Journal: Journal of Cell Science

Article Title: A PGAM5–KEAP1–Nrf2 complex is required for stress-induced mitochondrial retrograde trafficking

doi: 10.1242/jcs.203216

Figure Lengend Snippet: Nrf2 is not required for constitutive redox homeostasis. (A) Schematic of the mitochondrial PGAM5–KEAP1–Nrf2 complex, illustrating that a KEAP1 dimer bridges Nrf2 and PGAM5, and that the Neh2 domain of Nrf2 mediates binding to KEAP1. (B) 63× epifluorescence images confirming the expression and localization of the cytosolic roGFP and mitochondrial mito-roGFP reporter probes. Scale bar: 10 µm. (C) Representative excitation trace at 510 nm emission of RPE-1 cells stably expressing roGFP. Cells were treated with H2O (blue line) or 1 mM H2O2 (red line). (D) Same as in C but for mito-roGFP. (E) roGFP- or mito-roGFP-expressing RPE-1 cells (8000 cells/well) were transfected with siRNA, treated with DMSO or 10 µM MG132 for 2 h, or alternatively with 1 mM H2O2 or 1 mM dithiothreitol (DTT) for 30 min, and then analyzed at excitation wavelengths of 400 nm and 475 nm and emission wavelength of 510 nm. Values are normalized to siCON-transfected cells treated with vehicle (i.e. fold over control). Fluorescence excitation ratios >1.0 are oxidizing and those <1.0 are reducing. Data are mean±s.e.m. from five independent experiments. Statistical significance determined by two-way ANOVA with Tukey's post-hoc correction.

Article Snippet: Human retinal pigment epithelial cells transformed with telomerase (RPE-1) (CRL-4000, ATCC) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 1 g/l glucose supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), 1X nonessential amino acid cocktail (Life Technologies) and 10% fetal bovine serum (FBS, Life Technologies).

Techniques: Binding Assay, Expressing, Stable Transfection, Transfection, Fluorescence

Journal: Journal of Cell Science

Article Title: A PGAM5–KEAP1–Nrf2 complex is required for stress-induced mitochondrial retrograde trafficking

doi: 10.1242/jcs.203216

Figure Lengend Snippet: Nrf2 and PGAM5 are required for stress-induced mitochondrial retrograde trafficking. (A) RPE-1 cells transfected with siCON or siNrf2 were treated with DMSO or 10 µM MG132 for 2 h. Mitochondria are labeled with anti-Tom20 (red) and nuclei counterstained with DAPI (blue). Insets show higher magnification views of the boxed areas in panels a and c. (B) Quantification of mitochondrial clustering in siCON versus siNrf2 cells. Data are mean±s.d. from three independent experiments, in which >100 cells per condition were scored for each experiment. (C) Anti-Nrf2 and anti-β-tubulin loading control western blots to confirm the efficacy of Nrf2 knockdown and stabilization of Nrf2 by MG132. The asterisk denotes the nonspecific band, and the migration of molecular weight markers is indicated on the left. (D) RPE-1 cells transfected with control or siPGAM5 were treated with DMSO or 10 µM MG132 for 2 h. Mitochondria are labeled with anti-Tom20 (red) and nuclei with DAPI (blue). PGAM5 silencing demonstrated by loss of anti-PGAM5 immunoreactivity (green; panels f and h). (E) Data are mean±s.d. from four independent experiments, in which >100 cells per condition were scored for mitochondrial clustering per experiment. (F) Photomicrographs of siCON, siNrf2, siPGAM5 and siNrf2/siPGAM5 co-knockdown cells following exposure to DMSO or 10 µM MG132 for 2 h. Mitochondria and nuclei are labeled as in A. (G) Quantification of the mitochondrial clustering in F. Data are mean±s.d. from three independent experiments, in which >100 cells per condition were scored per experiment. (H) Western blots confirming knockdown of Nrf2 and PGAM5 in F. Scale bars: 10 µm. Statistical significance determined by two-way ANOVA with Sidak's or Tukey's post hoc correction.

Article Snippet: Human retinal pigment epithelial cells transformed with telomerase (RPE-1) (CRL-4000, ATCC) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 1 g/l glucose supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), 1X nonessential amino acid cocktail (Life Technologies) and 10% fetal bovine serum (FBS, Life Technologies).

Techniques: Transfection, Labeling, Western Blot, Migration, Molecular Weight

Journal: Journal of Cell Science

Article Title: A PGAM5–KEAP1–Nrf2 complex is required for stress-induced mitochondrial retrograde trafficking

doi: 10.1242/jcs.203216

Figure Lengend Snippet: Miro2 is required for mitochondrial retrograde trafficking. (A) Representative photomicrographs of RPE-1 cells treated with DMSO or the indicated proteasome inhibitors (10 µM MG132 or 1 µM epoxomicin) for 2 h. Mitochondria are labeled with anti-Tom20 (red) and nuclei with DAPI (blue). (B) The percentage of cells with clustered mitochondria as a function of treatment. Data are mean±s.d. from three independent experiments utilizing >100 cells per condition per experiment. (C) Confocal, 3D reconstruction of MitoTracker-labeled mitochondria (red) and microtubule stalk (green) exclusively observed in proteasome inhibitor-treated cells. (D) Representative photomicrographs of cells treated with DMSO or proteasome inhibitor (10 µM MG132 or 1 µM epoximicin) ± 4 µg/ml nocodazole. Mitochondria and nuclei are labeled as in A. (E) The % of cells with clustered mitochondria as a function of the treatments described in D. Data are mean±s.d. from three independent experiments, in which >100 cells per condition were scored for each experiment. (F) RPE-1 cells transfected with siCON or siMiro1 were treated with DMSO or 10 µM MG132 for 2 h. Mitochondria are labeled with anti-Tom20 (red) and nuclei with DAPI (blue). (G) Quantification of mitochondrial clustering in siCON versus siMiro1 cells. Data are mean±s.d. from three independent experiments, in which >100 cells per condition were scored for each experiment. (H) Representative western blot demonstrating that siMiro1 siRNA knocks down Miro1, but not Miro2. (I) RPE-1 cells transfected with siCON or siMiro2 were treated and processed as in F. (J) Quantification of mitochondrial clustering in siCON versus siMiro2 cells. Data are mean±s.d. from four independent experiments, in which >100 cells per condition were scored per experiment. (K) Representative western blot demonstrating Miro2 knockdown. Scale bars: 10 µm. Statistical significance determined by one-way (B) or two-way (E,G,J) ANOVA with Sidak's or Tukey's post hoc correction.

Article Snippet: Human retinal pigment epithelial cells transformed with telomerase (RPE-1) (CRL-4000, ATCC) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 1 g/l glucose supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), 1X nonessential amino acid cocktail (Life Technologies) and 10% fetal bovine serum (FBS, Life Technologies).

