Journal: Cell Death & Disease

Article Title: PLOD3 suppression exerts an anti-tumor effect on human lung cancer cells by modulating the PKC-delta signaling pathway

doi: 10.1038/s41419-019-1405-8

Figure Lengend Snippet: a R-H460 cells were transfected with 40 nM control siRNA (siCON) or PLOD3 siRNA (siPLOD3) for 24 h. Transcript and protein levels of PLOD3 were determined by western blotting (left) and qRT-PCR (right). ** P < 0.01. b Analysis of the viability of cells treated with or without 10 Gy radiation after transfection with siCON or siPLOD3. * P < 0.05; ** P < 0.01; *** P < 0.001. c Protein levels of PLOD3, cleaved PARP, and active caspase-3 (cell-death marker) as determined by western blotting. d Determination of cell death in R-H460 cells (treated as in b ) by AV/PI staining. *** P < 0.001. e Surviving fraction of cells treated with a single dose of radiation (0–8 Gy) after transfection with siCON or siPLOD3, measured two weeks after radiation treatment. * P < 0.05; ** P < 0.01

Article Snippet: Mouse monoclonal anti-PLOD3 antibody (Proteintech Group) together with rabbit polyclonal antibodies against PKCα, PKCβI, PKCβII, PKCγ, PKCδ, and PKCζ (Santa Cruz Biotechnology) were used for the proximity ligation reaction.

Techniques: Transfection, Control, Western Blot, Quantitative RT-PCR, Marker, Staining

Journal: Cell Death & Disease

Article Title: PLOD3 suppression exerts an anti-tumor effect on human lung cancer cells by modulating the PKC-delta signaling pathway

doi: 10.1038/s41419-019-1405-8

Figure Lengend Snippet: a Tumor volume was calculated at the indicated times using the formula: volume = (length × width 2 × 3.14)/6; ( n = 8). * P < 0.05; *** P < 0.001. b Tumors were excised and weighed at the end of the experiment (14 days). Representative images of tumors and tumor slides subjected to immunohistochemistry using an anti-PLOD3 antibody. c Effects of siRNA and radiation on tumor weights. Data shown are the means of each group. * P < 0.05; *** P < 0.001. d Body, liver, and lung tissues of mice were excised and weighed at the end of the experiment. All data are shown as the mean ± SD. e Five-year survival rate curves were obtained using Kaplan–Meier Plotter (www.kmplot.com) and display the survival probability based on data of 68 lung cancer patients treated with radiotherapy

Article Snippet: Mouse monoclonal anti-PLOD3 antibody (Proteintech Group) together with rabbit polyclonal antibodies against PKCα, PKCβI, PKCβII, PKCγ, PKCδ, and PKCζ (Santa Cruz Biotechnology) were used for the proximity ligation reaction.

Techniques: Immunohistochemistry

Journal: Cell Death & Disease

Article Title: PLOD3 suppression exerts an anti-tumor effect on human lung cancer cells by modulating the PKC-delta signaling pathway

doi: 10.1038/s41419-019-1405-8

Figure Lengend Snippet: a , b Cell death in cells transfected with 40 nM siPLOD3 as determined by AV/PI staining and western blotting at 12-h intervals. * P < 0.05. c R-H460 cells were treated with or without 40 µM Z-VAD-FMK (a pan-caspase inhibitor) after transfection with PLOD3 siRNA. Cell death was measured 48 h after treatment by AV/PI staining and western blotting. ** P < 0.01. d Analysis of caspase activity in R-H460 cells 48 h after transfection with PLOD3 siRNA by ELISA. Data were collected using a Multiskan EX at 405 nm. * P < 0.05; ** P < 0.01. e R-H460 cells were treated with Z-VAD-FMK (40 µM), Z-VDVAD-FMK (40 µM), Z-DEVD-FMK (40 µM), or Z-YVAD-FMK (40 µM) and then subjected to AV/PI staining and western blotting for active-caspase-3. ** P < 0.01; *** P < 0.001

Article Snippet: Mouse monoclonal anti-PLOD3 antibody (Proteintech Group) together with rabbit polyclonal antibodies against PKCα, PKCβI, PKCβII, PKCγ, PKCδ, and PKCζ (Santa Cruz Biotechnology) were used for the proximity ligation reaction.

Techniques: Transfection, Staining, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: PLOD3 suppression exerts an anti-tumor effect on human lung cancer cells by modulating the PKC-delta signaling pathway

doi: 10.1038/s41419-019-1405-8

Figure Lengend Snippet: a Cells were fixed with 4% paraformaldehyde and immunostained using antibodies targeting PLOD3 and Grp94 (ER marker). DNA was visualized by DAPI staining. b At 24-h post-transfection with siCON or siPLOD3, cell lysates were prepared and used for immunoblotting with antibodies against ER stress markers (ATF6, PERK, and IRE1α) and a DNA damage marker (γH2AX). c qRT-PCR analysis for IRE1α activation, spliced XBP-1 (sXBP-1), and unspliced X-box-binding protein 1 (unXBP-1). The GAPDH mRNA was utilized for normalization. * P < 0.05; *** P < 0.001. d R-H460 cells were pre-treated with APY29 (IRE1α inhibitor), and after transfection with siPLOD3, cell lysates were prepared and used for immunoblotting with antibodies against p-IRE1α, caspase-2, and caspase-4. e AV/PI staining of cells transfected with siCON or siPLOD3 and/or siIRE1α for 48 h. * P < 0.05. f R-H460 cells were treated with or without 40 µM Z-VAD-FMK after transfection with siPLOD3, and cell lysates were used for immunoblotting with antibodies against ATF6, p-PERK, p-IRE1α, and γH2AX. g R-H460 cells were transfected with 40 nM siCON or siPLOD3 for 36 h and then stained with 2′,7'-dichlorofluorescein diacetate. Intracellular levels of reactive oxygen species were observed by confocal microscopy

Article Snippet: Mouse monoclonal anti-PLOD3 antibody (Proteintech Group) together with rabbit polyclonal antibodies against PKCα, PKCβI, PKCβII, PKCγ, PKCδ, and PKCζ (Santa Cruz Biotechnology) were used for the proximity ligation reaction.

