Journal: Molecular and Cellular Biology

Article Title: The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation ▿The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation ▿ †

doi: 10.1128/MCB.01358-09

Figure Lengend Snippet: Cdh1 regulated cell motility via control of p190 abundance. (A) siRNA-transfected HeLa cells were placed on membranes in serum-free medium after 48 h of culture and were allowed to migrate in a Boyden chamber for 24 h, either in the absence of any stimuli or in the presence of 10% FBS. Membranes were then fixed and stained with 50% Giemsa solution in PBS. Data shown were representative of migrating cells transfected with the indicated siRNA oligonucleotides. (B) The levels of Cdh1 and p190 in cells subjected to a Boyden chamber assay were assessed by immunoblot analysis using anti-Cdh1 and anti-p190 antibodies. The corresponding α-tubulin levels are shown as a loading control. (C) Migration was assessed as the number of cells that invaded the membrane after 24 h of incubation. Data represented mean values ± standard errors of the means from measurements performed in triplicate from three independent experiments using HeLa cells (*, P

Article Snippet: The antibodies used in this study were anti-Myc (9E10; Santa Cruz Biotechnology), 1:1,000; anti-HA (12CA5; Santa Cruz Biotechnology), 1:1,000; anti-GFP (full-length; Santa Cruz Biotechnology), 1:500; anti-Cdh1 (DH01; Abcam), 1:500; anti-p190 (BD), 1:1,000; anti-α-tubulin (B-5-1-2; Sigma), 1:50,000; anti-RhoA, -B, and -C (Cell Signaling), 1:1,000; anti-Rac1 (Upstate Biotechnology), 1:1,000; anti-RhoGDI (Millipore), 1:500; anti-Skp2 (Zymed), 1:250; anti-cyclin B (GNS1; Santa Cruz), 1:200; and anti-Emi1 (Zymed), 1:200; anti-cdc27 (AF3.1; Santa Cruz Biotechnology), 1:500; and anti-His (MBL), 1:500.

Techniques: Transfection, Staining, Boyden Chamber Assay, Migration, Incubation

Journal: Molecular and Cellular Biology

Article Title: The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation ▿The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation ▿ †

doi: 10.1128/MCB.01358-09

Figure Lengend Snippet: APC/C Cdh1 -mediated ubiquitination of p190. (A) In vivo ubiquitination of p190. 293T cells were transfected with the GFP-full-length p190 expression plasmid or control vector, together with an HA-ubiquitin expression plasmid in the presence or absence of MG132. Lysates were immunoprecipitated (IP) with an anti-GFP antibody and were immunoblotted (Western blotting [WB]) using anti-HA (α-HA) and anti-GFP antibodies. (B) 293T cells transfected with either control or Cdh1 siRNA oligonucleotides for 48 h were subjected to an in vivo ubiquitination assay, as described for panel A. (C) Quantification of the ubiquitinated p190 protein in panel B was performed using densitometry. The value obtained for Cdh1 siRNA-transfected cells without MG132 treatment was set as 1. (D) Immortalized Cdh1 GT/GT MEFs were transfected with GFP-full-length p190, HA-ubiquitin (Ub), and full-length Cdh1 expression vectors. In vivo ubiquitination of p190 was evaluated as described for panels A and B. (E) In vitro ubiquitination assay. APC/C immunoprecipitated from HeLa cell lysates was conjugated with recombinant Cdh1 protein and was then subjected to an in vitro ubiquitination assay, as described in Materials and Methods. In vitro translated full-length p190 was used as a substrate. The reaction was terminated at the indicated time points. Ubiquitinated p190 was detected by immunoblotting with anti-p190 antibody (top panel). Recombinant His-Cdh1 protein used for APC/C binding was immunoblotted using anti-His antibody (middle). The presence of APC/C complex in each reaction product was confirmed by Western blotting against cdc27 (bottom). The input lane represents 0.5% of HeLa cell lysate used for immunoprecipitation.

Article Snippet: The antibodies used in this study were anti-Myc (9E10; Santa Cruz Biotechnology), 1:1,000; anti-HA (12CA5; Santa Cruz Biotechnology), 1:1,000; anti-GFP (full-length; Santa Cruz Biotechnology), 1:500; anti-Cdh1 (DH01; Abcam), 1:500; anti-p190 (BD), 1:1,000; anti-α-tubulin (B-5-1-2; Sigma), 1:50,000; anti-RhoA, -B, and -C (Cell Signaling), 1:1,000; anti-Rac1 (Upstate Biotechnology), 1:1,000; anti-RhoGDI (Millipore), 1:500; anti-Skp2 (Zymed), 1:250; anti-cyclin B (GNS1; Santa Cruz), 1:200; and anti-Emi1 (Zymed), 1:200; anti-cdc27 (AF3.1; Santa Cruz Biotechnology), 1:500; and anti-His (MBL), 1:500.

