Journal: Phytotherapy Research

Article Title: Dihydrotanshinone I Targets PGAM1 to Induce SYVN1 ‐Mediated Ubiquitination and Suppress Glycolysis in Hepatocellular Carcinoma

doi: 10.1002/ptr.70017

Figure Lengend Snippet: Proteomic analysis identifies PGAM1 as a key glycolytic target of DHT in HCC cells. (A) Principal component analysis (PCA) plot showing the overall protein expression profiles of the control and DHT‐treated groups. (B) Volcano plot displaying differentially expressed proteins between the control and DHT‐treated groups. (C) Heatmap showing the expression levels of glycolysis‐related proteins in the control and DHT‐treated groups. (D) Gene ontology (GO) analysis plot illustrating the functional categorization of differentially expressed proteins. (E) KEGG pathway enrichment analysis plot showing the enrichment of differentially expressed proteins in various pathways. Statistical analysis was performed using Student's t ‐test for differential expression and Fisher's exact test for GO/KEGG enrichment. p < 0.05 was considered statistically significant.

Article Snippet: The antibodies used included HK1 (#2024), HK2 (#2867), PKM1 (#7067), PKM2 (#4053), SYVN1 (#14773), DYKDDDDK‐Tag (#14793), HA‐Tag (#3724), and HRP‐labeled goat anti‐rabbit IgG (#7074) from Cell Signaling Technology, as well as PGAM1 (A4170) from Abclonal, and MYC tag antibody (60003‐2‐Ig) from Proteintech.

Techniques: Expressing, Control, Functional Assay, Quantitative Proteomics

Journal: Phytotherapy Research

Article Title: Dihydrotanshinone I Targets PGAM1 to Induce SYVN1 ‐Mediated Ubiquitination and Suppress Glycolysis in Hepatocellular Carcinoma

doi: 10.1002/ptr.70017

Figure Lengend Snippet: PGAM1 is upregulated in HCC and associated with glycolytic metabolism. (A) Distribution of PGAM1 expression in tumor and normal tissues shown as boxplots. The x‐axis represents sample groups, and the y ‐axis shows PGAM1 expression levels. (B) Stemness scores were evaluated in HCC samples with high PGAM1 expression compared to normal expression levels. (C) Single‐cell analysis based on GSE125449 (TISCH database): t‐SNE clustering of cells by type and PGAM1 expression distribution, with color intensity reflecting expression levels. A bar chart shows PGAM1 expression abundance across different cell types. (D) GO and KEGG enrichment analysis plots showing the functional roles and pathway involvement of differentially expressed PGAM1. (E) Heatmap showing the correlation between PGAM1 and metabolic/signaling pathway genes (HK1, HK2, MTOR, PKM, PRKAA1, PRKAA2, PRKAB1, PRKAG1). Color intensity represents correlation strength, with darker shades indicating stronger positive correlations. Statistical analysis was performed using the Wilcoxon rank‐sum test for expression comparisons, Fisher's exact test for GO/KEGG enrichment, and Spearman correlation for gene co‐expression analysis. p < 0.05 was considered statistically significant.

Article Snippet: The antibodies used included HK1 (#2024), HK2 (#2867), PKM1 (#7067), PKM2 (#4053), SYVN1 (#14773), DYKDDDDK‐Tag (#14793), HA‐Tag (#3724), and HRP‐labeled goat anti‐rabbit IgG (#7074) from Cell Signaling Technology, as well as PGAM1 (A4170) from Abclonal, and MYC tag antibody (60003‐2‐Ig) from Proteintech.

Techniques: Expressing, Single-cell Analysis, Functional Assay

Journal: Phytotherapy Research

Article Title: Dihydrotanshinone I Targets PGAM1 to Induce SYVN1 ‐Mediated Ubiquitination and Suppress Glycolysis in Hepatocellular Carcinoma