Techniques: Marker, Staining, Transfection, Western Blot, Quantitative RT-PCR, Activation Assay, Binding Assay, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: PLOD3 suppression exerts an anti-tumor effect on human lung cancer cells by modulating the PKC-delta signaling pathway

doi: 10.1038/s41419-019-1405-8

Figure Lengend Snippet: a R-H460 cells were fixed and incubated with a mouse anti-PLOD3 antibody together with rabbit antibodies against PKCs, followed by in situ proximity ligation assay analysis. Representative confocal images of cells with proximity ligation assay-positive signals (red dots). b , c R-H460 cells were transfected with HA-PLOD3, PKCα, or GFP-PKCδ. Cell lysates were immunoprecipitated with normal IgG (a negative control) or HA antibody, and then immunocomplexes were resolved by SDS-PAGE and immunoblotted with antibodies against HA, PKCα, and GFP. d Immunostaining analysis of the localization of PKCs in R-H460 cells. Cells were fixed with 4% paraformaldehyde and immunostained for PLOD3 (green), PKCα (red), and PKCδ (red). DNA was visualized by DAPI staining. e PLOD3 or control siRNA was transfected into R-H460 cells; 24-h post-transfection, cell lysates were prepared and used in immunoblotting with antibodies targeting PLOD3, p-PKCα, PKCδ, p-PKCδ, and β-actin. f Subcellular fraction analysis of R-H460 cells following siCON or siPLOD3 treatment. Proteins in each fraction were resolved by SDS-PAGE and immunoblotted with antibodies against PLOD3, PKCδ, lamin A/C, and α-tubulin. g R-H460 cells were transfected with 40 nM siCON or siPLOD3 and/or siPKCδ for 48 h. Cell death in R-H460 cells was determined by AV/PI staining (left). Protein levels of the indicated proteins were determined by western blotting (right). * P < 0.05. h R-H460 cells were transfected with 40 nM siCON or siPLOD3 and/or siPKCα for 48 h. Cell death in R-H460 cells was determined by AV/PI staining (left). Protein levels of the indicated proteins were determined by western blotting (right). * P < 0.05. i Protein levels of PLOD3, PKCδ, p-eIF2α, and p-IRE1 α were determined by western blotting. j Caspase-2, caspase-3, and caspase-4 activities in R-H460 cells (treated as in b ) were determined by caspase activity assay. Data were collected using the Multiskan EX at 405 nm. * P < 0.05; ** P < 0.01

Article Snippet: Mouse monoclonal anti-PLOD3 antibody (Proteintech Group) together with rabbit polyclonal antibodies against PKCα, PKCβI, PKCβII, PKCγ, PKCδ, and PKCζ (Santa Cruz Biotechnology) were used for the proximity ligation reaction.

Techniques: Incubation, In Situ, Proximity Ligation Assay, Transfection, Immunoprecipitation, Negative Control, SDS Page, Immunostaining, Staining, Control, Western Blot, Caspase Activity Assay

Journal: Cell Death & Disease

Article Title: PLOD3 suppression exerts an anti-tumor effect on human lung cancer cells by modulating the PKC-delta signaling pathway

doi: 10.1038/s41419-019-1405-8

Figure Lengend Snippet: a Cells were treated with cisplatin for 48 h. Proliferation was detected by cell viability assays. * P < 0.05; ** P < 0.01; *** P < 0.001. b A549 and R-H460 cells were transfected with 40 nM siCON or siPLOD3 for 24 h and then treated with cisplatin for another 48 h. The proliferation rate was determined by cell viability assays. * P < 0.05; ** P < 0.01; *** P < 0.001. c Cisplatin-treated cells (for 48 h) were analyzed by FACS after AV/PI staining. * P < 0.05; ** P < 0.01; *** P < 0.001. d Cell death (of cells treated as in b ) as analyzed by AV/PI staining. *** P < 0.001. e Protein levels of cleaved-PARP and active-caspase-3 as determined by western blotting after PLOD3 siRNA and cisplatin treatment. f R-H460 cells were transfected with siRNA in the absence or presence of 5 μM etoposide (ET) or 1 mM hydroxyurea (HU) or 0.1 μg/ml doxorubicin (DX). At 48 h after the drug treatments, the cells were analyzed by FACS after AV/PI staining. * P < 0.05

Article Snippet: Mouse monoclonal anti-PLOD3 antibody (Proteintech Group) together with rabbit polyclonal antibodies against PKCα, PKCβI, PKCβII, PKCγ, PKCδ, and PKCζ (Santa Cruz Biotechnology) were used for the proximity ligation reaction.

Techniques: Transfection, Staining, Western Blot

Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: High pY levels might affect the proliferation, migration and differentiation of lung fibroblasts. A , Changes in cell proliferation between hnLFs and hfLFs determined by CCK-8 kit (n = 7). *** P < 0.001. B-C , Assessing cell proliferation by EdU assay between hnLFs and hfLFs (n = 6). *** P < 0.001. D-G , Comparison of migration between hnLFs and hfLFs (n = 6). *** P < 0.001. H , Representative images of hnLFs and hfLFs under TEM. Arrows pointed to the lipid droplets in cytoplasm. I , Representative fluorescence images of Nile Red in hnLFs and hfLFs. J-K , The contractility of hnLFs and hfLFs in 3D collagen matrices (n = 6). *** P < 0.001. L , Phospho-RTK array analysis with 200 μg of lysates from hnLFs and hfLFs, which were serum-starved for 6 h. M , Quantification of phospho-RTK levels (average of two drops fold average of positive control). N-O , Western blot and quantification of p-VEGR2, p-PDGFRβ, p-EGFR and p-FGFR1 in hnLFs and hfLFs (n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001. P-Q , Western blot and quantification of p-PLCγ1, p-GAB1, p-SHC and p-SRC in hnLFs and hfLFs (n = 6). ** P < 0.01, *** P < 0.001. R-S , Western blot and quantification of p-AKT, p-ERK1/2 and p-STAT3 in hnLFs and hfLFs (n = 6). ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against FN (Cat#15613-1-AP), COL1A1 (Cat#67288-1-lg), α-SMA (Cat#55135-1-AP, Cat#67735-1-AP), FGFR1 (Cat#60325-1-lg), CD31 (Cat#60287-1-lg), CD45 (Cat#11265-1-lg), EpCAM (Cat#60287-1-lg), Vimentin (Cat#10366-1-AP) and GAPDH (Cat#60004-1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Migration, CCK-8 Assay, EdU Assay, Comparison, Fluorescence, Positive Control, Western Blot

Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: SH2 superbinder blocked multiple fibrosis associated pathways through interrupting pY-SH2 combination in pY mediated signal transmission. A-B , Phospho-RTK array analysis with 200 μg of lysates from hfLFs treated with 1 μM GST-SH2 WT or GST-SH2 TrM for 24 h. C , Western blot of p-VEGR2, p-PDGFRβ, p-EGFR and p-FGFR1 in hnLFs and hfLFs after GST, GST-SH2 WT and GST-SH2 TrM incubation. D , Western blot of p-PLCγ1, p-GAB1, p-SHC and p-SRC in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM incubation. E , Western blot of p-AKT, p-ERK1/2 and p-STAT3 in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM incubation. F , hnLFs and hfLFs were incubated with 1 μM GST, GST-SH2 WT or GST-SH2 TrM for 24 h. The immunoprecipitation of EGFR and SHC was detected. G , Representative fluorescence images of EGFR and SHC colocalization in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM treatment. H , Immunoprecipitations of GRB2 with GAB1, SHC, EGFR and PDGFRβ. I , GST pull down assay showed the binding capacity of SH2 superbinder with pY in hnLFs and hfLFs. J , Representative fluorescence images of GST tag and pY colocalization in hnLFs and hfLFs after incubation with GST, GST-SH2 WT or GST-SH2 TrM. K-L , GST pull down assay showed the binding capacity of SH2 superbinder with RTKs (such as VEGFR2, PDGFRβ, EGFR and FGFR1) and adaptor proteins (such as GAB1, SHC and SRC) in hfLFs and hfLFs.

Article Snippet: Antibodies against FN (Cat#15613-1-AP), COL1A1 (Cat#67288-1-lg), α-SMA (Cat#55135-1-AP, Cat#67735-1-AP), FGFR1 (Cat#60325-1-lg), CD31 (Cat#60287-1-lg), CD45 (Cat#11265-1-lg), EpCAM (Cat#60287-1-lg), Vimentin (Cat#10366-1-AP) and GAPDH (Cat#60004-1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Transmission Assay, Western Blot, Incubation, Immunoprecipitation, Fluorescence, Pull Down Assay, Binding Assay

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: ASC therapeutic effects on experimental periodontitis in rats. (A) The schematic diagram showed the induction of the experimental rat model of periodontitis for 21 days and ASC injection after the removal of ligatures. (B) Micro-CT imaging and bone parameters (CEJ-ABC distance, BV/TV, Tb. Th, and Tb. Sp) between healthy and experimental rats of periodontitis. Scale bar, 1 mm. (C) Micro-CT imaging displayed the alveolar bone of PBS-injected and ASC-injected rats on day 7, day 14, and day 21 Scale bar, 1 mm. (D) The CEJ-ABC distance and bone parameters (BV/TV, Tb. Th, and Tb. Sp) between the PBS-injected and ASC-injected rats. (E) Representative immunofluorescence staining of M1 phenotype macrophages (iNOS + ; Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in PBS-injected and ASC-injected rats. Scale bar, 20 μm. (F) The calculated ratio of iNOS + /CD206 + macrophages in PBS-injected and ASC-injected groups. CEJ-ABC, cementoenamel junction and alveolar bone crest. All data were expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Injection, Micro-CT, Imaging, Immunofluorescence, Staining

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: ASCs modulated macrophage polarization through NRF2. (A) Representative immunohistochemical staining of NRF2 in the PBS-injected and ASC-injected rats on day 7, day 14, and day 21. Scale bar, 50 μm. (B) Representative immunofluorescence staining of NRF2 (Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in PBS-injected and ASC-injected rats. Scale bar, 20 μm. (C) Schematic diagram of knocking down NRF2 in macrophages and macrophages cocultured with ASCs and LPS stimulation. (D, E) RT-qPCR and western blotting were used to measure the NRF2 siRNA or siNC in macrophages. (F, G) The mRNA expression and protein levels of M1 markers were increased, while those of M2 markers were decreased after silencing NRF2 in the cocultured groups. R, root; PDL, periodontal ligament; AB, alveolar bone; siRNA, small interfering RNA; NC, negative control. All data were expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Immunohistochemical staining, Staining, Injection, Immunofluorescence, Quantitative RT-PCR, Western Blot, Expressing, Small Interfering RNA, Negative Control

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: Inhibition of IDO activity reduced the therapeutic potential of ASCs in experimental periodontitis and downregulated NRF2 expression. (A) Three-dimensional reconstruction of maxillary alveolar bone in ASC-injected and 1-MT pretreated ASC-injected rats on day 7, day 14, and day 21. Scale bar, 1 mm. (B) Analysis of bone parameters. (C) Immunohistochemical staining of IL-1β, OCN, and NRF2 in the ASC-injected and 1-MT pretreated ASC-injected rats. Scale bar, 50 μm. (D) Representative immunofluorescence staining of M1 phenotype macrophages (iNOS + ; Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in ASC-injected and 1-MT pretreated ASC-injected rats. Scale bar, 20 μm. (E) The mean IOD of IL-1β, OCN, and NRF2 and the calculated ratio of iNOS + /CD206 + . IOD, integrated optical density.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Inhibition, Activity Assay, Expressing, Injection, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Redox Biology

Article Title: Chicoric acid prevents PDGF-BB-induced VSMC dedifferentiation, proliferation and migration by suppressing ROS/NFκB/mTOR/P70S6K signaling cascade

doi: 10.1016/j.redox.2017.11.012

Figure Lengend Snippet: CA abrogated PDGF-BB-induced VSMC dedifferentiation. VSMCs were pretreated with various concentrations (10, 50 and 100 μM) of CA for 6 h followed by stimulation with PDGF-BB (20 ng/mL) for 24 h. ( A ) Western blot was employed to quantitate the expression levels of contractile protein α-SMA, SMMHC, SM22α and synthetic proteins OPN. ( B ) Bar graph showing the relative protein level of α-SMA, SMMHC, SM22α and OPN. ( C ) Bar graph showing the relative mRNA level ofα-SMA, SMMHC, SM22α and OPN. Values are mean±SE. * P < 0.05 vs. Control, † P < 0.05 vs. PDGF-BB. n = 6 for each group.

Article Snippet: Antibodies against α-SMA, SM22α, PCNA, cyclin D1, P27 and horseradish peroxidase conjugated secondary antibodies were purchased from Proteintech Group, Inc (Wuhan, China).