Techniques: In Vivo, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Ubiquitin Assay, In Vitro, Recombinant, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation ▿The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation ▿ †

doi: 10.1128/MCB.01358-09

Figure Lengend Snippet: p190 was stabilized in Cdh1 -deficient cells. (A) Primary MEFs of the indicated genotypes were collected, lysed, and immunoblotted for endogenous p190, Cdh1, and Skp2. β-Actin levels are shown as a loading control. Each experiment was conducted in triplicate, and the immunoblots presented here are representative runs. (B) An abundance of p190 proteins was regulated posttranslationally. Total RNA was isolated from MEFs of the indicated genotypes and subjected to quantitative RT-PCR analysis of the p190 mRNA. Data were normalized to the levels of GAPDH mRNA and represent means ± standard deviations from three independent experiments. (C) Accumulation of p190 in Cdh1 -depleted HeLa cells. HeLa cells were transfected with either control or Cdh1 siRNA oligonucleotides. After 48 h of culture, cells were harvested and examined for the expression levels of p190, Cdh1, and α-tubulin using immunoblotting analysis. (D) HeLa cells were transfected with GFP-tagged full-length Cdh1 or DN-Cdh1 expression vectors. Cells were lysed and processed through immunoblotting using antibodies against p190, GFP, and α-tubulin. (E) Cdh1 activation caused a reduction in the levels of p190. HeLa cells transfected with control or Emi1 siRNA oligonucleotides were cultured for 48 h in the absence (−) or presence (+) of MG132. Cell lysates were subjected to SDS-PAGE and immunoblot analysis using the indicated antibodies. Emi1 depletion led to Cdh1 activation, as evidenced by the degradation of its target, cyclin B (arrow, middle lane). (F) Flow cytometric analysis of the cell cycle. Asynchronous MEFs with the indicated genotypes and HeLa cells that were transfected with either control or Cdh1 siRNA oligonucleotides for 24 h were stained with propidium iodide and were then subjected to flow cytometry. The percentage of cells in each phase of the cell cycle is shown.

Article Snippet: The antibodies used in this study were anti-Myc (9E10; Santa Cruz Biotechnology), 1:1,000; anti-HA (12CA5; Santa Cruz Biotechnology), 1:1,000; anti-GFP (full-length; Santa Cruz Biotechnology), 1:500; anti-Cdh1 (DH01; Abcam), 1:500; anti-p190 (BD), 1:1,000; anti-α-tubulin (B-5-1-2; Sigma), 1:50,000; anti-RhoA, -B, and -C (Cell Signaling), 1:1,000; anti-Rac1 (Upstate Biotechnology), 1:1,000; anti-RhoGDI (Millipore), 1:500; anti-Skp2 (Zymed), 1:250; anti-cyclin B (GNS1; Santa Cruz), 1:200; and anti-Emi1 (Zymed), 1:200; anti-cdc27 (AF3.1; Santa Cruz Biotechnology), 1:500; and anti-His (MBL), 1:500.

Techniques: Western Blot, Isolation, Quantitative RT-PCR, Transfection, Expressing, Activation Assay, Cell Culture, SDS Page, Flow Cytometry, Staining, Cytometry

Journal: Molecular and Cellular Biology

Article Title: The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation ▿The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation ▿ †

doi: 10.1128/MCB.01358-09

Figure Lengend Snippet: The middle domain of p190 interacted with Cdh1. (A) Schematic representations of the structure of p190 and its derived mutants. MD, middle domain; GBD, GTP-binding domain; GAP, GTPase-activating domain. (B) In vitro binding assay. The indicated GFP-p190 proteins expressed in 293T cells were immunopurified, immobilized on protein A beads (Dynal), and incubated with in vitro translated Cdh1. The resulting immunocomplex was analyzed using avidin-HRP to detect the bound biotin-Cdh1. The arrow indicates the MD-N mutant for p190. The arrowhead indicates IgG. (C) Lysates from 293T cells transfected with GFP-tagged full-length Cdh1 were immunoprecipitated using control rabbit IgG or an antibody (Ab) against p190. These immunoprecipitates were then fractionated by SDS-PAGE and immunoblotted using an anti-GFP antibody (top) or an anti-p190 antibody (bottom). (D) In vivo coimmunoprecipitation of p190 with Cdh1. Lysates from 293T cells were immunoprecipitated with rabbit preimmune serum or serum against Cdh1. These immunoprecipitates were then fractionated by SDS-PAGE and immunoblotted using anti-p190 (top) or anti-Cdh1 (bottom) antibodies. IP: immunoprecipitation; WB, Western blotting.