doi: 10.1002/ptr.70017

Figure Lengend Snippet: DHT suppresses HCC progression by targeting PGAM1‐mediated glycolysis. (A) CCK8 assay showing relative proliferation rates of OE‐PGAM1, KD‐PGAM1, and control HCC cells (Huh7, PLC, Hep3B) at 24, 48, and 72 h ( n = 3). (B) Quantification of glucose consumption, lactate production, and ATP levels in OE‐PGAM1, KD‐PGAM1, and control cells ( n = 3). (C) Wound healing assay showing migration distances of OE‐PGAM1, KD‐PGAM1, and control cells at 0 h, 12, and 24 ( n = 3). (D) Transwell invasion assay of OE‐PGAM1, KD‐PGAM1, and control HCC cells at 12 and 24 h ( n = 3). (E) Protein expression levels of key glycolytic enzymes (HK1, HK2, PKM1, PKM2, PGAM1) in HCC cells following DHT treatment ( n = 3). (F) Changes in glucose consumption, lactate production, and ATP levels in HCC cells treated with different concentrations of DHT ( n = 3). Data are presented as mean ± SD. Statistical analysis was performed using one‐way ANOVA or Student's t‐test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies used included HK1 (#2024), HK2 (#2867), PKM1 (#7067), PKM2 (#4053), SYVN1 (#14773), DYKDDDDK‐Tag (#14793), HA‐Tag (#3724), and HRP‐labeled goat anti‐rabbit IgG (#7074) from Cell Signaling Technology, as well as PGAM1 (A4170) from Abclonal, and MYC tag antibody (60003‐2‐Ig) from Proteintech.

Techniques: CCK-8 Assay, Control, Wound Healing Assay, Migration, Transwell Invasion Assay, Expressing

Journal: Phytotherapy Research

Article Title: Dihydrotanshinone I Targets PGAM1 to Induce SYVN1 ‐Mediated Ubiquitination and Suppress Glycolysis in Hepatocellular Carcinoma

doi: 10.1002/ptr.70017

Figure Lengend Snippet: Rescue of DHT‐induced anti‐proliferative and glycolysis inhibition effects in HCC by PGAM1 overexpression. (A, B) WB analysis showing PGAM1 protein levels in control, DHT‐treated, OE‐PGAM1, and OE‐PGAM1 + DHT groups in Huh7 (A) and PLC (B) cells. β‐actin served as loading control. (C, D) CCK8 assay showing relative proliferation rates of control, DHT‐treated, OE‐PGAM1, and OE‐PGAM1 + DHT groups in Huh7 (C) and PLC (D) cells at 24, 48, and 72 h ( n = 3). (E, F) Quantification of extracellular glucose concentration (left panels), intracellular lactate content (middle panels), and intracellular ATP levels (right panels) in Control, DHT‐treated, OE‐PGAM1, and OE‐PGAM1 + DHT groups in Huh7 (E) and PLC (F) cells. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 3).

Article Snippet: The antibodies used included HK1 (#2024), HK2 (#2867), PKM1 (#7067), PKM2 (#4053), SYVN1 (#14773), DYKDDDDK‐Tag (#14793), HA‐Tag (#3724), and HRP‐labeled goat anti‐rabbit IgG (#7074) from Cell Signaling Technology, as well as PGAM1 (A4170) from Abclonal, and MYC tag antibody (60003‐2‐Ig) from Proteintech.

Techniques: Inhibition, Over Expression, Control, CCK-8 Assay, Concentration Assay

Journal: Phytotherapy Research

Article Title: Dihydrotanshinone I Targets PGAM1 to Induce SYVN1 ‐Mediated Ubiquitination and Suppress Glycolysis in Hepatocellular Carcinoma

doi: 10.1002/ptr.70017

Figure Lengend Snippet: DHT directly binds to PGAM1 with high affinity and stability. (A) Proposed docking model of DHT and PGAM1, with 2D and 3D representations highlighting the key binding sites. (B) Root mean square deviation (RMSD) trajectory showing system stability. (C) Root mean square fluctuation (RMSF) profile indicating residue flexibility. (D) Time evolution of hydrogen bond interactions between DHT and PGAM1. (E) Free energy landscape, with red low‐energy regions indicating that the binding of DHT to PGAM1 is stable and favorable. (F) Binding free energy decomposition of the PGAM1‐DHT complex, showing contributions from gas‐phase energy (GGAS), solvation energy (GSOLV), and total energy (TOTAL). (G) Surface plasmon resonance (SPR) sensorgram showing the interaction between PGAM1 and DHT at various concentrations (15.6 to 1000 nM). (H) Dose–response curve derived from the SPR data, with a calculated dissociation constant (KD) of 697 nM.