Techniques: Western Blot, Expressing, Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu

doi: 10.1152/ajpcell.00538.2018

Figure Lengend Snippet: Autophagosome formation and autophagy markers in podocytes expressing apolipoprotein L1 (APOL1) nonrisk and risk alleles. A: vector- (V-), G0-, G1-, and G2-podocytes grown on coverslips were stained with acridine orange (n = 6). Cells were examined under a confocal microscope. Representative fluoromicrographs (autophagosomes displaying orange fluorescence) are shown. Scale bar, 50 µm. B: cumulative distribution data of vacuole accumulation (orange fluorescence) in control and experimental conditions (n = 6) are shown in a dot plot. *P < 0.05 vs. V. **P < 0.01 vs. V and G0. C: HEK cells grown on coverslips were transfected with V (VHEK), APOL1G0 (G0HEK), and APOL1G1 (G1HEK). After 48 h, cells labeled for light chain 3-II (LC3-II; green fluorescence) and nuclei were stained with DAPI (blue fluorescence); subsequently, cells were examined under a confocal microscope. Representative fluoromicrographs are displayed. Scale bar, 50 µm. D: podocytes stably overexpressing APOL1G0 (G0) and V were differentiated (n = 4–5). Protein blots from different cellular lysates of V and G0 were probed for beclin-1, p62, and APOL1; the same blots were reprobed for GAPDH. Gels from different cellular lysates are shown. i: Distribution densitometric data of beclin-1/GAPDH ratios from D are shown in a dot plot. *P < 0.05 vs.V. ii: Distribution densitometric data of p62/GAPDH ratios from D are shown in a dot plot. *P < 0.05 vs. V. iii: Distribution densitometric data of APOL1/GAPDH ratios from D are shown in a dot plot. *P < 0.05 vs. V. E: RNAs were extracted from different sets of V- and G0-podocytes (n = 5) and assayed for miR193a. Distribution data are shown in a dot plot. *P < 0.05 vs. V.

Article Snippet: Total protein lysed extracts (30 μg/lane) were loaded onto a 10% polyacrylamide (PAGE) precast gels (Bio-Rad, Hercules, CA) and after transfer onto PVDF membranes were processed for immunostaining with primary antibodies against APOL1 (cat. no. 66124-I-IG, Proteintech), ATG14L (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"Ab139727","term_id":"62160308","term_text":"AB139727"}} Ab139727 , Abcam), UVRAG (cat. no. 19571-I-AP, Proteintech), p62 (cat. no. SC-28359, Santa Cruz Biotechnology), LC3-II (cat. no. 2775S, Cell Signaling), PI3KC3 (cat. no. I3857S, Cell Signaling), phosphorylated (p-)ULK1 (cat. no. SC-390904, Santa Cruz), p-PI3KC3 (cat. no. 13857S, Cell Signaling), p-mTOR (cat. no. 2971S, Cell Signaling), CD2AP (cat. no. SC-25272; Santa Cruz Biotechnology), nephrin (cat. no. ABb235903, Abcam), WT1 (cat. no. AB15249, Abcam), podocalyxin (cat. no. PA-1-46170, Invitrogen), Rubicon (cat. no. 7151-S, Santa Cruz Biotechnology), podocin (cat. no. SC-22296, Santa Cruz Biotechnology), and beclin-1 (cat. no. 3738S, Cell Signaling), followed by treatment with horseradish peroxidase-labeled appropriate secondary antibodies.

Techniques: Expressing, Plasmid Preparation, Staining, Microscopy, Fluorescence, Transfection, Labeling, Stable Transfection

Journal: American Journal of Physiology - Cell Physiology

Article Title: Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu

doi: 10.1152/ajpcell.00538.2018

Figure Lengend Snippet: Formation of autophagy complexes utilizing bioinformatics studies. A: beclin1-Bcl2 homodimer: beclin-1 (bright orange) and Bcl2 (raspberry) form a homodimer in which Bcl2 binds at the BH3 domain of beclin-1. ATG14L complex I consists of beclin-1 (bright orange), APOL1G0 (marine blue), PI3KC3 (wheat), and ATG14L (magenta). UVRAG complex II consists of beclin-1 (bright orange), APOL1G0 (marine blue), PI3KC3 (wheat). and UVRAG (cyan). B: schema of protein-protein interaction is shown. In brief, beclin-1 dimerizes with Bcl2 to form an inactive complex. Activation of JNK1 phosphorylates Bcl2, causing release of beclin-1. Oligomerization of beclin-1 allows it to form PI3KC3 complexes along with ATG14 (complex I) or UVRAG (complex II). C: Rubicon complex IIIa consists of APOL1G1 (marine blue), PI3KC3 (wheat), UVRAG (magenta), and Rubicon (brown). Rubicon complex IIIb consists of APOL1G2, PI3KC3 (wheat), UVRAG (magenta), and Rubicon (brown). D: schema of Rubicon complex III is shown. Enhanced expression of Rubicon in APOL1G1 and APOL1G2 milieus lead to their binding with UVRAG and PI3KC3 in a relatively lower concentration of beclin-1. APOL1, apolipoprotein L1; BH3, Bcl-2 homology domain 3; CCD, coiled-coil domain; ECD, evolutionarily conserved domain; PI3KC3, class III phosphatidylinositol 3-kinase (PI3KC3); Rubicon, run domain beclin-1-interacting and cysteine-rich domain-containing protein; ATG14L, autophagy-related gene 14L; UVRAG, UV radiation resistance-associated gene; JNK1, c-Jun NH2-terminal protein kinase-1.

Article Snippet: Total protein lysed extracts (30 μg/lane) were loaded onto a 10% polyacrylamide (PAGE) precast gels (Bio-Rad, Hercules, CA) and after transfer onto PVDF membranes were processed for immunostaining with primary antibodies against APOL1 (cat. no. 66124-I-IG, Proteintech), ATG14L (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"Ab139727","term_id":"62160308","term_text":"AB139727"}} Ab139727 , Abcam), UVRAG (cat. no. 19571-I-AP, Proteintech), p62 (cat. no. SC-28359, Santa Cruz Biotechnology), LC3-II (cat. no. 2775S, Cell Signaling), PI3KC3 (cat. no. I3857S, Cell Signaling), phosphorylated (p-)ULK1 (cat. no. SC-390904, Santa Cruz), p-PI3KC3 (cat. no. 13857S, Cell Signaling), p-mTOR (cat. no. 2971S, Cell Signaling), CD2AP (cat. no. SC-25272; Santa Cruz Biotechnology), nephrin (cat. no. ABb235903, Abcam), WT1 (cat. no. AB15249, Abcam), podocalyxin (cat. no. PA-1-46170, Invitrogen), Rubicon (cat. no. 7151-S, Santa Cruz Biotechnology), podocin (cat. no. SC-22296, Santa Cruz Biotechnology), and beclin-1 (cat. no. 3738S, Cell Signaling), followed by treatment with horseradish peroxidase-labeled appropriate secondary antibodies.