Article Snippet: The antibodies used in this study were anti-Myc (9E10; Santa Cruz Biotechnology), 1:1,000; anti-HA (12CA5; Santa Cruz Biotechnology), 1:1,000; anti-GFP (full-length; Santa Cruz Biotechnology), 1:500; anti-Cdh1 (DH01; Abcam), 1:500; anti-p190 (BD), 1:1,000; anti-α-tubulin (B-5-1-2; Sigma), 1:50,000; anti-RhoA, -B, and -C (Cell Signaling), 1:1,000; anti-Rac1 (Upstate Biotechnology), 1:1,000; anti-RhoGDI (Millipore), 1:500; anti-Skp2 (Zymed), 1:250; anti-cyclin B (GNS1; Santa Cruz), 1:200; and anti-Emi1 (Zymed), 1:200; anti-cdc27 (AF3.1; Santa Cruz Biotechnology), 1:500; and anti-His (MBL), 1:500.

Techniques: Derivative Assay, Binding Assay, In Vitro, Incubation, Avidin-Biotin Assay, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, In Vivo, Western Blot

Journal: Molecular and Cellular Biology

Article Title: The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation ▿The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation ▿ †

doi: 10.1128/MCB.01358-09

Figure Lengend Snippet: Developmental defects of Cdh1 GT/GT embryos. (A) Hematoxylin-eosin staining of sections of wild-type, Cdh1 GT/GT , or tetraploid complementation-rescued placentas at 12.5 dpc. The asterisk indicates thrombus in the labyrinth layer. D, decidua; Sp, spongiotrophoblast layer; L, labyrinth layer. (B) Percentage of placentas with thrombus formation at the indicated dpc. Placentas with thrombus that had a diameter larger than 500 μm (longest diameter) were judged as positive. The number of placentas analyzed was as follows: at 11.5 dpc, Cdh1 +/+ , 5; Cdh1 GT/+ , 8; and Cdh1 GT/GT , 7; at 12.5 dpc, Cdh1 +/+ , 6; Cdh1 GT/+ , 15; and Cdh1 GT/GT , 10; at 13.5 dpc, Cdh1 +/+ , 8; Cdh1 GT/+ , 8; and Cdh1 GT/GT , 10. (C) The eyes of wild-type and Cdh1 GT/GT mice at 18.5 dpc were obtained from tetraploid complementation. The arrows indicate the margin of the eyelid. (D) Hematoxylin-eosin staining of transverse eye sections from 18.5-dpc embryos, as in panel C. The arrows indicate the margin of the eyelid. (E) Scanning electron microgr aphs of the eyes of wild-type or Cdh1 GT/GT rescue embryos at the indicated dpc. The arrowheads indicate eyelid fusion in wild-type embryos. (F) Percentage of embryos with eyelids open at the indicated dpc. The number of embryos analyzed was as follows: at 14.5 dpc, Cdh1 +/+ , 2; and Cdh1 GT/GT , 2; at 15.5 dpc, Cdh1 +/+ , 8; Cdh1 GT/GT , 5; at 16.5 dpc, Cdh1 +/+ , 8; and Cdh1 GT/GT , 3; at 18.5 dpc, Cdh1 +/+ , 9; and Cdh1 GT/GT , 10. (G) Immunohistochemical staining of p190 in transverse sections of the eyelid of wild-type and Cdh1 GT/GT rescue embryos at 18.5 dpc. The arrows indicate the margin of the eyelid. (H) Sagittal sections of rescue embryos at 18.5 dpc were stained with an anti-p190 antibody. The arrows indicate p190 staining in the central nervous system of Cdh1 GT/GT embryos. Bars, 50 μm (D) and 100 μm (E).

Article Snippet: The antibodies used in this study were anti-Myc (9E10; Santa Cruz Biotechnology), 1:1,000; anti-HA (12CA5; Santa Cruz Biotechnology), 1:1,000; anti-GFP (full-length; Santa Cruz Biotechnology), 1:500; anti-Cdh1 (DH01; Abcam), 1:500; anti-p190 (BD), 1:1,000; anti-α-tubulin (B-5-1-2; Sigma), 1:50,000; anti-RhoA, -B, and -C (Cell Signaling), 1:1,000; anti-Rac1 (Upstate Biotechnology), 1:1,000; anti-RhoGDI (Millipore), 1:500; anti-Skp2 (Zymed), 1:250; anti-cyclin B (GNS1; Santa Cruz), 1:200; and anti-Emi1 (Zymed), 1:200; anti-cdc27 (AF3.1; Santa Cruz Biotechnology), 1:500; and anti-His (MBL), 1:500.