Article Snippet: The antibodies used included HK1 (#2024), HK2 (#2867), PKM1 (#7067), PKM2 (#4053), SYVN1 (#14773), DYKDDDDK‐Tag (#14793), HA‐Tag (#3724), and HRP‐labeled goat anti‐rabbit IgG (#7074) from Cell Signaling Technology, as well as PGAM1 (A4170) from Abclonal, and MYC tag antibody (60003‐2‐Ig) from Proteintech.

Techniques: Binding Assay, Residue, SPR Assay, Derivative Assay

Journal: Phytotherapy Research

Article Title: Dihydrotanshinone I Targets PGAM1 to Induce SYVN1 ‐Mediated Ubiquitination and Suppress Glycolysis in Hepatocellular Carcinoma

doi: 10.1002/ptr.70017

Figure Lengend Snippet: DHT promotes PGAM1 degradation through SYVN1‐mediated ubiquitination. (A) Huh7 and PLC cells were treated with DHT (5 μM) for 24 h. Total RNA was extracted, and qPCR was performed to analyze mRNA expression levels. (B, C) Cells were treated with MG‐132 (2.5 μM) for 1 h, followed by treatment with DHT (5 μM) for 24 h in Huh7 and PLC cells. Western blot was performed using the indicated antibodies. (D) WB analysis of PGAM1 protein levels at different time points (0, 1, 3, 6, 12, 24 h) after treatment with CHX and co‐treatment with CHX and DHT. (E) Cells were transfected with FLAG‐PGAM1 and HA‐Ub for 24 h, treated with 5 μM DHT for 24 h, with MG‐132 (2.5 μM) added during the final 3 h of the DHT treatment. After lysis, immunoprecipitation was conducted as indicated. (F) WB analysis of FLAG‐PGAM1 in Huh7 and PLC cells following DHT (3 μM) treatment. Cells were transfected with FLAG‐PGAM1 and HA‐Ub and treated with K48 or K63 ubiquitin mutants as indicated. Immunoprecipitation using FLAG antibody was performed to pull down FLAG‐PGAM1, followed by detection of ubiquitination by HA antibody. The lysate controls show the expression levels of FLAG‐PGAM1 and HA‐Ub. (G) Protein–protein docking of PGAM1 (green) and SYVN1 (purple) using GRAMM rigid docking, with a binding free energy of −35.8 kcal/mol. Residues involved in the binding interface are highlighted, illustrating the interaction sites between PGAM1 and SYVN1. (H) After transfection with FLAG‐PGAM1 and HA‐Ub for 24 h, cells were exposed to 5 μM DHT for another 24 h. Following treatment, cells were lysed, and the resulting lysates were used for immunoprecipitation as described. (I) Cells were initially transfected with siSYVN1 for 24 h, then subjected to a second transfection with FLAG‐PGAM1 and HA‐Ub for an additional 24 h. Subsequently, the cells were exposed to 5 μM DHT for 24 h, lysed, and the resulting lysates were used for immunoprecipitation as described. Data are presented as mean ± SD. Statistical significance was assessed using Student's t ‐test or one‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies used included HK1 (#2024), HK2 (#2867), PKM1 (#7067), PKM2 (#4053), SYVN1 (#14773), DYKDDDDK‐Tag (#14793), HA‐Tag (#3724), and HRP‐labeled goat anti‐rabbit IgG (#7074) from Cell Signaling Technology, as well as PGAM1 (A4170) from Abclonal, and MYC tag antibody (60003‐2‐Ig) from Proteintech.

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Transfection, Lysis, Immunoprecipitation, Binding Assay

Journal: Phytotherapy Research

Article Title: Dihydrotanshinone I Targets PGAM1 to Induce SYVN1 ‐Mediated Ubiquitination and Suppress Glycolysis in Hepatocellular Carcinoma