Techniques: Activation Assay, Expressing, Binding Assay, Concentration Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu

doi: 10.1152/ajpcell.00538.2018

Figure Lengend Snippet: Analysis of composition of autophagy complexes by immunoprecipitation (IP) studies. A: protein blots of cellular lysates (n = 3) from differentiated vector- (V-) and G0-podocytes were probed for ATG14L and reprobed for UVRAG, beclin-1, PI3KC3 (VPS34), APOL1, and GAPDH. Representative gels are displayed. i: Distribution densitometric data of ATG14L/GAPDH ratios from A are shown in a dot plot. ii: Distribution densitometric data of UVRAG/GAPDH ratios from A are shown in a dot plot. iii: Distribution densitometric data of beclin-1/GAPDH ratios from A are shown in a dot plot. iv: Distribution densitometric data of PI3KC3/GAPDH ratios from A are shown in a dot plot. v: Distribution densitometric data of APOL1/GAPDH ratios from A are shown in a dot plot. *P < 0.05 vs. V. B: cellular lysates were immunoprecipitated with an anti-ATG14L antibody (n = 6). Protein blots of IP fractions were probed for ATG14L and reprobed for beclin-1, PI3KC3, and APOL1. IgG expression was used as a loading marker. Gels from three different IP fractions are displayed. i: Distribution densitometric data of ATG14L/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V. ii: Distribution densitometric data of beclin-1/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V. iii: Distribution densitometric data of PI3KC3/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V; iv: Distribution densitometric data of APOL1/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V. C: cellular lysates of V- and G0-podocytes from A were immunoprecipitated with anti-UVRAG antibody (n = 6). Protein blots were probed for UVRAG, beclin-1, PI3KC3, APOL1, and IgG. Gels from three independent IP fractions are shown. i: Distribution densitometric data of UVRAG/IgG ratios from C are shown in a dot plot. *P < 0.05 vs. V. ii: Distribution densitometric data of beclin-1/IgG ratios from C are shown in a dot plot. *P < 0.05 vs. V. iii: Distribution densitometric data of PI3KC3/IgG ratios from 3C are shown in a dot plot. *P < 0.05 vs. V. iv: Distribution densitometric data of APOL1/IgG ratios from C are shown in a dot plot. *P < 0.05 vs. V. D: protein blots of cellular lysates of V- and G0-podocytes were probed for APOL1, PI3KC3, and beclin-1 and reprobed for actin (n = 6). Cellular lysates from three independent lysates are shown. i: Distribution densitometric data of APOL1/actin ratios from D are shown in a dot plot. *P < 0.05 vs. V. ii: Distribution densitometric data of PI3KC3/actin ratios from D are shown in a dot plot. *P < 0.05 vs. V. iii: Distribution densitometric data of beclin-1/actin ratios from D are shown in a dot plot. *P < 0.05 vs. V. E: cellular lysates of V- and G0-podocytes were immunoprecipitated with anti-APOL1 antibody. IP fractions were probed for APOL1, PI3KC3, beclin-1, and IgG. Gels from three independent IP fractions are displayed. i: Distribution densitometric data of APOL1/IgG in ratios from E are shown in a dot plot. *P < 0.05 vs. V. ii: Distribution densitometric data of PI3KC3/IgG ratios from 3E are shown in a dot plot. iii: Distribution densitometric data of beclin-1/IgG ratios from E are shown in a dot plot. *P < 0.05 vs. V. APOL1, apolipoprotein L1; PI3KC3, class III phosphatidylinositol 3-kinase; ATG14L, autophagy-related gene 14L; UVRAG, UV radiation resistance-associated gene.

Article Snippet: Total protein lysed extracts (30 μg/lane) were loaded onto a 10% polyacrylamide (PAGE) precast gels (Bio-Rad, Hercules, CA) and after transfer onto PVDF membranes were processed for immunostaining with primary antibodies against APOL1 (cat. no. 66124-I-IG, Proteintech), ATG14L (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"Ab139727","term_id":"62160308","term_text":"AB139727"}} Ab139727 , Abcam), UVRAG (cat. no. 19571-I-AP, Proteintech), p62 (cat. no. SC-28359, Santa Cruz Biotechnology), LC3-II (cat. no. 2775S, Cell Signaling), PI3KC3 (cat. no. I3857S, Cell Signaling), phosphorylated (p-)ULK1 (cat. no. SC-390904, Santa Cruz), p-PI3KC3 (cat. no. 13857S, Cell Signaling), p-mTOR (cat. no. 2971S, Cell Signaling), CD2AP (cat. no. SC-25272; Santa Cruz Biotechnology), nephrin (cat. no. ABb235903, Abcam), WT1 (cat. no. AB15249, Abcam), podocalyxin (cat. no. PA-1-46170, Invitrogen), Rubicon (cat. no. 7151-S, Santa Cruz Biotechnology), podocin (cat. no. SC-22296, Santa Cruz Biotechnology), and beclin-1 (cat. no. 3738S, Cell Signaling), followed by treatment with horseradish peroxidase-labeled appropriate secondary antibodies.

Techniques: Immunoprecipitation, Plasmid Preparation, Expressing, Marker

Journal: American Journal of Physiology - Cell Physiology

Article Title: Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu

doi: 10.1152/ajpcell.00538.2018

Figure Lengend Snippet: APOL1 risk milieus induce Rubicon complex formation in podocytes. A: protein blots of cellular lysates of vector- (V-), G0-, G1-, and G2-podocytes were probed for UVRAG, Rubicon, PI3KC3,and APOL1 and reprobed for GAPDH. Gels from three independent lysates are displayed. i: Distribution densitometric data of UVRAG/GAPDH ratios from A are shown in a dot plot. ii: Distribution densitometric data of Rubicon/GAPDH ratios from A are shown in a dot plot. *P < 0.05 vs. G0. iii: Distribution densitometric data of PI3KC3/GAPDH ratios from A are shown in a dot plot. *P < 0.05 vs. G0. iv: distribution densitometric data of APOL1/GAPDH ratios from A are shown in a dot plot. *P < 0.05 compared vs. V. B: cellular lysates of V-, G0-, G1-, and G2-podocytes from 4A were immunoprecipitated with an anti-Rubicon antibody. Immunoprecipitation (IP) fractions were probed for UVRAG, Rubicon, PI3KC3, and APOL1. IgG was used as a loading marker. Gels from three independent IP fractions are displayed. i: Distribution densitometric data of UVRAG/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V and G0. ii: Distribution densitometric data of Rubicon/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V and G0. iii: Distribution densitometric data of PI3KC3/IgG ratios from B are shown in a dot plot. iv: Distribution densitometric data of APOL1/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V. C: RNAs were extracted from different sets of V-, G0-, G1-, and G2-podocytes (n = 5). cDNAs were amplified with a specific primer for Rubicon. Cumulative data are shown in a dot plot. ***P < 0.001 vs. V and G. APOL1, apolipoprotein L1; PI3KC3, class III phosphatidylinositol 3-kinase; Rubicon, run domain beclin-1-interacting and cysteine-r UVRAG, UV radiation resistance-associated gene.

Article Snippet: Total protein lysed extracts (30 μg/lane) were loaded onto a 10% polyacrylamide (PAGE) precast gels (Bio-Rad, Hercules, CA) and after transfer onto PVDF membranes were processed for immunostaining with primary antibodies against APOL1 (cat. no. 66124-I-IG, Proteintech), ATG14L (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"Ab139727","term_id":"62160308","term_text":"AB139727"}} Ab139727 , Abcam), UVRAG (cat. no. 19571-I-AP, Proteintech), p62 (cat. no. SC-28359, Santa Cruz Biotechnology), LC3-II (cat. no. 2775S, Cell Signaling), PI3KC3 (cat. no. I3857S, Cell Signaling), phosphorylated (p-)ULK1 (cat. no. SC-390904, Santa Cruz), p-PI3KC3 (cat. no. 13857S, Cell Signaling), p-mTOR (cat. no. 2971S, Cell Signaling), CD2AP (cat. no. SC-25272; Santa Cruz Biotechnology), nephrin (cat. no. ABb235903, Abcam), WT1 (cat. no. AB15249, Abcam), podocalyxin (cat. no. PA-1-46170, Invitrogen), Rubicon (cat. no. 7151-S, Santa Cruz Biotechnology), podocin (cat. no. SC-22296, Santa Cruz Biotechnology), and beclin-1 (cat. no. 3738S, Cell Signaling), followed by treatment with horseradish peroxidase-labeled appropriate secondary antibodies.

Techniques: Plasmid Preparation, Immunoprecipitation, Marker, Amplification

Journal: American Journal of Physiology - Cell Physiology

Article Title: Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu

doi: 10.1152/ajpcell.00538.2018

Figure Lengend Snippet: Expression of APOL1 risk alleles is associated with autophagy blockade in podocytes. A: protein blots of independent sets of differentiated vector- (V-), G0-, G1-, and G2-podocytes were probed for phospho (p)-mTOR, LC3-II, p62, PI3KC3, and GAPDH (n = 6). Gels from two independent lysates are shown. i: Densitometric distribution data of p-mTOR/GAPDH ratios from A are shown in a dot plot. *P < 0.05 vs. V and G0. ii: Densitometric distribution data of PI3KC3/GAPDH ratios from A are shown in a dot plot. *P < 0.05 and **P < 0.01 vs. V. iii: Densitometric distribution data of p62/GAPDH ratios from A are shown in a dot plot. *P < 0.05 vs. V. iv: Densitometric distribution data of LC3-I/GAPDH ratios from A are shown in a dot plot. **P < 0.01 and ***P < 0.001 vs. V. v: Densitometric distribution data of LC3-II/GAPDH ratios from A are shown in a dot plot. *P < 0.05 and **P < 0.01 vs. V. B: cellular lysates from A were probed for APOL1 and reprobed for GAPDH. Gels from two independent cellular lysates are displayed. i: Densitometric distribution data of APOL1/GAPDH ratios from B are shown in a dot plot. **P < 0.01 and ***P < 0.001 vs. V. C: protein blots from independent sets of V-, G0-, G1-, and G2-podocytes were probed for p-ULK1 and reprobed for p-PI3KC3, and GAPDH (n = 6). Gels from three independent lysates are displayed. i: Densitometric distribution data of p-ULK/GAPDH ratios from C are shown in a dot plot. *P < 0.05 compared with other variables. ii: Densitometric distribution data of p-PI3KC3/GAPDH ratios from C are shown in a dot plot. *P < 0.05 vs. other variables. APOL1, apolipoprotein L1; LC3, light chain 3; PI3KC3, class III phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; ULK1, Unc-51 like autophagy-activating kinase-1.

Article Snippet: Total protein lysed extracts (30 μg/lane) were loaded onto a 10% polyacrylamide (PAGE) precast gels (Bio-Rad, Hercules, CA) and after transfer onto PVDF membranes were processed for immunostaining with primary antibodies against APOL1 (cat. no. 66124-I-IG, Proteintech), ATG14L (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"Ab139727","term_id":"62160308","term_text":"AB139727"}} Ab139727 , Abcam), UVRAG (cat. no. 19571-I-AP, Proteintech), p62 (cat. no. SC-28359, Santa Cruz Biotechnology), LC3-II (cat. no. 2775S, Cell Signaling), PI3KC3 (cat. no. I3857S, Cell Signaling), phosphorylated (p-)ULK1 (cat. no. SC-390904, Santa Cruz), p-PI3KC3 (cat. no. 13857S, Cell Signaling), p-mTOR (cat. no. 2971S, Cell Signaling), CD2AP (cat. no. SC-25272; Santa Cruz Biotechnology), nephrin (cat. no. ABb235903, Abcam), WT1 (cat. no. AB15249, Abcam), podocalyxin (cat. no. PA-1-46170, Invitrogen), Rubicon (cat. no. 7151-S, Santa Cruz Biotechnology), podocin (cat. no. SC-22296, Santa Cruz Biotechnology), and beclin-1 (cat. no. 3738S, Cell Signaling), followed by treatment with horseradish peroxidase-labeled appropriate secondary antibodies.

Techniques: Expressing, Plasmid Preparation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu

doi: 10.1152/ajpcell.00538.2018

Figure Lengend Snippet: Autophagy blockade causes dedifferentiation in podocytes. A: independent sets of differentiated podocytes were treated with vehicle (C) or 3-MA (3 mM) for 48 h under starved conditions (n = 6). Protein blots were probed for phospho (p)-PI3KC3, podocalyxin, and podocin and reprobed for GAPDH. Gels from two independent lysates are displayed. B: cellular lysates from A were probed for WT1 and reprobed for GAPDH (n = 6). Gels from two independent lysates are displayed. i: Densitometric distribution of p-PI3CK3/GAPDH ratios from A are shown in a dot plot. ***P < 0.001 vs. C. ii: Densitometric distribution of podocalyxin/GAPDH ratios from A are shown in a dot plot. ***P < 0.001 vs. C. iii: Densitometric distribution of podocin/GAPDH ratios from A are shown in a dot plot. ***P < 0.001 vs. C. iv: Densitometric distribution of WT1/GAPDH ratios from B are shown in a dot plot. ***P < 0.001 vs. C. C: independent sets of differentiated G0-podocytes were treated with vehicle or 3-MA for 48 h under starved conditions (n = 6). Protein blots of vector (V-), G0- and G0 + 3MA podocytes were probed with APOL1, nephrin, CD2AP, and podocalyxin and reprobed for actin. Gels from three independent lysates are displayed. i: Densitometric distribution of APOL1/GAPDH ratios from C are shown in a dot plot. ii: Densitometric distribution of nephrin/actin ratios from C are shown in a dot plot. *P < 0.05 vs. Vs and G0 + 3MA. iii: Densitometric distribution of CD2AP/actin ratios from C are shown in a dot plot. iii: Densitometric distribution of podocalyxin/actin ratios from C are shown in a dot plot. *P < 0.05 vs. Vs and G0 + 3MA. PI3KC3, class III phosphatidylinositol 3-kinase; 3-MA, 3-methyladenine; mTOR, mammalian target of rapamycin; WT1, Wilms tumor type 1; CD2AP, CD2-associated protein.

Article Snippet: Total protein lysed extracts (30 μg/lane) were loaded onto a 10% polyacrylamide (PAGE) precast gels (Bio-Rad, Hercules, CA) and after transfer onto PVDF membranes were processed for immunostaining with primary antibodies against APOL1 (cat. no. 66124-I-IG, Proteintech), ATG14L (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"Ab139727","term_id":"62160308","term_text":"AB139727"}} Ab139727 , Abcam), UVRAG (cat. no. 19571-I-AP, Proteintech), p62 (cat. no. SC-28359, Santa Cruz Biotechnology), LC3-II (cat. no. 2775S, Cell Signaling), PI3KC3 (cat. no. I3857S, Cell Signaling), phosphorylated (p-)ULK1 (cat. no. SC-390904, Santa Cruz), p-PI3KC3 (cat. no. 13857S, Cell Signaling), p-mTOR (cat. no. 2971S, Cell Signaling), CD2AP (cat. no. SC-25272; Santa Cruz Biotechnology), nephrin (cat. no. ABb235903, Abcam), WT1 (cat. no. AB15249, Abcam), podocalyxin (cat. no. PA-1-46170, Invitrogen), Rubicon (cat. no. 7151-S, Santa Cruz Biotechnology), podocin (cat. no. SC-22296, Santa Cruz Biotechnology), and beclin-1 (cat. no. 3738S, Cell Signaling), followed by treatment with horseradish peroxidase-labeled appropriate secondary antibodies.

Techniques: Plasmid Preparation, Wilms Tumor Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu

doi: 10.1152/ajpcell.00538.2018

Figure Lengend Snippet: APOL1 risk alleles are associated with dedifferentiation of molecular phenotype in podocytes. A: protein blots of independent sets of vector (V-), G0-, G1-, and G2-podocytes were probed with APOL1 and reprobed for CD2AP, nephrin, and GAPDH (n = 6). Gels from three independent lysates are displayed. i: Densitometric distribution of CD2AP/GAPDH ratios from A are shown in a dot plot. **P < 0.01, ***P < 0.001 vs. V. ii: Densitometric distribution of nephrin/GAPDH ratios from A are shown in a dot plot. *P < 0.05, **P < 0.01 vs. V. iii: Densitometric distribution of APOL1/GAPDH ratios from from A are shown in a dot plot. *P < 0.05, **P < 0.01 vs. V. APOL1, apolipoprotein L1; CD2AP, CD2-associated protein.

Article Snippet: Total protein lysed extracts (30 μg/lane) were loaded onto a 10% polyacrylamide (PAGE) precast gels (Bio-Rad, Hercules, CA) and after transfer onto PVDF membranes were processed for immunostaining with primary antibodies against APOL1 (cat. no. 66124-I-IG, Proteintech), ATG14L (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"Ab139727","term_id":"62160308","term_text":"AB139727"}} Ab139727 , Abcam), UVRAG (cat. no. 19571-I-AP, Proteintech), p62 (cat. no. SC-28359, Santa Cruz Biotechnology), LC3-II (cat. no. 2775S, Cell Signaling), PI3KC3 (cat. no. I3857S, Cell Signaling), phosphorylated (p-)ULK1 (cat. no. SC-390904, Santa Cruz), p-PI3KC3 (cat. no. 13857S, Cell Signaling), p-mTOR (cat. no. 2971S, Cell Signaling), CD2AP (cat. no. SC-25272; Santa Cruz Biotechnology), nephrin (cat. no. ABb235903, Abcam), WT1 (cat. no. AB15249, Abcam), podocalyxin (cat. no. PA-1-46170, Invitrogen), Rubicon (cat. no. 7151-S, Santa Cruz Biotechnology), podocin (cat. no. SC-22296, Santa Cruz Biotechnology), and beclin-1 (cat. no. 3738S, Cell Signaling), followed by treatment with horseradish peroxidase-labeled appropriate secondary antibodies.

