (ML) PACE4 inhibitor, or 50 μM PEGylated cell-impermeable ML (PEG8-ML) treatment of DU145 (***P<0.001, n = 3) ( G , H ) or LNCaP ( I , J ) (**P<0.01, n = 3) cells, respectively. Beta-Actin (β-actin) and TLP were used as loading controls. Data are presented as the mean ± SEM. Statistical tests were conducted using Student’s t test.

Article Snippet: Western blots were performed using the following antibodies: PTEN (#9559, Cell Signaling), furin (#43996, Cell Signaling), Actin (A2228, Sigma), PRR (GTX114169, GeneTex), and PACE4 (ab151562, Abcam).

Techniques: Western Blot, Expressing, Infection, Control, shRNA, Plasmid Preparation, Recombinant, Incubation, High Performance Liquid Chromatography, Mass Spectrometry, Sequencing