Techniques: Staining, Mouse Assay, Immunohistochemistry

Journal: The American Journal of Pathology

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

doi: 10.1016/j.ajpath.2018.07.025

Figure Lengend Snippet: Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n  = 6 controls and 8 patients with HIVAN. ∗∗∗ P ÂÂ

Article Snippet: Cells were incubated with the following primary antibodies: APOL1 (1G12D11; 1.57 μg/100 μL; Proteintech, Chicago, IL), rabbit (rb)-PAX2 (EP3251; 0.145 μg/100 μL, Abcam, Cambridge, UK), rb–claudin 1 (0.175 μg/100 μL, Abcam), rb–Wilms tumor 1 (WT1) (0.36 μg/100 μL, Abcam), rb-podocalyxin (0.53 μg/100 μL, catalog number PAI-46170; Life Technologies, Carlsbad, CA), synaptopodin (P-19, 0.36 μg/100 μL, Santa Cruz Biotechnology Inc., Santa Cruz, TX), CD2AP (B-4, 0.36 μg/100 μL, Santa Cruz Biotechnology), and mouse-α-actinin (H-2; 0.1 μg/100 μL, Santa Cruz Biotechnology) for overnight at 4°C.

Techniques: Expressing, Labeling, Fluorescence, Software

Journal: The American Journal of Pathology

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

doi: 10.1016/j.ajpath.2018.07.025

Figure Lengend Snippet: The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n  = 4 ( A and C–F ). ∗ P ÂÂ

Article Snippet: Cells were incubated with the following primary antibodies: APOL1 (1G12D11; 1.57 μg/100 μL; Proteintech, Chicago, IL), rabbit (rb)-PAX2 (EP3251; 0.145 μg/100 μL, Abcam, Cambridge, UK), rb–claudin 1 (0.175 μg/100 μL, Abcam), rb–Wilms tumor 1 (WT1) (0.36 μg/100 μL, Abcam), rb-podocalyxin (0.53 μg/100 μL, catalog number PAI-46170; Life Technologies, Carlsbad, CA), synaptopodin (P-19, 0.36 μg/100 μL, Santa Cruz Biotechnology Inc., Santa Cruz, TX), CD2AP (B-4, 0.36 μg/100 μL, Santa Cruz Biotechnology), and mouse-α-actinin (H-2; 0.1 μg/100 μL, Santa Cruz Biotechnology) for overnight at 4°C.

Techniques: Expressing, Incubation, Wilms Tumor Assay, Generated, Amplification, Labeling, Microscopy, Fluorescence

Journal: The American Journal of Pathology

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

doi: 10.1016/j.ajpath.2018.07.025

Figure Lengend Snippet: Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n  = 4. ∗∗∗ P ÂÂ

Article Snippet: Cells were incubated with the following primary antibodies: APOL1 (1G12D11; 1.57 μg/100 μL; Proteintech, Chicago, IL), rabbit (rb)-PAX2 (EP3251; 0.145 μg/100 μL, Abcam, Cambridge, UK), rb–claudin 1 (0.175 μg/100 μL, Abcam), rb–Wilms tumor 1 (WT1) (0.36 μg/100 μL, Abcam), rb-podocalyxin (0.53 μg/100 μL, catalog number PAI-46170; Life Technologies, Carlsbad, CA), synaptopodin (P-19, 0.36 μg/100 μL, Santa Cruz Biotechnology Inc., Santa Cruz, TX), CD2AP (B-4, 0.36 μg/100 μL, Santa Cruz Biotechnology), and mouse-α-actinin (H-2; 0.1 μg/100 μL, Santa Cruz Biotechnology) for overnight at 4°C.

Techniques: Expressing, Transgenic Assay, Mouse Assay, Labeling, Fluorescence, Software

Journal: The American Journal of Pathology

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

doi: 10.1016/j.ajpath.2018.07.025

Figure Lengend Snippet: HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n  = 4 ( A and B ); n  = 3 ( E and F ). ∗ P ÂÂ

Article Snippet: Cells were incubated with the following primary antibodies: APOL1 (1G12D11; 1.57 μg/100 μL; Proteintech, Chicago, IL), rabbit (rb)-PAX2 (EP3251; 0.145 μg/100 μL, Abcam, Cambridge, UK), rb–claudin 1 (0.175 μg/100 μL, Abcam), rb–Wilms tumor 1 (WT1) (0.36 μg/100 μL, Abcam), rb-podocalyxin (0.53 μg/100 μL, catalog number PAI-46170; Life Technologies, Carlsbad, CA), synaptopodin (P-19, 0.36 μg/100 μL, Santa Cruz Biotechnology Inc., Santa Cruz, TX), CD2AP (B-4, 0.36 μg/100 μL, Santa Cruz Biotechnology), and mouse-α-actinin (H-2; 0.1 μg/100 μL, Santa Cruz Biotechnology) for overnight at 4°C.

Techniques: Expressing, Incubation, Wilms Tumor Assay, Transduction, Plasmid Preparation, Amplification, Labeling, Fluorescence