Techniques: Plasmid Preparation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu

doi: 10.1152/ajpcell.00538.2018

Figure Lengend Snippet: Effect of Rubicon-silencing on dedifferentiation and autophagy blockade in APOL1 risk milieu. A: independent sets of vector (V-), G0-, G1-, and G2-podocytes were transfected with scrambled (SCR, 25 nM) or Rubicon-small interfering (si)RNA (n = 5). After 48 h, protein blots were probed for WT1 and podocalyxin; the same blots were reprobed for GAPDH. Representative gels are displayed. i: Densitometric distribution of podocalyxin/GAPDH ratios from A are shown in a dot plot. **P < 0.01 vs. V only; #P < 0.05 vs. G2 only; ###P < 0.001 vs. G1 only. ii: Densitometric distribution of WT1/GAPDH ratios from A are shown in a dot plot. **P < 0.01 vs. V only; ***P < 0.001 vs. G1 and G2 only; #P < 0.05 vs. respective V and G0 only; ###P < 0.001 vs. G1 and G2 only. B: independent sets of HEK cells were transfected with vector (VHEK), APOL1G0 (G0HEK), APOL1G1 (G1HEK), and APOL1G2 (G2HEK) (n = 6). After 48 h, protein blots from independent cellular lysates of VHEKs, G0HEKs, G1HEKs, and G2HEKs were probed for Rubicon and p62 and reprobed for GAPDH. Gels from three independent cellular lysates are displayed. i: Densitometric distribution data of Rubicon/GAPDH ratios from B are shown in a dot plot. **P < 0.01 vs. VHEKs and G0HEKs. ii: Densitometric distribution data of p62/GAPDH ratios from B are shown in a dot plot. ***P < 0.001 vs. VHEKs and G0HEKs. Rubicon, run domain beclin-1-interacting and cysteine-rich domain-containing protein; APOL1, apolipoprotein L1; WT1, Wilms tumor type 1.

Article Snippet: Total protein lysed extracts (30 μg/lane) were loaded onto a 10% polyacrylamide (PAGE) precast gels (Bio-Rad, Hercules, CA) and after transfer onto PVDF membranes were processed for immunostaining with primary antibodies against APOL1 (cat. no. 66124-I-IG, Proteintech), ATG14L (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"Ab139727","term_id":"62160308","term_text":"AB139727"}} Ab139727 , Abcam), UVRAG (cat. no. 19571-I-AP, Proteintech), p62 (cat. no. SC-28359, Santa Cruz Biotechnology), LC3-II (cat. no. 2775S, Cell Signaling), PI3KC3 (cat. no. I3857S, Cell Signaling), phosphorylated (p-)ULK1 (cat. no. SC-390904, Santa Cruz), p-PI3KC3 (cat. no. 13857S, Cell Signaling), p-mTOR (cat. no. 2971S, Cell Signaling), CD2AP (cat. no. SC-25272; Santa Cruz Biotechnology), nephrin (cat. no. ABb235903, Abcam), WT1 (cat. no. AB15249, Abcam), podocalyxin (cat. no. PA-1-46170, Invitrogen), Rubicon (cat. no. 7151-S, Santa Cruz Biotechnology), podocin (cat. no. SC-22296, Santa Cruz Biotechnology), and beclin-1 (cat. no. 3738S, Cell Signaling), followed by treatment with horseradish peroxidase-labeled appropriate secondary antibodies.

Techniques: Plasmid Preparation, Transfection, Wilms Tumor Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu

doi: 10.1152/ajpcell.00538.2018

Figure Lengend Snippet: Schematic diagram showing the role of APOL1 risk alleles in blockade autophagy in podocytes. APOL1G1 and APOL1G2 enhance expression of Rubicon, which forms a Rubicon complex inhibiting phosphorylation of UVRAG and attenuates generation of PI3P, resulting in inhibition of fusion of autophagosomes with lysosomes. Disruption of APOL1-miR193a axis in APOL1 risk milieu enhances expression of miR193a. miR193a attenuates transcription of PIK3C3′s regulatory unit (PIK3R3) and compromises maturation of autophagosomes. miR193a-induced attenuated transcription of mTOR is inhibiting the reformation of lysosomes required for fusion with autophagosomes. APOL1, apolipoprotein L1; PI3P, phosphatidyinositol-3-phosphate; PI3K, phosphatidylinositol 3-kinase; Pi3KC3, class III phosphatidylinositol 3-kinase; PIK3E3, PI3KC3′s regulatory unit; mTOR, mammalian target of rapamycin; Rubicon, run domain beclin-1-interacting and cysteine-rich domain-containing protein; UVRAG, UV radiation resistance-associated gene.

Article Snippet: Total protein lysed extracts (30 μg/lane) were loaded onto a 10% polyacrylamide (PAGE) precast gels (Bio-Rad, Hercules, CA) and after transfer onto PVDF membranes were processed for immunostaining with primary antibodies against APOL1 (cat. no. 66124-I-IG, Proteintech), ATG14L (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"Ab139727","term_id":"62160308","term_text":"AB139727"}} Ab139727 , Abcam), UVRAG (cat. no. 19571-I-AP, Proteintech), p62 (cat. no. SC-28359, Santa Cruz Biotechnology), LC3-II (cat. no. 2775S, Cell Signaling), PI3KC3 (cat. no. I3857S, Cell Signaling), phosphorylated (p-)ULK1 (cat. no. SC-390904, Santa Cruz), p-PI3KC3 (cat. no. 13857S, Cell Signaling), p-mTOR (cat. no. 2971S, Cell Signaling), CD2AP (cat. no. SC-25272; Santa Cruz Biotechnology), nephrin (cat. no. ABb235903, Abcam), WT1 (cat. no. AB15249, Abcam), podocalyxin (cat. no. PA-1-46170, Invitrogen), Rubicon (cat. no. 7151-S, Santa Cruz Biotechnology), podocin (cat. no. SC-22296, Santa Cruz Biotechnology), and beclin-1 (cat. no. 3738S, Cell Signaling), followed by treatment with horseradish peroxidase-labeled appropriate secondary antibodies.

Techniques: Expressing, Inhibition

Journal: eLife

Article Title: A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis

doi: 10.7554/eLife.66222

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-ARG1 (Rabbit polyclonal) , Proteintech , RRID: AB_2289842 , IHC (1:750).

Techniques: Transfection, Construct, shRNA, Recombinant, Plasmid Preparation, Liposomes, Enzyme-linked Immunosorbent Assay