doi: 10.1002/ptr.70017

Figure Lengend Snippet: DHT exhibits potent antitumor activity in HCC models without observable short‐term toxicity. (A) Schematic representation of the experimental protocol for evaluating the efficacy of DHT in an orthotopic HCC model established by hydrodynamic tail vein injection of AKT/NRAS ( n = 6). (B) Representative macroscopic images of liver tissues and quantitative analysis of terminal liver weights across control and treatment groups in the orthotopic model. (C) Time course analysis of body weight changes during the treatment period across experimental groups. (D) Representative photographs of excised tumors from subcutaneous xenograft model at study endpoint across four treatment groups: Vehicle control, DHT (10 mg/kg), DHT (20 mg/kg), and sorafenib (30 mg/kg) ( n = 5). (E) Tumor growth curves showing the evolution of tumor volume over time across all four groups. Statistical comparisons were conducted at the final time point (Day 27). (F) Quantitative comparison of terminal tumor weights among the four experimental groups. (G) Serum biochemical analysis of liver and kidney function markers (ALT, AST, CRE, and BUN) across all treatment groups. (H) H&E staining and immunohistochemical (IHC) analysis of Ki67, TUNEL, and PGAM1 in liver tissues, illustrating the effects of DHT on cell proliferation, apoptosis, and PGAM1 expression in HCC. (I) WB analysis of liver tissues showing expression levels of HK1, HK2, PKM1, PKM2, and PGAM1 in the three groups of mice ( n = 3). Data are presented as mean ± SD. Statistical significance was assessed using Student's t ‐test or one‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies used included HK1 (#2024), HK2 (#2867), PKM1 (#7067), PKM2 (#4053), SYVN1 (#14773), DYKDDDDK‐Tag (#14793), HA‐Tag (#3724), and HRP‐labeled goat anti‐rabbit IgG (#7074) from Cell Signaling Technology, as well as PGAM1 (A4170) from Abclonal, and MYC tag antibody (60003‐2‐Ig) from Proteintech.

Techniques: Activity Assay, Injection, Control, Comparison, Staining, Immunohistochemical staining, TUNEL Assay, Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Interactions between CLC-5 and KIF3B subunits. A : CLC-5 consists of 18 helices (A to R) with the NH 2 - and COOH-terminus (C-term) domains being cytoplasmic (membrane boundaries indicated by arrows) ( , ). B : yeast 2-hybrid using the CLC-5 C-term, which contains 2 CBS domains to screen a renal cDNA library, identified KIF3B as a putative interactor. Interaction between large T and p53 provided a positive control. C : 35 S-methionine-labeled CLC-5 C-term was incubated with GST-tagged KIF3B coiled coil (CC)/globular domain (GD). Resin-bound proteins were resolved by SDS-PAGE and detected by autoradiography. Results from 2 independent in vitro translation experiments using 2 different GST-tagged KIF3B clones are shown. The larger band at ∼24.5 kDa represents the 216 amino acid Myc-tagged CLC-5 C-term, whereas the smaller band at ∼18.5 kDa likely represents another translated form of CLC-5 C-term that originated from use of an alternative methionine at codon 583 and hence lacked the NH 2 terminal 32 amino acids. This smaller CLC-5 C-term, which was less efficiently translated, had both CBS domains and hence was pulled down by the GST-tagged KIF3B CC/GD. D : endogenous coimmunoprecipitation using wild-type mouse whole kidney extracts and a KIF3B monoclonal antibody (mAb). KIF3B, KIF3A, and KAP3 mAb were used to confirm endogenous coimmunoprecipitation of these proteins which form a trimeric complex. It is important to note that the interaction between CLC-5 and KIF3B is likely to be transient, as endosomes will be released to continue recycling, and that only a small proportion of the total cellular CLC-5 will be present, at a given time point, on the surface of endosomes that are being trafficked anterogradely; these features are consistent with the relatively low amount of KIF3B that is immunopreciptiated by CLC-5. E : coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-5 and KIF3B domains. Lane 1 : input; lanes 2 , 3 , and 4 : pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively. F : summary of KIF3B domains showing presence (+) or absence (−) of interactions with CLC-5. G : coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-4 and KIF3B, and CLC-7 and KIF3B. Lane 1 : input; lanes 2 , 3 , and 4 : pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: cDNA Library Assay, Positive Control, Labeling, Incubation, SDS Page, Autoradiography, In Vitro, Clone Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Confocal microscopy shows colocalization of CLC-5 and KIF3B. HEK293 cells transfected with fluorescently tagged CLC-5 and KIF3B (CLC-5-RFP and KIF3B-GFP) and costained with anti-α-tubulin or anti-Kinesin-2. A - C : Z-stack dual wavelength confocal microscopy. For each stack, images were obtained in 2 channels over 15 horizontal planes that were 0.7 μm apart. A : HEK293 cells transfected with KIF3B-GFP and CLC-5-RFP. Green signals: KIF3B; red signals: CLC-5; yellow signals: colocalized KIF3B and CLC-5 (merge). Confocal Z-stack images and intensity profile line scans demonstrated that KIF3B-GFP and CLC-5-RFP are partially colocalized, as evidenced by the yellow signals or by an overlap of <300 nm, which is the estimated diameter of a proximal tubular endosomal vesicle , in the red and green peaks (confocal images 1 to 5). The intensity profile line scans were performed between the white crosses as indicated on confocal images 1 to 5, with the intensity measured in arbitrary units (A.U.). Scale bar top : 10 μm; scale bar confocal images 1 to 5: 2 μm. B : HEK293 cells transfected with CLC-5-RFP and costained with anti-α-tubulin. Red signals: CLC-5; green signals: tubulin; yellow signals: colocalized CLC-5 and tubulin (merge). CLC-5-RFP was present at the plasma membrane and in punctate structures throughout the cytoplasm, where it was localized with tubulin, indicating its association with microtubules. Scale bars: 10 μm. C : HEK293 cells transfected with CLC-5-RFP and costained with anti-Kinesin-2 antibody. Red signals: CLC-5; green signals: Kinesin-2; yellow signals: colocalized CLC-5 and Kinesin-2 (merge). Scale bars: 10 μm. D and E : live cell imaging using dual-wavelength confocal microscopy of HEK293 cells cotransfected with full-length CLC-5-RFP and full-length KIF3B-GFP. Green signals: KIF3B; red signals: CLC-5; yellow signals: colocalized CLC-5 and KIF3B. Scale bar: 10 μm. A proportion of CLC-5-RFP-labeled vesicles was transported along KIF3B-labeled cytoskeletal network structures. E : progress of a vesicle (arrowed), over 10 s, is illustrated. The vesicles were estimated to move at an approximate rate of 0.42 μm/s. Scale bar: 5 μm.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: Confocal Microscopy, Transfection, Live Cell Imaging, Labeling