Techniques: Labeling, Transfection, Western Blot

Journal: Journal of Cell Science

Article Title: A PGAM5–KEAP1–Nrf2 complex is required for stress-induced mitochondrial retrograde trafficking

doi: 10.1242/jcs.203216

Figure Lengend Snippet: Unconstrained mitochondrial KEAP1 abrogates mitochondrial clustering. (A) Photomicrographs of siCON and siKEAP1 cells treated with DMSO or 10 µM MG132 for 2 h. Mitochondria are visualized with anti-Tom20 (red) and nuclei with DAPI (blue). (B) Data are mean±s.d. from five independent experiments, quantifying mitochondrial clustering in >100 cells per condition per experiment in A. (C) Representative western blot showing KEAP1 knockdown (lanes 3 and 4) and Nrf2 stabilization by siKEAP1 and MG132 (lanes 2 and 4). The asterisk indicates nonspecific band. (D) RPE-1 cells transfected with siCON or siNrf2/siKEAP1 combination were treated with DMSO or 10 µM MG132 for 2 h. Mitochondria are labeled with anti-Tom20 (red) and nuclei with DAPI (blue). (E) Quantification of mitochondrial clustering from D. Data are mean±s.d. from three independent experiments, in which >100 cells per condition were scored per experiment. (F) Anti-Nrf2 western blot demonstrating the efficacy of siNrf2 and siKEAP1 treatments. The anti-β-tubulin blot is shown as a loading control and migration of molecular weight markers is indicated on the left. (G) Photomicrographs of control and siPGAM5/siKEAP1 co-treated RPE-1 cells following exposure to DMSO or 10 µM MG132. Mitochondria and nuclei are labeled as in D. Endogenous PGAM5 (green) was detected with an anti-PGAM5 antibody. (H) Data are mean±s.d. from three independent experiments in G quantifying mitochondrial clustering and utilizing >100 cells per condition per experiment. (I) Anti-Nrf2 western blot demonstrating the impact of siPGAM5/siKEAP1 co-knockdown on Nrf2 levels. Scale bars: 10 µm. Statistical significance determined by two-way ANOVA with Tukey's post hoc correction.

Article Snippet: Human retinal pigment epithelial cells transformed with telomerase (RPE-1) (CRL-4000, ATCC) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 1 g/l glucose supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), 1X nonessential amino acid cocktail (Life Technologies) and 10% fetal bovine serum (FBS, Life Technologies).

Techniques: Western Blot, Transfection, Labeling, Migration, Molecular Weight

Journal: Journal of Cell Science

Article Title: A PGAM5–KEAP1–Nrf2 complex is required for stress-induced mitochondrial retrograde trafficking

doi: 10.1242/jcs.203216

Figure Lengend Snippet: The nontranscriptional, Neh2 domain of Nrf2 restores mitochondrial clustering. (A) MEFs from wild-type and strain-matched Nrf2−/− mice treated with DMSO or 5 μM MG132 for 4 h or 6 h. Mitochondria are labeled with MitoTracker (red), Nrf2 with an anti-Nrf2 antibody (green) and nuclei with DAPI (blue). Arrowheads indicate Nrf2−/− MEFs showing anti-Nrf2 immunoreactivity; asterisks indicate examples of cells scored as having clustered mitochondria. (B) Diagrams of wild-type protein (Nrf2+/+) and the Neh2-β-galactosidase fusion produced in Nrf2−/− mice. (C) Data are mean±s.d. from four independent experiments showing mitochondrial clustering in MEFs, with >50 cells analyzed per condition per experiment. Data are stratified for all cells (white bars), cells with Nrf2-immunoreactivity (gray bars) and cells without Nrf2-immunoreactivity (diagonally striped bars). (D) Anti-Nrf2, anti-GFP and anti-β-tubulin western blots of RPE-1 cells stably expressing siRNA-resistant Neh2-YFP. Cells were transfected with siCON or siNrf2 siRNA and treated with DMSO or 10 µM MG132 for 2 h. Endogenous (endog.) Nrf2 and Neh2-YFP are marked. The asterisk indicates nonspecific band detected by anti-Nrf2 antibody, and the migration of molecular weight markers is indicated on the left. (E) Neh2-YFP-expressing RPE-1 cells treated as indicated; mitochondria are labeled with anti-Tom20 (red) and nuclei with DAPI (blue). Examples of Neh2-YFP-expressing cells scored as having clustered mitochondria are indicated with asterisks. (F) Mitochondrial clustering quantified from E and stratified based on YFP-positive cells (white bars) and YFP-negative cells (gray bars). Data are mean±s.d. from three independent experiments, in which >100 cells per condition were analyzed for each experiment. (G) Photomicrographs of RPE-1 cells treated with vehicle, 10 µM MG132, 1 µM Actinomycin D or MG132+Actinomycin D. Mitochondria are labeled with anti-Tom20 (red), endogenous Nrf2 with anti-Nrf2 (green) and nuclei with DAPI (blue). (H) Quantification of % of cells with clustered mitochondria in G. Data are mean±s.d. from three independent experiments utilizing >100 cells per condition per experiment. (I) Anti-Nrf2, anti-Hsp70 and anti-β-tubulin western blots confirming the efficacy of Actinomycin D (ActD) in blocking Hsp70 induction by MG132. Scale bars: 10 µm. Statistical significance determined by three-way ANOVA with Tukey's or Sidak's post hoc correction (C,H) or two-way ANOVA with Tukey's post hoc correction (F).

Article Snippet: Human retinal pigment epithelial cells transformed with telomerase (RPE-1) (CRL-4000, ATCC) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 1 g/l glucose supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), 1X nonessential amino acid cocktail (Life Technologies) and 10% fetal bovine serum (FBS, Life Technologies).

Techniques: Labeling, Produced, Western Blot, Stable Transfection, Expressing, Transfection, Migration, Molecular Weight, Blocking Assay

Journal: Journal of Cell Science

Article Title: A PGAM5–KEAP1–Nrf2 complex is required for stress-induced mitochondrial retrograde trafficking

doi: 10.1242/jcs.203216

Figure Lengend Snippet: Nrf2 and p62 cooperatively suppress aberrant KEAP1 activity. (A) RPE-1 cells transfected with siCON, siNrf2, sip62 or siNrf2/sip62 combination were treated with DMSO or 10 µM MG132 for 2 h. Mitochondria are labeled with MitoTracker (red), and nuclei with DAPI (blue). (B) Quantification of clustering in A. Data are mean±s.d. from three independent experiments utilizing >100 cells per condition per experiment. (C) Anti-Nrf2, anti-p62 and anti-β-tubulin western blots to demonstrate the efficacy of knockdowns in A and B. (D) RPE-1 cells transfected with siCON or siNrf2/sip62/siKEAP1 combination were treated and processed as in A. (E) Quantification of clustering from D, performed as described in B. (F) Anti-Nrf2, anti-p62, anti-KEAP1 and anti-β-tubulin western blots to demonstrate the efficacy of knockdowns in D and E. (G) RPE-1 cells transfected with siCON or siNrf2/sip62/siPGAM5 combination were treated with DMSO or 10 µM MG132 for 2 h. Mitochondria labeled with anti-Tom20 (red), endogenous PGAM5 with anti-PGAM5 (green) and nuclei with DAPI (blue). (H) Quantification of clustering in G. Data are mean±s.d. from three independent experiments utilizing >100 cells per condition per experiment. (I) Representative western blot from G demonstrating knockdown of Nrf2 and p62. Scale bars: 10 µm. Statistical significance was determined by two-way ANOVA with Tukey's post hoc correction.

Article Snippet: Human retinal pigment epithelial cells transformed with telomerase (RPE-1) (CRL-4000, ATCC) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 1 g/l glucose supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), 1X nonessential amino acid cocktail (Life Technologies) and 10% fetal bovine serum (FBS, Life Technologies).

Techniques: Activity Assay, Transfection, Labeling, Western Blot

Journal: Journal of Cell Science

Article Title: A PGAM5–KEAP1–Nrf2 complex is required for stress-induced mitochondrial retrograde trafficking

doi: 10.1242/jcs.203216

Figure Lengend Snippet: Unconstrained KEAP1 promotes loss of Miro2. (A) RPE-1 cells were transfected with the indicated siRNAs and treated with DMSO or 10 µM MG132 for 2 h. Western blots show knockdown of targeted proteins and levels of Miro1 and Miro2. (B) Anti-Miro western blots of lysates from Nrf2 knockout (Nrf2−/−) and age-matched, wild-type (Nrf2+/+) brains. Numbers indicate the fraction of Miro2 present compared to wild-type control for each age tested (wild type set at 1.0). Left and center panels from 11- to 12-month-old mice and right panel from 22- to 23-month-old mice. (C) Cells were transfected with siRNA and treated as in A. (D) RPE-1 cells were transfected with the indicated siRNAs for 12 h followed by exposure to DMSO or 1 µM MG132 for 24 h prior to western blotting with the indicated antibodies. (E) Densitometric quantification from D. Data are mean±s.e.m. from four independent experiments. (F) Graph of mean fluorescence intensity of MitoTracker Green (MTG) uptake in cells transfected with the indicated siRNAs and treated with ethanol or 5 µM FCCP prior to FACS analysis. MTG uptake is a proxy for mitochondrial content in cells and the acute FCCP treatment was used to dissipate the membrane potential to demonstrate that MTG uptake is independent of mitochondrial membrane potential. Data are mean±s.e.m. from five independent experiments. (G) Mean fluorescence intensity calculated by dividing the TMRE signal by the MTG signal with the siCON control set at a value of 1.0. Cells were treated as in F. Data are mean±s.e.m. from five independent FACS experiments utilizing >20,000 cell counts per condition per experiment. (H) Graph of FACS data pooled from three independent experiments demonstrating that Nrf2 depletion does not alter basal mitophagic flux. RPE-1 cells stably expressing mt-mKeima and YFP-parkin were treated with the indicated siRNAs and exposed to DMSO (black bars) or 10 µM MG132 (red bars) for 4 h. Excitation profiles of mt-mKeima at 440 nm (neutral pH) and at 586 nm (acidic pH) in siCON- and siNrf2-depleted RPE-1 cells were quantified by FACS analysis and used to define mitochondria as being mitochondrial (neutral) or lysosomal (acidic). (I) Schematic showing the roles of Nrf2 and p62 in suppressing aberrant degradation of Miro2 by the KEAP1-Cul3 E3 Ub ligase. Parkin mediates the degradation of Miro1 to halt mitochondrial motility following irreparable loss of mitochondrial membrane potential (Birsa et al., 2014; Kazlauskaite et al., 2014; Klosowiak et al., 2016; Wang et al., 2011b). Statistical significance was determined by two-way ANOVA with Tukey's post hoc correction (E,H) and by one-way ANOVA (F,G).

Article Snippet: Human retinal pigment epithelial cells transformed with telomerase (RPE-1) (CRL-4000, ATCC) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 1 g/l glucose supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), 1X nonessential amino acid cocktail (Life Technologies) and 10% fetal bovine serum (FBS, Life Technologies).

Techniques: Transfection, Western Blot, Knock-Out, Fluorescence, Stable Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The endoplasmic reticulum–localized enzyme zDHHC6 mediates S-acylation of short transmembrane constructs from multiple type I and II membrane proteins

doi: 10.1016/j.jbc.2023.105201

Figure Lengend Snippet: A minimal transmembrane domain (TMD) of type II transmembrane proteins is sufficient for the S-acylation of their proximal cysteines by zDHHC6. A , schematic of the constructs used in the analysis. A small Myc cytoplasmic tag (EQKLISEEDL) was fused at the N-terminal part of the TMDs (and proximal region) of several type II transmembrane proteins; the theoretical region spanning the bilayer is underlined . A GFP tag was fused at the C-terminal part of the constructs following the transmembrane segments. Type II proteins chosen here are TMPRSS2, BST2, and transferrin receptor (TfR). B – E , HEK293T cells were cotransfected with a plasmid encoding each of the constructs described in A together with a plasmid encoding HA-DHHC6 (HA-6) or the empty control plasmid (pEF). Six hours post-transfection, cells were metabolically labeled overnight with either palmitate (−Az-C16:0) or palmitate azide (+Az-C16:0) followed by click chemistry using alkyne-PEG. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed first with an anti-GFP antibody (revealed with secondary antibodies coupled to the infrared dye IR800) followed by an anti-HA antibody (and IR680-coupled secondary antibodies). Positions of molecular weight markers (in kilodalton) are shown on the right-hand side of the membranes, whereas numbers on the left (0, 1, and 2) relate to the number of modified cysteines within the transmembrane fusion proteins. The graph below each panel shows mean ± SEM of normalized substrate S-acylation by HA-tagged DHHC6; filled circles represent individual samples (n = 4–5 cell samples from two independent experiments). Statistical analysis (Student’s t test) was performed to reveal significant S-acylation of the substrates following the expression of exogenous HA-DHHC6 versus its acylation by endogenous enzymes (pEF samples) (∗∗∗ p < 0.001). BST2, bone marrow stromal antigen 2; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; TMPRSS2, transmembrane serine protease 2; TfR, transferrin receptor.

Article Snippet: Mouse GFP antibody (Clontech; clone JL8, used at 1:4000 dilution) was obtained from Takara.

Techniques: Construct, Plasmid Preparation, Transfection, Metabolic Labelling, Labeling, SDS Page, Molecular Weight, Modification, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The endoplasmic reticulum–localized enzyme zDHHC6 mediates S-acylation of short transmembrane constructs from multiple type I and II membrane proteins

doi: 10.1016/j.jbc.2023.105201

Figure Lengend Snippet: Proximal cysteines of the transmembrane domains of type I membrane proteins can also be S-acylated by zDHHC6. A , schematic of the constructs used in the analysis. The signal peptide (SigPep) of azurocidin was inserted upstream of the coding sequence of GFP to allow for its translocation into the lumen of the endoplasmic reticulum. The transmembrane domains and proximal cysteines of several type I membrane proteins were fused to GFP at their N terminus and to the Myc tag (EQKLISEEDL) at their C terminus. The theoretical regions spanning the bilayer are underlined . Type I proteins chosen here are influenza (H7N1) hemagglutinin, cluster of differentiation 4 (CD4), the glycoprotein of the vesicular stomatitis virus (Indiana strain) (VSV-G), Ebola glycoprotein (Ebola GP), and human calnexin (CANX). B – F , HEK293T cells were cotransfected with a plasmid encoding each of the constructs described in A together with a plasmid encoding HA-DHHC6 (HA-6) or the empty control plasmid (pEF). Six hours post-transfection, cells were metabolically labeled overnight with either palmitate (−Az-C16:0) or palmitate azide (+Az-C16:0) followed by click chemistry using alkyne-PEG. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed first with an anti-GFP antibody (revealed with secondary antibodies coupled to the infrared dye IR800) followed by an anti-HA antibody (and IR680-coupled secondary antibodies). Positions of molecular weight markers (in kilodalton) are shown on the right-hand side of the membranes, whereas numbers on the left (0, 1, and 2) relate to the number of modified cysteines within the transmembrane fusion proteins. The graph on the right-hand side of each panel shows mean ± SEM of normalized substrate S-acylation by HA-tagged zDHHC6; filled circles represent individual samples (n = 6–10 different cell samples from two independent experiments). Statistical analysis (Student’s t test) was performed to reveal significant S-acylation of the substrates following the expression of exogenous HA-DHHC6 versus its acylation by endogenous enzymes (pEF samples) (∗∗∗ p < 0.001; ∗∗ p < 0.01). HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line.

Article Snippet: Mouse GFP antibody (Clontech; clone JL8, used at 1:4000 dilution) was obtained from Takara.

Techniques: Membrane, Construct, Sequencing, Translocation Assay, Virus, Plasmid Preparation, Transfection, Metabolic Labelling, Labeling, SDS Page, Molecular Weight, Modification, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The endoplasmic reticulum–localized enzyme zDHHC6 mediates S-acylation of short transmembrane constructs from multiple type I and II membrane proteins

doi: 10.1016/j.jbc.2023.105201

Figure Lengend Snippet: S-acylation of proximal cysteines by selected endoplasmic reticulum–resident zDHHC enzymes. HEK293T cells were cotransfected with a plasmid encoding GFP-tagged constructs of the transmembrane domains of type II (BST2 [ A ] and TMPRSS2 [ B ]) or a type I (HA [ C ]) membrane protein together with the empty control plasmid (pEF) or plasmids encoding selected HA-tagged DHHC enzymes (HA-4, -6, -10, -11, -13, -14, or -16). Six hours post-transfection, cells were metabolically labeled overnight with either palmitate (−Az-C16:0) or palmitate azide (+Az-C16:0) followed by click chemistry using alkyne-PEG. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed first with an anti-GFP antibody (revealed with secondary antibodies coupled to the infrared dye IR800) followed by an anti-HA antibody (and IR680-coupled secondary antibodies). Positions of molecular weight markers (in kilodalton) are shown on the right-hand side of the membranes, whereas numbers on the left (0, 1, and 2) relate to the number of modified cysteines within the transmembrane fusion proteins. The graph on the right-hand side of each panel shows mean ± SEM of normalized substrate S-acylation by HA-tagged zDHHC enzymes; filled circles represent individual samples (n = 4 different cell samples from two independent experiments). Statistical analysis (Student’s t test) was performed to reveal significant S-acylation of the substrates following the expression of exogenous HA-DHHC enzymes versus its acylation by endogenous enzymes (pEF samples) (∗∗∗ p < 0.001; ∗∗ p < 0.01; and ∗ p < 0.05). BST2, bone marrow stromal antigen 2; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; TMPRSS2, transmembrane serine protease 2.

Article Snippet: Mouse GFP antibody (Clontech; clone JL8, used at 1:4000 dilution) was obtained from Takara.

Techniques: Plasmid Preparation, Construct, Membrane, Transfection, Metabolic Labelling, Labeling, SDS Page, Molecular Weight, Modification, Expressing

Journal: iScience

Article Title: NFAT signaling in human mesenchymal stromal cells affects extracellular matrix remodeling and antifungal immune responses

doi: 10.1016/j.isci.2021.102683

Figure Lengend Snippet: Phagocytosis of zymosan by Ad-MSCs leads to cytokines expression (A) IL-8 , IL-6 , IL12p40 , IL-1B , IDO and IL-2 mRNA expression in Ad-MSCs stimulated with 10 μg/mL zymosan (ZYM) at different time points. The data represent the relative expression compared to the housekeeping gene (GAPDH). The data are representative of two independent experiments. (B) Confocal images showing pHrodo-labeled zymosan internalization by hTERT-MSCs. Fluorescence emission indicates phagosome acidification. (C) Flow cytometry analysis showing hTERT-MSCs ability to phagocytose pHrodo-zymosan compared with untreated cells (middle and left panels, respectively). Right panel shows decreased phagocytic ability for cells pretreated with cytochalasin D. (D) Confocal images showing hTERT-MSC-mediated endocytosis of dextran chains in the tested range 3–40 kDa. (E) Flow cytometry analysis showing the ability of hTERT-MSCs to internalize dextran chains at the indicated molecular weight (kDa). Gray shading represents the negative control, where endocytic processes were inhibited at 4°C. ∗∗p < 0.01, ∗∗∗p < 0.005; two-tailed unpaired t test.

Article Snippet: ASC52telo, hTERT immortalized adipose derived Mesenchymal stem cells , ATCC , SCRC-4000.

Techniques: Expressing, Labeling, Fluorescence, Flow Cytometry, Molecular Weight, Negative Control, Two Tailed Test

Journal: iScience

Article Title: NFAT signaling in human mesenchymal stromal cells affects extracellular matrix remodeling and antifungal immune responses

doi: 10.1016/j.isci.2021.102683

Figure Lengend Snippet: Ad-MSCs engage a PRR-driven response to zymosan leading to CN-NFAT activation (A) Immunofluorescent dectin-1 labeling in hTERT-MSCs. (B) Western blot analysis of zymosan-induced Syk phosphorylation (p-Syk) and total Syk expression (Syk) in hTERT-MSCs. β-Actin was used as a loading control. (C) Western blot quantification showing the band intensity ratio for pSYK/Syk. (D) Live calcium imaging in Ad-MSCs stimulated with zymosan (ZYM), ionomycin (Iono), ATP, or no treatment (NT). A calcium tracker was used. The calcium dynamics are presented as fluorescence intensity plotted against time, where the values of fluorescence are normalized to the fluorescence signal at t = 0 s. (E) Calcium flux quantification. The graph represents the area under the curve (AUC) for the untreated (NT), zymosan (ZYM), ionomycin (Iono) and ATP groups. The data are representative of three independent experiments. The data were analyzed against NT by Kruskal-Wallis test followed by Dunn's multiple comparison test. (F) Schematic of dectin1-NFAT signaling cascade with inhibitors. (G) Representative confocal images of NFAT1 subcellular localization in Ad-MSCs untreated, WGP-stimulated, or stimulated with zymosan in presence and absence of FK506, dectin-1 neutralizing antibody (Dectin Neu ), Piceatannol (PIC) or BAPTA AM. (H) Quantification of NFAT1 nuclear/cytoplasmic ratio (expressed as percentage). A total of 30 images from three independent experiments were analyzed and compared by one-way ANOVA followed by Sidak's multiple comparison test. ∗∗∗∗p < 0.0001.

Article Snippet: ASC52telo, hTERT immortalized adipose derived Mesenchymal stem cells , ATCC , SCRC-4000.

Techniques: Activation Assay, Labeling, Western Blot, Phospho-proteomics, Expressing, Control, Imaging, Fluorescence, Comparison

Journal: iScience

Article Title: NFAT signaling in human mesenchymal stromal cells affects extracellular matrix remodeling and antifungal immune responses

doi: 10.1016/j.isci.2021.102683

Figure Lengend Snippet: Zymosan elicits a PRR-driven response in Ad-MSCs that culminates in NF-κB activation (A–D) RNA-sequencing analysis of zymosan-treated (ZYM) and untreated (NT) Ad-MSCs (fold change ≥ |1.5| and a p value ≤ 0.05). (A) A heatmap representing differentially expressed genes (DEGs) between NT and ZYM. Colors indicate the row-scaled log2FC. (B) A volcano plot representing all the genes as a function of log2 fold changes (x axis, log2FC) and p value (y axis, log P). Genes highlighted in red are DEGs with log2FC > 0.58 and a p value < 0.05. Genes highlighted in blue are DEGs with log2FC < −0.58 and a p value < 0.05. (C) Gene ontology analysis on biological processes (BP) of significantly upregulated genes. (D) Gene set enrichment analysis (GSEA) performed on an unfiltered gene list.

Article Snippet: ASC52telo, hTERT immortalized adipose derived Mesenchymal stem cells , ATCC , SCRC-4000.

Techniques: Activation Assay, RNA Sequencing

Journal: iScience

Article Title: NFAT signaling in human mesenchymal stromal cells affects extracellular matrix remodeling and antifungal immune responses

doi: 10.1016/j.isci.2021.102683

Figure Lengend Snippet: Zymosan induces the activation of NF-κB, which controls IL-6 and IL-8 protein expression (A) Western blot analyses of TLR2, MALT1, CARD9, TRAF6, Myd88, Bcl10, and Ras in hTERT-MSCs, HEK-293, THP-1, and THP-1-derived macrophages. β-Actin was used as loading control. (B) NF-κB p65 subunit expression in nuclear extracts of hTERT-MSCs untreated (NT) or stimulated for 10 min with 10 μg/mL zymosan (ZYM). The data are representative of four independent experiments and were analyzed by two-tailed unpaired t-tests. (C) The relative luminescence units (RLU) of MSC NF-κB reporter cells stimulated for 18 h with zymosan in the presence or absence of TPCA-1 inhibitor. The data are representative of three independent experiments and were analyzed by two-tailed unpaired t test. ∗p < 0.05, ∗∗p < 0.01. (D) IL8 protein expression in supernatants of Ad-MSCs untreated or stimulated with zymosan, zymosan + FK506 or zymosan + TPCA-1 for 18h. The data represent the means of three independent experiments and were analyzed by one-way ANOVA followed by Sidak's multiple comparison test. (E) IL6 protein expression in supernatants of Ad-MSCs untreated or stimulated with zymosan, zymosan + FK506 or zymosan + TPCA-1 for 18h. The data represent the means of three independent experiments and were analyzed by one-way ANOVA followed by Sidak's multiple comparison test. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: ASC52telo, hTERT immortalized adipose derived Mesenchymal stem cells , ATCC , SCRC-4000.

Techniques: Activation Assay, Expressing, Western Blot, Derivative Assay, Control, Two Tailed Test, Comparison

Journal: iScience

Article Title: NFAT signaling in human mesenchymal stromal cells affects extracellular matrix remodeling and antifungal immune responses

doi: 10.1016/j.isci.2021.102683

Figure Lengend Snippet: VIVIT-MSCs line characterization (A) Schematic of the VIVIT-TET expression vectors and the strategy used to obtain the stable line. (B) Mechanism of action of VIVIT peptide. (C) FACS analysis of VIVIT expression in MSC after 0.125–2 μg/mL doxycyline (DOX) treatment. (D) VIVIT-mediated inhibition of NFAT nuclear translocation was visualized by confocal microscopy. The top panel shows untreated cells or cells stimulated with zymosan in the absence of DOX (MSC-VIVIT-). The bottom panel shows the same conditions after DOX stimulation (MSC-VIVIT+). (E) Quantification of the NFAT1 nuclear signal. A total of 30 images from three independent experiments were used for the analysis. The data were analyzed by one-way ANOVA followed by Sidak's multiple comparison test. ∗∗∗∗p < 0.0001. (F) HAS1 and CEMIP mRNA expression in MSC-VIVIT + or MSC-VIVIT-stimulated with 10 μg/mL zymosan. The data represent the relative expression compared with the housekeeping gene ( ACTB ). The data are representative of three independent experiments. The data were analyzed by Mann-Whitney test. ∗p < 0.5.

Article Snippet: ASC52telo, hTERT immortalized adipose derived Mesenchymal stem cells , ATCC , SCRC-4000.

Techniques: Expressing, Inhibition, Translocation Assay, Confocal Microscopy, Comparison, MANN-WHITNEY

Journal: iScience

Article Title: NFAT signaling in human mesenchymal stromal cells affects extracellular matrix remodeling and antifungal immune responses

doi: 10.1016/j.isci.2021.102683

Figure Lengend Snippet: Selective inhibition of NFAT in MSCs impacts PBMCs antifungal response (A) Schematic of the peripheral blood mononuclear cell (PBMC) coculture experimental design. (B) A heatmap representing the cytokine expression triggered by zymosan in PBMCs, cocultured PBMCs and VIVIT-MSCs without doxycycline (PBMC/MSC-VIVIT-) and cocultured PBMCs and MSC with doxycycline (PBMC/MSC VIVIT+). (C) Graphs comparing cytokine expression detected in conditioned media (CM) of untreated PBMCs (NT) and PBMCs treated with zymosan (ZYM). The data were analyzed by repeated-measures (RM) one-way ANOVA with the Greenhouse-Geisser correction followed by the Sidak's multiple comparison test. ∗adj. p < 0.05, ∗∗ adj. p < 0.01. (D) Cytokine expression in “PBMC/MSC-VIVIT-" and “PBMC/MSC-VIVIT+” CM. The data were analyzed by RM one-way ANOVA with the Greenhouse-Geisser correction followed by the Sidak's multiple comparison test. ∗adj. p < 0.05. (E) Schematic of the decellularization experimental design. (F) IL8 protein levels in supernatants of zymosan-treated (10μg/mL) PBMC/dVIVIT+ and PBMC/dVIVIT-. PBMCs isolated from 4 different donors were used for the analysis. The data were analyzed by Paired t test. ∗p < 0.05.

Article Snippet: ASC52telo, hTERT immortalized adipose derived Mesenchymal stem cells , ATCC , SCRC-4000.

Techniques: Inhibition, Expressing, Comparison, Isolation

Journal: iScience

Article Title: NFAT signaling in human mesenchymal stromal cells affects extracellular matrix remodeling and antifungal immune responses

doi: 10.1016/j.isci.2021.102683

Figure Lengend Snippet:

Article Snippet: ASC52telo, hTERT immortalized adipose derived Mesenchymal stem cells , ATCC , SCRC-4000.

Techniques: Produced, Control, Recombinant, Lysis, Western Blot, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Derivative Assay, Software

Journal: Journal of the American Society of Nephrology : JASN

Article Title: ApoL1 Overexpression Drives Variant-Independent Cytotoxicity

doi: 10.1681/ASN.2016121322

Figure Lengend Snippet: Comparable expression of APOL1-G0, -G1, and -G2 can be achieved in stable cell lines. APOL1 expression was assayed by immunoblotting 293 cell lines that conditionally express APOL1-G0, -G1, and -G2 (G0, G1, and G2) with tetracycline induction. Three clones of each genotype were studied (labeled A, B, C for each genotype). (A) Band density was quantitated using ImageJ software, and the expression of APOL1 normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown for each clone tested before tetracycline treatment (0 hour) and 4 and 8 hours after the addition of tetracycline (1 µg/ml). (B) APOL1 expression, determined by immunoblotting, after 0, 4, and 8 hours of tetracycline induction for three G0, G1, and G2 clones each. Blots were stripped and reprobed for GAPDH expression as loading controls. (C) One clone for each genotype (G0 C, G1 C, G2 B) judged to have comparable levels of APOL1 expression by immunoblotting was examined with confocal immunofluorescent microscopy for APOL1 expression before and 5 hours after the addition of tetracycline (1 μg/ml). Scale bars=25 µm.

Article Snippet: Antibodies and Reagents Antibodies for immunoblotting include a rabbit polyclonal anti-APOL1 (HPA01885; Sigma) at 1:4000 or rabbit monoclonal anti-APOL1 (ab108315, lot#GR8593–2; AbCam) at 1:4000, rabbit monoclonal anti-LC3II (3868; Cell Signaling) at 1:1000, rabbit polyclonal anti-p62 (P0067, lot#108K4764; Sigma) at 1:2000, rabbit monoclonal anti-GAPDH (2118; Cell Signaling) at 1:2000, mouse monoclonal anti–Na/K-ATPase (Cat#sc-21712, Lot#G1615; Santa Cruz Biotech) at 1:200, and rabbit monoclonal anti-tubulin (2128; Cell Signaling) at 1:1000.

Techniques: Expressing, Stable Transfection, Western Blot, Clone Assay, Labeling, Software, Microscopy

Journal: Journal of the American Society of Nephrology : JASN

Article Title: ApoL1 Overexpression Drives Variant-Independent Cytotoxicity

doi: 10.1681/ASN.2016121322

Figure Lengend Snippet: Stable expression of APOL1 does not induce autophagy. The protein abundance of LC3II, p62, and GAPDH was assessed by immunoblotting in three clones of each genotype (G0, G1, and G2) after tetracycline induction (1 μg/ml). The bar graphs show the normalized expression of (A–D) LC3II or (E and F) p62, two biochemical markers of autophagy, 8 or 16 hours after tetracycline addition. (A and B) The results for three clones of each genotype, demonstrating genotype-independent, clonal variability in LC3II abundance. (C–F) Bar graphs of normalized LC3II and p62 abundance stratified by APOL1 genotype (mean±SD). Conditions include no treatment (Cont), treatment with bafilomycin A (Baf, 100 nM) alone to induce an autophagic block, treatment with tetracycline alone (Tet, 1 μg/ml), and treatment with tetracycline and bafilomycin A (Tet+Baf). Cells were treated with tetracycline for 8 or 16 hours, as indicated. Bafilomycin was added for the last 3 hours. Within-genotype treatment differences were not statistically significant (single-factor ANOVA).

Article Snippet: Antibodies and Reagents Antibodies for immunoblotting include a rabbit polyclonal anti-APOL1 (HPA01885; Sigma) at 1:4000 or rabbit monoclonal anti-APOL1 (ab108315, lot#GR8593–2; AbCam) at 1:4000, rabbit monoclonal anti-LC3II (3868; Cell Signaling) at 1:1000, rabbit polyclonal anti-p62 (P0067, lot#108K4764; Sigma) at 1:2000, rabbit monoclonal anti-GAPDH (2118; Cell Signaling) at 1:2000, mouse monoclonal anti–Na/K-ATPase (Cat#sc-21712, Lot#G1615; Santa Cruz Biotech) at 1:200, and rabbit monoclonal anti-tubulin (2128; Cell Signaling) at 1:1000.

Techniques: Expressing, Western Blot, Clone Assay, Blocking Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: ApoL1 Overexpression Drives Variant-Independent Cytotoxicity

doi: 10.1681/ASN.2016121322

Figure Lengend Snippet: APOL1-induced cytotoxicity is variant-independent. (A) Fluorescent assay demonstrating no difference in the average cytotoxicity-to-viability ratio, normalized to control untreated cells, in 293 cells stimulated with tetracycline to express APOL1-G0, -G1, or G2 (G0, G1, G2). Each bar represents mean±SD of three independent experiments with one to two independent clones, with two to four technical replicates (n=12–14). (B) MTT assays after 8 hours of tetracycline (1 μg/ml) induction demonstrated no loss of viability in G0, G1, or G2 cells compared with uninduced cells (mean±SD, n=2–3 independent experiments, each with three technical replicates, for each APOL1 genotype). (C) In contrast, at 16 hours of tetracycline (1 μg/ml) induction, cytotoxicity is marked in G0, G1, and G2 cells compared with uninduced control cells (mean±SD, n=2 independent experiments, each with two to three technical replicates, for each APOL1 genotype). MTT assays in control cells of each genotype were normalized to 100% viability in each independent experiment.

Article Snippet: Antibodies and Reagents Antibodies for immunoblotting include a rabbit polyclonal anti-APOL1 (HPA01885; Sigma) at 1:4000 or rabbit monoclonal anti-APOL1 (ab108315, lot#GR8593–2; AbCam) at 1:4000, rabbit monoclonal anti-LC3II (3868; Cell Signaling) at 1:1000, rabbit polyclonal anti-p62 (P0067, lot#108K4764; Sigma) at 1:2000, rabbit monoclonal anti-GAPDH (2118; Cell Signaling) at 1:2000, mouse monoclonal anti–Na/K-ATPase (Cat#sc-21712, Lot#G1615; Santa Cruz Biotech) at 1:200, and rabbit monoclonal anti-tubulin (2128; Cell Signaling) at 1:1000.

Techniques: Variant Assay, Fluorescence, Clone Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: ApoL1 Overexpression Drives Variant-Independent Cytotoxicity

doi: 10.1681/ASN.2016121322

Figure Lengend Snippet: Clonogenic survival assays demonstrate variant-independent cell death after APOL1 expression. Bar graph shows the fraction of surviving cells in the test condition normalized to untreated control cells (mean+SD, n=3). The vertical axis indicates the APOL1 genotype and treatments. The normalized surviving fraction of 1.0 is highlighted in red. Respective APOL1 genotypes are abbreviated G0, G1, or G2. C-Del338+Tet, 16 hours after plating the cells tetracycline 1 µg/ml was added to media to induce expression in a stable 293 cell line of an APOL1 transgene with a deletion of carboxy terminal amino acids, 339–398; Dig, digoxin (1 nM, 6 hours); Gly, glycine (5 mM glycine added to culture media throughout assay); 2HrTet, tetracycline (1 μg/ml, 2 hours); LowTet, 16 hours after plating the cells tetracycline was added at 5 ng/ml for APOL1-G0 and APOL1-G1 and 10 ng/ml for APOL1-G2; NoTx, no additions; Tet, tetracycline (1 μg/ml, 16 hours); v., versus; Wort, wortmanin (50 nM, 16 hours).

Article Snippet: Antibodies and Reagents Antibodies for immunoblotting include a rabbit polyclonal anti-APOL1 (HPA01885; Sigma) at 1:4000 or rabbit monoclonal anti-APOL1 (ab108315, lot#GR8593–2; AbCam) at 1:4000, rabbit monoclonal anti-LC3II (3868; Cell Signaling) at 1:1000, rabbit polyclonal anti-p62 (P0067, lot#108K4764; Sigma) at 1:2000, rabbit monoclonal anti-GAPDH (2118; Cell Signaling) at 1:2000, mouse monoclonal anti–Na/K-ATPase (Cat#sc-21712, Lot#G1615; Santa Cruz Biotech) at 1:200, and rabbit monoclonal anti-tubulin (2128; Cell Signaling) at 1:1000.

Techniques: Variant Assay, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: ApoL1 Overexpression Drives Variant-Independent Cytotoxicity

doi: 10.1681/ASN.2016121322

Figure Lengend Snippet: APOL1-G0, -G1, -G2 cause a time-dependent loss of cellular K+ and a gain of cellular Na+. Cell-associated (A) K+ and (C) Na+ were measured as described in the Concise Methods in G0, G1, or G2 cells without or with tetracycline (1 µg/ml) for 4 hours (mean±SD, n=5–6). Cellular (B) K+ and (D) Na+ content as a function of time after tetracycline addition is shown, normalized to time 0 (mean±SD, n=3). (B) Hatches between the time 0 and the 5-hour time measurement added for clarity, illustrating this line connects these two time points and does not represent additional interval measurements. Tet, tetracycline.

Article Snippet: Antibodies and Reagents Antibodies for immunoblotting include a rabbit polyclonal anti-APOL1 (HPA01885; Sigma) at 1:4000 or rabbit monoclonal anti-APOL1 (ab108315, lot#GR8593–2; AbCam) at 1:4000, rabbit monoclonal anti-LC3II (3868; Cell Signaling) at 1:1000, rabbit polyclonal anti-p62 (P0067, lot#108K4764; Sigma) at 1:2000, rabbit monoclonal anti-GAPDH (2118; Cell Signaling) at 1:2000, mouse monoclonal anti–Na/K-ATPase (Cat#sc-21712, Lot#G1615; Santa Cruz Biotech) at 1:200, and rabbit monoclonal anti-tubulin (2128; Cell Signaling) at 1:1000.

Techniques:

Journal: Journal of the American Society of Nephrology : JASN

Article Title: ApoL1 Overexpression Drives Variant-Independent Cytotoxicity

doi: 10.1681/ASN.2016121322

Figure Lengend Snippet: APOL1 expression is associated with the appearance of cation-selective and pH-sensitive whole-cell membrane currents. (A) Whole-cell membrane currents were recorded in an APOL1-G2–expressing 293 cell 6 hours after addition of tetracycline (1 µg/ml). Voltage ramps were applied every 6 seconds and the outward current at +60 mV (red) and inward current at −80 mV (black) during each ramp are plotted as a function of time after rupture of the patch for whole-cell recording. At the time indicated by the horizontal bar (top), the bath solution was changed from pH 7.2 to pH 5.1 and subsequently returned to pH 7.2. (B) Summary of inward (−80) and outward (+60) current amplitudes recorded at pH 7.2 at 4–5 or 6–7 hours after tetracycline induction. Values are mean±SD, n=3–5. (C) Representative current-voltage relationships (I-V) of the cell shown in (A) obtained in bath solution of the indicated pH. Note that the blue and black traces are virtually superimposable. (D) Summary of inward (−80) and outward (+60) current amplitudes at the different pH values; mean±SD, n=3. (E) Representative I-Vs showing the inhibition by reduced pH and the shift in reversal potential upon replacement of extracellular Na+ with NMDG (n=3). (F) Experiments were performed as in (A). The I-Vs show inhibition by reduced pH in APOL1-G0–expressing 293 cells (representative of n=3). Note that the blue and black traces are superimposed. G0, APOL1-G0; G2, APOL1-G2; TET, tetracycline.

Article Snippet: Antibodies and Reagents Antibodies for immunoblotting include a rabbit polyclonal anti-APOL1 (HPA01885; Sigma) at 1:4000 or rabbit monoclonal anti-APOL1 (ab108315, lot#GR8593–2; AbCam) at 1:4000, rabbit monoclonal anti-LC3II (3868; Cell Signaling) at 1:1000, rabbit polyclonal anti-p62 (P0067, lot#108K4764; Sigma) at 1:2000, rabbit monoclonal anti-GAPDH (2118; Cell Signaling) at 1:2000, mouse monoclonal anti–Na/K-ATPase (Cat#sc-21712, Lot#G1615; Santa Cruz Biotech) at 1:200, and rabbit monoclonal anti-tubulin (2128; Cell Signaling) at 1:1000.

Techniques: Expressing, Inhibition

Journal: Journal of the American Society of Nephrology : JASN

Article Title: ApoL1 Overexpression Drives Variant-Independent Cytotoxicity

doi: 10.1681/ASN.2016121322

Figure Lengend Snippet: APOL1-G0, -G1, and -G2 proteins, induced with low doses of tetracycline, do not localize to plasma membrane, cause cell death, or stimulate p38 MAPK phosphorylation. (A) APOL1-G0, -G1, or -G2 clones were cultured without tetracycline (0); with low (L) concentrations of tetracycline adjusted to induce equivalent amounts of G0, G1, and G2 protein (G0, 5 ng/ml; G1, 5 ng/ml; G2, 10 ng/ml); or with high (H) tetracycline concentrations (1 μg/ml, all genotypes). Cell surface proteins were biotinylated and cells were collected in lysis buffer. APOL1 was detectable in the input lysates of tetracycline-stimulated but not untreated cells (left panel). Biotinylated APOL1 was only identified in membranes from cells treated with high concentrations of tetracycline (right panel, labeled IP). (B) Fluorogenic cytotoxicity/viability assay demonstrates dose-dependent cytotoxicity with increasing tetracycline doses across APOL1 genotypes. An increased cytotoxicity/viability ratio was observed in APOL1-G0 and APOL1-G1 clones at the “1×” tetracycline dose compared with the no tetracycline control, despite using doses chosen to provide matched APOL1 expression. Cells were treated for 24 hours with no tetracycline (0), or increasing doses of tetracycline (1×–5×); these doses correspond to 5, 10, 15, 20, and 25 ng/ml for APOL1-G0 and -G1; and, 10, 20, 30, 40, and 50 ng/ml for APOL1-G2, respectively. (C) MTT assays also show no significant cytotoxicity after treatment with low concentrations of tetracycline. Bars represent mean±SD (n=2). (D) Immunoblots of APOL1, phosphorylated p38 MAPK, total p38 MAPK, and GAPDH from one of three experiments yielding similar results. Lanes 1–3, lysates from 293 cells used to generate the stable cell lines (293 No. Tet); lanes 4–6, lysates from G0 (0), G1 (1), and G2 (2) cells treated for 24 hours with low concentrations of tetracycline, as above; lanes 7–9, lysates from G0 (0), G1 (1), and G2 (2) cells treated for 8 hours with tetracycline, 1 μg/ml. IB, immunoblot; IP, immunoprecipitate; M.W., molecular weight; Phospho-p38, phosphorylated-p38 mitogen-activated protein kinase; Tet, tetracycline.

Article Snippet: Antibodies and Reagents Antibodies for immunoblotting include a rabbit polyclonal anti-APOL1 (HPA01885; Sigma) at 1:4000 or rabbit monoclonal anti-APOL1 (ab108315, lot#GR8593–2; AbCam) at 1:4000, rabbit monoclonal anti-LC3II (3868; Cell Signaling) at 1:1000, rabbit polyclonal anti-p62 (P0067, lot#108K4764; Sigma) at 1:2000, rabbit monoclonal anti-GAPDH (2118; Cell Signaling) at 1:2000, mouse monoclonal anti–Na/K-ATPase (Cat#sc-21712, Lot#G1615; Santa Cruz Biotech) at 1:200, and rabbit monoclonal anti-tubulin (2128; Cell Signaling) at 1:1000.

Techniques: Clone Assay, Cell Culture, Lysis, Labeling, Viability Assay, Expressing, Western Blot, Stable Transfection, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy

doi: 10.1016/j.jbc.2024.107493

Figure Lengend Snippet: Protein expression changes of FABP1, Acox2, Hmgcs2, and Acaa1b, PLTP. A , immunohistochemical staining of FABP1, Acox2, Hmgcs2, and Acaa1b, PLTP and quantification of average fluorescence intensity (n = 3, 20× magnification, three fields of view were selected for each kidney section for quantification). B , information on changes in abundance of five proteins in label-free proteome quantitation. For comparisons involving multiple groups, one-way ANOVA followed by Dunnett's post hoc test was employed. Significance levels are denoted as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. FAB1, fatty acid–binding protein 1.

Article Snippet: Rabbit monoclonal FABP1 antibody (diluted 1:4000; Arkham) was used as the primary antibody and horseradish peroxidase anti-rabbit IgG (Boster) as the secondary antibody.

Techniques: Expressing, Immunohistochemical staining, Staining, Fluorescence, Quantitation Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy

doi: 10.1016/j.jbc.2024.107493

Figure Lengend Snippet: Visualization and fluorescence quantification of HK-2 cell surface HS and intracellular FABP1. A , superpositively charged green fluorescent protein (ScGFP) labels highly negatively charged components on the cell surface. The green color diminished after incubation with heparinase ( bottom ), suggesting that HS is the dominant negatively charged species in HK-2. The scale bar represents 20 μm. B , visualization and fluorescence quantification of HS on the cell surface after high-glucose-high-fat treatment and high-glucose-high-fat + LMWH treatment with HK-2. The scale bar represents 20 μm. C , results of quantification of HS on the surface of HK-2 cells by LC-MRM (n = 3). D , composition of HS disaccharides on the surface of HK-2 cells (n = 3, ΔIS: ΔGlcA2S-GlcNS6S, ΔIIS: ΔGlcA-GlcNS6S, ΔIIIS: ΔGlcA2S-GlcNS, ΔIVS: ΔGlcA-GlcNS, ΔIA: ΔGlcA2S-GlcNAc6S, ΔIIA: ΔGlcA-GlcNAc6S, ΔIIA: ΔGlcA2S-GlcNAc6S, ΔIIIA: ΔGlcA2S-GlcNAc, ΔIVA: ΔGlcA-GlcNAc). E , visualization and fluorescence quantification of HK-2 endocytosis FABP1. The change of red fluorescence revealed that LMWH could reverse the endocytosis of FABP1 by HK-2 in a high-glucose-high-fat environment to a certain extent (n = 3, three fields of view were selected and the fluorescence of all cells in each field of view was quantified). The scale bar represents 20 μm. F , assess the effect of altered intracellular FABP1 levels on PPAR signaling using luciferase reporter assays. G , Western blot analysis of target gene proteins downstream of PPAR. H , analysis of the effect of high-glucose-high-fat on PPAR activation. The data from the two cohorts were subjected to analysis using the independent samples t test. For comparisons involving multiple groups, one-way ANOVA followed by Dunnett's post hoc test was employed. Significance levels are denoted as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. FAB1, fatty acid–binding protein 1; HS, heparan sulfate; LC, liquid chromatography; LMWH, low molecular weight heparin; MRM, multiple reaction monitoring; PPAR, peroxisome proliferator–activated receptor.

Article Snippet: Rabbit monoclonal FABP1 antibody (diluted 1:4000; Arkham) was used as the primary antibody and horseradish peroxidase anti-rabbit IgG (Boster) as the secondary antibody.

Techniques: Fluorescence, Incubation, Luciferase, Western Blot, Activation Assay, Binding Assay, Liquid Chromatography, Molecular Weight, Targeted Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy

doi: 10.1016/j.jbc.2024.107493

Figure Lengend Snippet: Effect of silencing FABP1 on apoptosis of HK-2 cells in a high-glucose-high-fat environment. For comparisons involving multiple groups, one-way ANOVA followed by Dunnett's post hoc test was employed. Significance levels are denoted as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. FAB1, fatty acid–binding protein 1.

Article Snippet: Rabbit monoclonal FABP1 antibody (diluted 1:4000; Arkham) was used as the primary antibody and horseradish peroxidase anti-rabbit IgG (Boster) as the secondary antibody.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy

doi: 10.1016/j.jbc.2024.107493

Figure Lengend Snippet: Validation of FABP1–HS interactions and characterization of oligosaccharide structures involved in the interactions. A , BLI. B , FABP1 affinity chromatography separated affinity LMWH and SEC analysis affinity LMWH polymerization degree changes. C , schematic of complete enzymatic hydrolysis and HONO degradation of LMWH. D , comparison of the relative content of disaccharides after HONO degradation of LMWH-dp8 and affinity-dp8. E , comparison of the relative content of disaccharides after complete enzymatic hydrolysis of LMWH-dp8 and affinity-dp8. F , TOP20 sequence obtained by Sep-GAG software. G , SAX chromatogram of affinity-dp8. H , MS/MS sequencing results of the five major components of affinity-dp8. I , Sep-GAG combined with MS/MS sequencing to obtain sequences of the five components of affinity-dp8. BLI, biolayer interferometry; FAB1, fatty acid–binding protein 1; GAG, glycosaminoglycan; HONO, nitrous acid; HS, heparan sulfate; LMWH, low molecular weight heparin; MS/MS, tandem mass spectrometry; SAX, strong anion exchange chromatography; SEC, size-exclusion chromatography.

Article Snippet: Rabbit monoclonal FABP1 antibody (diluted 1:4000; Arkham) was used as the primary antibody and horseradish peroxidase anti-rabbit IgG (Boster) as the secondary antibody.

Techniques: Biomarker Discovery, Affinity Chromatography, Comparison, Sequencing, Software, Tandem Mass Spectroscopy, Binding Assay, Molecular Weight, Mass Spectrometry, Chromatography, Size-exclusion Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy

doi: 10.1016/j.jbc.2024.107493

Figure Lengend Snippet: Molecular docking of dp6 and FABP1. Left panel shows the complex of the FABP1 (shown in part) and the hexasaccharide, visualized using AutoDock simulation. The right panel demonstrates the contributions of the protein and oligosaccharide binding motifs and the types of interactions ( orange dashed arrows represent electrostatic attractions, and green dashed arrows represent hydrogen bonds). FAB1, fatty acid–binding protein 1.

Article Snippet: Rabbit monoclonal FABP1 antibody (diluted 1:4000; Arkham) was used as the primary antibody and horseradish peroxidase anti-rabbit IgG (Boster) as the secondary antibody.

Techniques: Binding Assay