Journal: American Journal of Physiology - Renal Physiology

Article Title: CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Electrophysiological, cell surface expression, and albumin uptake studies in nonpolarized HEK293 cells. A-C : representative current recording from a HEK293 cell expressing CLC-5 ( A ), CLC-5 and KIF3B ( B ), and an untransfected HEK293 cell ( C ). D : maximum conductance measured from HEK293 cells cotransfected with CLC-5 and expression plasmids as indicated. Coexpression of KIF3B (FL) significantly increased conductance. E : densitometric analysis of CLC-5 in cell surface fractions. Coexpression of KIF3B (FL) significantly increased the cell surface expression of CLC-5. F : microtubule orientation in nonpolarized HEK293 cells. G : representative Western blot analysis for KIF3B, KIF3A, and tubulin expression in cell lysates obtained from HEK293 cells transfected with control or KIF3B siRNA. KIF3B siRNA suppressed endogenous KIF3B expression, whereas there was no effect on KIF3A and tubulin expression. H : densitometric analysis of KIF3B expression in cell lysates obtained from HEK293 cells transfected with control or KIF3B siRNA. KIF3B siRNA significantly reduced expression of endogenous KIF3B. I : maximum conductance measured from HEK293 cells cotransfected with CLC-5 and KIF3B siRNA or control siRNA plasmids as indicated. KIF3B siRNA significantly decreased conductance compared with coexpression of CLC-5 with control siRNA. J : densitometric analysis of CLC-5 in cell surface fractions. KIF3B siRNA significantly decreased the cell surface expression of CLC-5, compared with coexpression of CLC-5 with control siRNA ( n = 6). K : RT-PCR analysis of cubilin, megalin, KIF3B, calmodulin (positive control), and water blank (negative control) in HEK293 cells and control human kidney (hKid). L : albumin uptake in HEK293 cells transfected with KIF3B constructs. KIF3B (FL) significantly increased albumin uptake, whereas KIF3B siRNA significantly decreased albumin uptake, compared with their respective controls. M : competition assays for albumin uptake. HEK293 cells were transfected with KIF3B constructs, in the absence (no competition) or presence (albumin competition) of excess unlabeled albumin, and the uptake of fluorescently labeled albumin measured ( n = 6); the uptake of fluorescently labeled albumin without competition was taken as the reference and set at 100% and compared with that obtained with competition using unlabeled albumin. Endocytosis of the fluorescently labeled albumin was significantly reduced by competition with unlabeled albumin. Means ± 1 SE and P values calculated by Student's unpaired, 2-tailed t -test are shown. * P < 0.02, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: Expressing, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Construct, Labeling

Journal: American Journal of Physiology - Renal Physiology

Article Title: CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Cell surface expression, albumin endocytosis, and transferrin endocytosis studies in polarized opossum kidney (OK) cells. A : microtubule orientation in polarized OK cells [apical membrane (APM) and basolateral membrane (BLM)]. B : confocal microscopy of polarized OK cells stained with ZO1 (green) and EB1 (red). Top : XY image. Bottom : single orthogonal view of ZO1 and EB1. OK cells were polarized in culture as evidenced by the presence of tight junctions (green). In addition, numerous EB1-labeled microtubules are observed below the tight junctions demonstrating that the growing ends of microtubules are predominantly at the basolateral surface of polarized OK cells. Scale bar = 10 μm. C : RT-PCR analysis of KIF3B and calmodulin (positive control) expression in OK and HEK293 cells. A water blank was used as the negative control. OK cells have previously been shown to express CLC-5, megalin, and cubilin ( , ). D : representative Western blot analysis for KIF3B, KIF3A, and tubulin expression in cell lysates obtained from OK cells transfected with control or KIF3B siRNA. KIF3B siRNA suppressed endogenous KIF3B expression, whereas there was no effect on KIF3A and tubulin expression. E : densitometric analysis of KIF3B expression in cell lysates obtained from OK cells transfected with control or KIF3B siRNA. KIF3B siRNA significantly reduced expression of endogenous KIF3B to 55 ± 4.3%, compared with control, i.e., nonsense siRNA ( n = 4). F : confocal microscopy of polarized OK cells transfected with CLC-5-Myc and stained with anti-Myc (red) and anti-ZO1 (green). Top : XY image. Bottom : single orthogonal view of CLC-5-Myc and ZO1. CLC-5-Myc-labeled structures can be observed above the tight junctions (green) demonstrating expression of CLC-5-Myc predominantly at the apical surface of polarized OK cells. Scale bar = 10 μm. G : CLC-5 plasma membrane expression in OK cells (densitometric analysis after surface biotinylation). KIF3B (FL) significantly decreased the cell surface expression of CLC-5, compared with coexpression of CLC-5 with pCMV-HA alone (HA; n = 9). H : albumin uptake in OK cells transfected with KIF3B constructs. KIF3B (FL) significantly decreased albumin uptake ( n = 6), compared with expression of pCMV-HA alone (HA), whereas KIF3B siRNA significantly increased albumin uptake, compared with expression of control siRNA ( n = 12). I : transferrin uptake in OK cells transfected with KIF3B constructs. KIF3B (FL) significantly decreased transferrin uptake ( n = 6), compared with expression of pCMV-HA alone (HA), whereas KIF3B siRNA significantly increased transferrin uptake ( n = 12), compared with expression of control siRNA. J : competition assays for albumin uptake. OK cells were transfected with KIF3B constructs, in the absence (no competition) or presence (competition) of excess unlabeled albumin and transferrin, and the uptake of fluorescently labeled albumin was measured. Endocytosis of fluorescently labeled albumin was significantly reduced by competition with either unlabeled albumin or unlabeled transferrin ( n = 6). K : competition assays for transferrin uptake. OK cells were transfected with KIF3B constructs, in the absence (no competition) or presence (competition) of excess unlabeled albumin and transferrin, and the uptake of fluorescently labeled transferrin was measured. Endocytosis of fluorescently labeled transferrin was significantly reduced by competition with either unlabeled albumin or unlabeled transferrin ( n = 6). Means ± 1 SE and P values calculated by Student's unpaired, 2-tailed t -test are shown; * P < 0.05, ** P < 0.02, $ P < 0.001.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: Expressing, Confocal Microscopy, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Western Blot, Transfection, Construct

Journal: American Journal of Physiology - Renal Physiology

Article Title: CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Expression level and subcellular distribution of Kif3b in Clcn5 Y/+ and Clcn5 Y/− mouse kidneys. A : quantitative PCR (qPCR) of mRNA in Clcn5 Y/+ and Clcn5 Y/− kidneys ( n = 4 in each group). Kif3b mRNA levels (adjusted to GAPDH mRNA) are significantly higher in Clcn5 Y/− than control kidneys (155 ± 11%; * P < 0.05). B : representative Western blot for Kif3b in Clcn5 Y/+ and Clcn5 Y/− kidneys ( n = 4 in each group), using 20 μg of protein. C : densitometric analysis normalized to β-actin levels confirms that Kif3b protein is more abundant in Clcn5 Y/− kidneys (153 ± 19% of Clcn5 Y/+ level; * P < 0.05). D : representative Western blot for Kif3b in low-speed (LS), high-speed (HS) membrane, and cytosolic (C) fractions of Clcn5 Y/+ and Clcn5 Y/− kidneys. The samples were loaded at 20 μg total protein per lane, as appropriate for subcellular fractionations . Kif3b is detected in the cytosol of Clcn5 Y/− samples but not in controls. E and F : density distribution after centrifugation of the HS (membrane) fraction, using a Percoll gradient, is presented compared with the initial concentration (C/Ci), so that values >1 reflect organelle enrichment and values <1 reflect organelle depletion. This gradient resolves a low-density peak ( fractions 2–4 ), including early endosomes (Rab5a, V-ATPase), from a high-density peak ( fractions 9–10 ), enriched in lysosomes (cathepsin D). Kif3b partially overlaps with V-ATPase E1 subunit (low-density fractions 2–3 ) and Clc-5 ( fraction 3 ), which supports their association with endosomes ( F ). Means ± 1 SE and P values were calculated by the Student's unpaired, 2-tailed t -test.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Centrifugation, Concentration Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Endogenous Kif3b expression and albumin endocytosis in mouse proximal renal tubular primary cell cultures (mPTCs). A : endogenous expression of Kif3b in mPTC cultures from Clcn5 Y/+ and Clcn5 Y/− mice. The expression of Kif3b was determined by qPCR and was found to be significantly higher in the Clcn5 Y/− mice and consistent with the observations from mouse whole kidney extracts ( Fig. 5 A ). B : expression of human KIF3B (hKIF3B) in transfected mPTCs from Clcn5 Y/+ ( n = 3) and Clcn5 Y/− ( n = 3) mice was assessed by qPCR and this showed an ∼3,000-fold higher expression of the exogenous human KIF3B, compared with the endogenous expression of Kif3b [Kif3b(m)] and Kif3a . C : uptake of FITC-albumin in mPTC cultures from Clcn5 Y/+ and Clcn5 Y/− mice in mock-transfected cells, in the absence or presence of competition by excess unlabeled albumin and transferrin, or after energy depletion by deoxyglucose (DOG) and sodium azide (NaN 3 ), or in KIF3B-transfected cells. In Clcn5 Y/+ mPTCs, overexpression of hKIF3B decreases specific albumin endocytic uptake. In Clcn5 Y/− mPTCs, albumin uptake is fully abrogated (to the level of metabolic inhibition and albumin competition) by hKIF3B overexpression. D : RT-PCR analyses of caveolin-1 (Cav-1) and caveolin-2 (Cav-2) in HEK293 cells, OK cells, Clcn5 Y/+ and Clcn5 Y/− whole mouse kidneys, and Clcn5 Y/+ and Clcn5 Y/− mPTCs. GAPDH and a water blank were used as positive and negative controls, respectively. Means ± 1 SE and P values were calculated by the Student's unpaired, 2-tailed t -test.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: Expressing, Transfection, Over Expression, Inhibition, Reverse Transcription Polymerase Chain Reaction

Journal: American Journal of Physiology - Renal Physiology

Article Title: CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Schematic representation of a working model for endosomal transport involving CLC-5 and KIF3B interaction in polarized cells. CLC-5 and KIF3B are expressed in polarized renal proximal tubular cells which reabsorb proteins and solutes via the receptor-mediated endocytic pathway that uses megalin and cubilin as the apical plasma membrane (APM) receptors ( , , ). Ligands bind to these receptors at the APM where CLC-5 and interacting proteins Nedd4-2, NHERF2, and cofilin are also present ( , , ). The receptor-ligand complex is internalized in vesicles, containing CLC-5 and CLC-4 , which are anterogradely transported along microtubules via the Kinesin-2 complex . These vesicles fuse with early endosomes where acidification involves the vacuolar H + -ATPase, with counter ions being provided by CLC-5 . In nonpolarized cells, the orientation of the microtubules is reversed ( Fig. 3 F ), and CLC-5 vesicles would be transported to the cell surface.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: