| 1997 |
CLIC4 (p64H1) localizes to the endoplasmic reticulum when expressed in HEK-293 and HT-4 cells; incorporation of HEK-293 ER vesicles into planar lipid bilayers produced intermediate-conductance, outwardly rectifying anion channels. Protein kinase C phosphorylation of p64H1 increased its apparent molecular weight from ~29 kDa to ~43 kDa. |
In vitro expression, immunolocalization, planar lipid bilayer electrophysiology, in vitro PKC phosphorylation assay |
The Journal of biological chemistry |
High |
9295337
|
| 2001 |
CLIC4 directly binds dynamin I and 14-3-3ζ (confirmed by gel overlay and reverse pull-down), and also associates with α-tubulin, β-actin, creatine kinase, and two 14-3-3 isoforms in rat brain (confirmed by affinity chromatography, mass spectrometry, and co-immunoprecipitation). CLIC4 partially co-localizes with caveolin and functional caveolae in HEK-293 cells, implicating it in caveolar endocytosis. |
Affinity chromatography, mass spectrometry, co-immunoprecipitation, gel overlay, reverse pull-down, immunofluorescence |
The Biochemical journal |
High |
11563969
|
| 2002 |
CLIC4 (mtCLIC) associates with the inner mitochondrial membrane. Overexpression reduces mitochondrial membrane potential, releases cytochrome c, activates caspases, and induces apoptosis. CLIC4 is transcriptionally regulated by p53 and TNF-α. CLIC4 antisense prevents p53-induced apoptosis but not Bax-induced apoptosis, placing CLIC4 in an independent proapoptotic pathway converging on mitochondria. |
Subcellular fractionation, transient transfection overexpression, mitochondrial membrane potential assay, cytochrome c release assay, caspase activation assay, antisense knockdown, genetic epistasis with Bax |
Molecular and cellular biology |
High |
11997498
|
| 2002 |
Overexpressed CLIC4 in stably transfected HEK-293 cells forms novel low-conductance (~1 pS) plasma membrane anion channels with mild outward rectification, sensitive to IAA (IC50 ~100 µM). Anti-CLIC4 antibodies applied to the cytoplasmic face (but not external face) inhibit these channels, establishing that the C-terminus of the integral membrane form of CLIC4 faces the cytoplasm. |
Stable transfection, patch-clamp electrophysiology (whole-cell and single-channel), antibody inhibition from cytoplasmic vs. external face |
Biochemical and biophysical research communications |
High |
12237120
|
| 2002 |
TGF-β1 specifically upregulates CLIC4 (>16-fold) during fibroblast-to-myofibroblast conversion, an effect not shared by CLIC1, CLIC2, CLIC3, or CLIC5. Conditional expression of CLIC4 in MEF/3T3 fibroblasts inhibits cell motility by 27% in a migration assay. |
Differential display mRNA profiling, RT-PCR, tetracycline-regulated conditional expression, migration assay |
The American journal of pathology |
Medium |
12163372
|
| 2003 |
Multiple stress inducers (DNA damage, apoptotic stimuli) cause translocation of cytoplasmic CLIC4 to the nucleus. CLIC4 associates with Ran, NTF2, and Importin-α nuclear import complexes. Deletion or mutation of the C-terminal nuclear localization signal abolishes nuclear translocation; N-terminal deletion enhances it. Nuclear-targeted CLIC4 accelerates apoptosis and induces apoptosis even in Apaf-null fibroblasts or Bcl-2-overexpressing keratinocytes. |
Immunogold EM, confocal microscopy, co-immunoprecipitation, deletion/mutation analysis, adenoviral nuclear targeting, apoptosis assays in Apaf-null and Bcl-2-overexpressing cells |
The Journal of biological chemistry |
High |
14610078
|
| 2003 |
CLIC4 colocalizes with AKAP350 at the centrosome and midbody in cultured mammalian cells, and with AKAP350 and the tight junction protein ZO-1 in the apical region of polarized epithelial cells. CLIC4 is enriched in mitochondria, cortical actin-based structures, and the nuclear matrix, and associates with microtubule cytoskeletal proteins biochemically. |
Immunofluorescence microscopy, subcellular fractionation, biochemical co-sedimentation |
Cell motility and the cytoskeleton |
Medium |
14569596
|
| 2005 |
Crystal structure of CLIC4 resolved at 1.8 Å by X-ray crystallography. CLIC4 is monomeric and adopts a GST fold, similar to CLIC1 but with differences in helix 2 of the glutaredoxin-like N-terminal domain. Purified recombinant CLIC4 binds artificial lipid bilayers, induces chloride efflux when associated with liposomes, and forms a 30 pS ion channel in artificial bilayers. Oxidation enhances membrane binding; no channels were observed under reducing conditions. |
X-ray crystallography (1.8 Å), lipid bilayer reconstitution, chloride efflux assay, tip-dip electrophysiology, oxidation/reduction manipulation |
The FEBS journal |
High |
16176272
|
| 2005 |
CLIC4 protein levels are upregulated by c-Myc; Myc binds directly to the CLIC4 gene promoter and activates its transcription (by quantitative proteomics and ChIP). Suppression of CLIC4 by RNAi inhibits Myc-induced apoptosis under stress conditions and abolishes cooperative induction of apoptosis by Myc and Bax. |
Isotope-coded affinity tag quantitative proteomics, chromatin immunoprecipitation, RNAi knockdown, apoptosis assays |
The Journal of biological chemistry |
High |
16316993
|
| 2005 |
CLIC4 expression decreases during VEGF-A-induced endothelial cell tubular morphogenesis. siRNA- or antisense-mediated suppression of CLIC4 arrests tubular morphogenesis in vitro, establishing a required role for CLIC4 in endothelial tube/lumen formation. |
2D proteomics, antisense and siRNA knockdown, in vitro tubulogenesis assay |
The Journal of biological chemistry |
Medium |
16239224
|
| 2007 |
Reconstituted recombinant CLIC4 in planar lipid bilayers forms redox-regulated, poorly selective ion channels (maximum ~15 pS in KCl). A truncated version comprising only the N-terminal 61 residues (containing the predicted transmembrane domain) also forms non-selective channels with reduced conductance that retain trans-redox sensitivity and can be blocked by trans (not cis) thiol-reactive DTNB, suggesting the predicted TMD forms oligomeric pores and the trans cysteine is at the external pore entrance. |
Planar lipid bilayer reconstitution, site-specific truncation mutant analysis, redox manipulation, thiol-reactive DTNB block from trans/cis sides |
Molecular membrane biology |
High |
17453412
|
| 2007 |
CLIC1 and CLIC5 channel activity in planar bilayers is strongly and reversibly inhibited by F-actin; CLIC4 channels are NOT inhibited by F-actin under the same conditions, demonstrating differential actin regulation among CLIC family members. |
Planar lipid bilayer reconstitution with addition of F-actin; cytochalasin reversal experiment |
The FEBS journal |
High |
18028448
|
| 2007 |
CLIC4 expression is reduced in multiple human epithelial cancers and excluded from the nucleus in cancer cells. In xenografts, adenoviral introduction of CLIC4 or nuclear-targeted CLIC4 into breast cancer cells inhibits tumor growth, whereas overexpression of CLIC4 in stromal cells enhances tumor growth. |
Tissue microarray, adenoviral transduction into xenografts, CLIC4 overexpression in stromal cells, in vivo tumor growth assay |
Clinical cancer research |
Medium |
17200346
|
| 2008 |
CLIC4 physically interacts with the C-terminus of the histamine H3 receptor, confirmed by in vitro pull-down, co-immunoprecipitation from rat brain lysate, and immunofluorescence co-localization in rat cerebellar neurons. CLIC4 enhances cell surface expression of H3R, but not a mutant H3R that cannot interact with CLIC4, as measured by flow cytometry, radioligand binding, and cell-based ELISA. |
In vitro pull-down, co-immunoprecipitation from rat brain, immunofluorescence, flow cytometry, radioligand binding assay, cell-based ELISA |
Biochemical and biophysical research communications |
High |
18302930
|
| 2009 |
TGF-β promotes CLIC4 and Schnurri-2 expression, their cytoplasmic association, and their co-translocation to the nucleus. In the nucleus, CLIC4 associates with phospho-Smad2 and phospho-Smad3, protecting them from dephosphorylation by nuclear phosphatases, thereby sustaining TGF-β signaling. In the absence of CLIC4 or Schnurri-2, TGF-β signaling is abrogated; direct nuclear targeting of CLIC4 removes the requirement for Schnurri-2. |
Co-immunoprecipitation, nuclear fractionation, siRNA knockdown, adenoviral nuclear targeting, TGF-β signaling reporter assays, phospho-Smad stabilization assay |
Nature cell biology |
High |
19448624
|
| 2009 |
Cytosolic CLIC4 undergoes rapid but transient translocation to discrete domains at the plasma membrane upon stimulation of Gα13-coupled, RhoA-activating receptors (LPA, thrombin, S1P). This translocation is strictly dependent on Gα13-mediated RhoA activation and F-actin integrity but not Rho kinase activity. Mutational analysis reveals dependence on at least six conserved residues including reactive Cys35. Membrane-targeted CLIC4 does not modulate transmembrane chloride currents. |
Live-cell imaging, pharmacological inhibitors (Y-27632, cytochalasin), dominant-negative/constitutively active RhoA constructs, site-directed mutagenesis, electrophysiology |
Molecular biology of the cell |
High |
19776349
|
| 2010 |
CLIC4 is directly S-nitrosylated on a cysteine residue (detected by biotin switch assay), and this modification induces conformational unfolding (CD spectra, trypsinolysis) and enhanced association with importin-α and Ran, promoting nuclear translocation. TNF-α-induced nuclear translocation of CLIC4 depends on nitric oxide synthase activity, and NOS inhibition blocks TNF-α-induced CLIC4 nitrosylation and nuclear import. Cysteine mutants show altered nitrosylation, nuclear residence, and stability. |
Biotin switch assay (S-nitrosylation detection), CD spectroscopy, limited trypsinolysis, co-immunoprecipitation with importin-α/Ran, NOS inhibition, site-directed mutagenesis |
The Journal of biological chemistry |
High |
20504765
|
| 2011 |
CLIC4-null macrophages show reduced accumulation of phosphorylated IRF3 upon LPS stimulation, while CLIC4 overexpression enhances LPS-mediated IRF3 phosphorylation. CLIC4-null mice are protected from LPS-induced death with reduced serum inflammatory cytokines, and are impaired in Listeria monocytogenes clearance. Deletion of CLIC4 had little effect on MAPK and NF-κB activation, placing CLIC4 specifically in the IRF3 arm of LPS signaling. |
CLIC4-null mouse generation, LPS challenge in vivo, Western blot for phospho-IRF3/MAPK/NF-κB, stable CLIC4-overexpressing macrophage lines, Listeria infection assay |
European journal of immunology |
High |
21469130
|
| 2012 |
CLIC4-null mice exhibit delayed wound reepithelialization and corneal wound healing, reduced β4 integrin and p21 expression in wounded skin, reduced TGF-β-induced phospho-Smad2 in CLIC4-null keratinocytes, slower keratinocyte migration, and failure to increase migration in response to TGF-β, placing CLIC4 upstream of TGF-β signaling in epidermal wound healing. |
CLIC4-null mouse (C57Bl/6 background), full-thickness skin wound and corneal wound assays, Western blot, keratinocyte migration assay, TGF-β stimulation of cultured keratinocytes |
The American journal of pathology |
High |
22613027
|
| 2012 |
In metabolically stressed keratinocytes, CLIC4 is S-nitrosylated and translocates to the nucleus, where it enhances TGF-β signaling by protecting phospho-Smad2/3 from dephosphorylation. Inhibiting antioxidant defense in tumor cells increases S-nitrosylation and nuclear CLIC4 translocation. Adenoviral nuclear targeting of CLIC4 in squamous cancer cells enhances TGF-β transcriptional activity and inhibits growth in vitro and in orthograft tumors. |
Adenoviral nuclear targeting, TGF-β reporter assay, S-nitrosylation assay, tumor orthograft model, transgenic epidermis overexpression model |
Carcinogenesis |
High |
22387366
|
| 2013 |
CLIC4 is required for TGF-β-induced activation of p38 MAPK in stromal fibroblasts, and this requirement involves interaction of CLIC4 with PPM1a, the selective phosphatase of activated p38. Genetic ablation of CLIC4 in primary fibroblasts prevents TGF-β-induced expression of α-SMA and extracellular matrix components. Conditioned media from CLIC4-overexpressing fibroblasts increases tumor cell migration/invasion and promotes EMT in a TGF-β-dependent manner. |
CLIC4 knockout primary fibroblasts, co-immunoprecipitation (CLIC4–PPM1a), p38 MAPK phosphorylation assay, conditioned medium experiments, migration/invasion assays |
Oncogene |
High |
23416981
|
| 2014 |
CLIC4 knockdown in HeLa and MDA-MB-231 cells decreases cell-matrix adhesion, cell spreading, and integrin signaling, while increasing cell motility. LPA stimulates recruitment of CLIC4 to β1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both internalization and serum/LPA-induced recycling of β1 integrin (but not EGFR). CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of β1 integrin trafficking. |
siRNA knockdown, live-cell imaging, co-immunoprecipitation, integrin internalization/recycling assays, Rab35 activity assay |
Journal of cell science |
High |
25344254
|
| 2015 |
Clic4 silencing or β-cell-specific knockout reduces cytokine-induced apoptosis, associated with increased expression and stability of Bcl-2, Bad, and phosphorylated Bad. Mass spectrometry of co-immunoprecipitated proteins found no direct association of CLIC4 with Bcl-2 family proteins, but CLIC4 co-purified with proteasome components, suggesting CLIC4 regulates Bcl-2/Bad stability via proteasomal degradation. |
siRNA knockdown, β-cell-specific knockout mice, co-immunoprecipitation with mass spectrometry, Bcl-2/Bad half-life measurement, cytokine-induced apoptosis assay |
Molecular metabolism |
Medium |
25830089
|
| 2016 |
CLIC4 is required for apical exocytosis and lumen formation in renal tubulogenesis. CLIC4-null embryos have impaired renal tubulogenesis; in MDCK 3D cultures, CLIC4 localizes to early endosomes, recycling endosomes, and apical transport carriers before reaching apical membrane at steady state. CLIC4 suppression impairs apical vesicle coalescence and lumen formation, rescued by Rab8 and Cdc42. CLIC4 selectively modulates retromer-mediated apical transport by negatively regulating branched actin formation on early endosomes. |
CLIC4-null mouse embryos, MDCK 3D culture lumenogenesis assay, live confocal imaging, Rab8/Cdc42 rescue experiments, retromer knockdown, actin branching analysis |
Nature communications |
High |
26786190
|
| 2016 |
Elevated CLIC4 induces expression of Smad7Δ, a novel truncated/alternatively spliced form of Smad7 missing 94 bp in exon 4 (predicted to lack the TGF-β inhibitory MH2 domain). Smad7Δ acts as a dominant-negative inhibitor of full-length Smad7, thereby further enhancing TGF-β signaling. TGF-β treatment also enhances Smad7Δ expression, amplified by CLIC4. |
CLIC4 overexpression in multiple cell types, RT-PCR splice variant identification, TGF-β reporter assay, proliferation assay, Smad phosphorylation analysis |
PloS one |
Medium |
27536941
|
| 2016 |
CLIC4 activates ERM proteins (ezrin, radixin, moesin) in glomerular endothelial cells; CLIC4 silencing reduces ERM phosphorylation and cytoskeletal association, and exogenous CLIC4 rescues ERM dephosphorylation. Mice lacking both CLIC4 and CLIC5 show profound reduction of glomerular EC ERM phosphorylation and develop glomerular capillary architectural defects, proteinuria, and glomerular cell proliferation. |
CLIC4/CLIC5 knockout mice (single and double), CLIC4 siRNA in cultured glomerular EC, exogenous CLIC4 expression rescue, ERM phosphorylation assay, histopathology |
American journal of physiology. Renal physiology |
High |
27582103
|
| 2017 |
Upon LPS stimulation of macrophages, CLIC4 translocates to the nucleus and cellular membrane (detected by confocal microscopy and cell fractionation). siRNA knockdown of CLIC4 in BMDMs impairs IL-1β transcription, ASC speck formation, and secretion of mature IL-1β in LPS/ATP-stimulated cells, showing CLIC4 participates in both the priming signal (IL-1β transcription) and the NLRP3 inflammasome activation step. |
Confocal microscopy, cell fractionation, siRNA knockdown, IL-1β transcription assay, ASC speck formation assay, ELISA for mature IL-1β |
The Journal of biological chemistry |
High |
28576828
|
| 2018 |
CLIC4 translocation to the plasma membrane upon LPA or EGF stimulation requires RhoA activation via the RhoA effector mDia2 and depends on F-actin polymerization. CLIC4 binds the G-actin-binding protein profilin-1 via residues required for CLIC4 trafficking. Profilin-1 silencing impairs agonist-induced CLIC4 trafficking and mDia2-dependent filopodium formation. CLIC4 knockdown increases filopodium formation (rescued by wild-type CLIC4 but not trafficking-incompetent CLIC4(C35A)), and CLIC4 accelerates LPA-induced filopodium retraction. |
Live-cell imaging, siRNA knockdown, co-immunoprecipitation, profilin-1 pull-down, mDia2 overexpression, filopodium quantification, CLIC4 mutant rescue |
The Journal of biological chemistry |
High |
30381396
|
| 2019 |
CLIC4 localizes to the cytokinetic cleavage furrow and midbody in a RhoA-dependent manner. Mutations of GST activity-related residues (C35A, F37D) abolish cell-cycle-dependent CLIC4 localization. CLIC4 interacts with ezrin, anillin, and ALIX at the cleavage furrow and midbody. CLIC4 facilitates ezrin activation at the cleavage furrow; conversely, inhibition of ezrin activation reduces CLIC4 translocation. CLIC4 and CLIC1 double knockout causes abnormal polar cortex blebbing and cleavage furrow regression, resulting in multinucleated cells. |
Live-cell imaging, immunofluorescence, CLIC1/CLIC4 knockout, site-directed mutagenesis (C35A, F37D), co-immunoprecipitation identifying ezrin/anillin/ALIX, ezrin phosphorylation assay |
Life science alliance |
High |
31879279
|
| 2019 |
Proteomic analysis of CLIC4-interacting proteins in pulmonary artery endothelial cells identified Arf6 GTPase-activating proteins and clathrin as binding partners. CLIC4 overexpression promotes Arf6-mediated reduction of gyrating clathrin and increased lysosomal targeting of BMPRII, reducing BMPRII expression and signaling. Arf6 siRNA, Arf inhibitor SecinH3, and clathrin-mediated endocytosis inhibitors restore BMPRII expression, but chloride channel inhibitor IAA-94 does not, placing CLIC4 in an Arf6-dependent trafficking pathway independent of its channel activity. |
Proteomic interactome (Co-IP/MS), Arf6 siRNA, pharmacological inhibitors (SecinH3, IAA-94, clathrin inhibitors), BMPRII expression and signaling assays, sugen/hypoxia and monocrotaline animal models |
Circulation research |
High |
30582444
|
| 2020 |
CLIC4 localizes to the cytokinetic cleavage furrow and is required for successful completion of mitotic cell division. CLIC4 recruits MST4 kinase (STK26) to the cleavage furrow and regulates ezrin phosphorylation, thereby remodeling the sub-plasma-membrane actomyosin network during cytokinesis. |
Live-cell imaging, CLIC4 knockdown, MST4 kinase recruitment assay, ezrin phosphorylation assay, co-immunoprecipitation |
Journal of cell science |
Medium |
32184265
|
| 2020 |
CLIC4 silencing enhances filopodium formation induced by constitutively active mDia2 mutants. CLIC4 binds the actin-regulatory region of mDia2 in vitro (pull-down assay), suggesting CLIC4 modulates the activity of the open conformation of mDia2 to inhibit filopodium formation. |
siRNA knockdown, in vitro pull-down (CLIC4 with mDia2 actin-regulatory region), filopodium quantification with constitutively active mDia2 mutants |
FEBS letters |
Medium |
32145706
|
| 2021 |
CLIC1 and CLIC4 in endothelial cells are required for S1P-induced activation of Rac1 downstream of S1PR1. CLIC1 and CLIC4 transiently translocate to the plasma membrane in response to S1P. Only CLIC1 (not CLIC4) was essential for S1P-induced RhoA activation downstream of S1PR2/S1PR3. CLIC1 and CLIC4 are not functionally interchangeable and are critical for S1P-induced endothelial barrier function. |
siRNA knockdown of CLIC1/CLIC4, live-cell imaging of membrane translocation, Rac1/RhoA GTPase activation assays, endothelial barrier assay, rescue experiments |
Science signaling |
High |
33879602
|
| 2022 |
CLIC4 is present in mitochondrial-associated membranes (MAMs) of cardiomyocytes. CLIC4 loss increases myocardial infarction and reduces cardiac function after ischemia-reperfusion injury. CLIC4-null cardiomyocytes show increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation. CLIC4 modulates ER and mitochondrial calcium homeostasis under physiological and pathological conditions. |
MAM fractionation, CLIC4-null mouse IR injury model, echocardiography, cardiomyocyte hypoxia-reoxygenation assay, calcium imaging, apoptosis assay |
Science advances |
High |
36269835
|
| 2022 |
FTO-mediated m6A demethylation stabilizes CLIC4 mRNA; FTO depletion increases m6A methylation on CLIC4 mRNA and reduces its stability, leading to decreased CLIC4 protein. |
MeRIP-RT-qPCR, mRNA stability assay, RNA-seq, FTO knockdown/overexpression |
Cell death discovery |
Medium |
35397614
|
| 2022 |
CLIC4 deletion from murine breast tumor cells using CRISPR enhances ROS accumulation, sensitizes cells to H2O2-induced apoptosis, and is associated with mitochondrial hyperactivity (increased membrane potential, organelle enlargement, increased superoxide). In the absence of CLIC4, H2O2-induced apoptosis involves degradation of Bcl2 and UCP2. CLIC4 therefore maintains redox homeostasis and mitochondrial function, consistent with a glutaredoxin-like enzymatic activity. |
CRISPR knockout, ROS measurement, mitochondrial membrane potential assay, apoptosis assay, transcriptomic profiling, Bcl2/UCP2 protein analysis |
The Journal of biological chemistry |
Medium |
35863434
|
| 2023 |
CLIC4 is required for thrombin/PAR1-mediated RhoA activation, ERM phosphorylation, and endothelial barrier disruption. Thrombin promotes CLIC4 (but not CLIC1) relocalization to HUVEC membranes. Endothelial-specific CLIC4 deletion in mice reduces lung edema and microvascular permeability induced by PAR1 activating peptide. CLIC1 was not required for thrombin-mediated barrier disruption but contributed to barrier recovery. |
CLIC4/CLIC1 siRNA knockdown, endothelial-specific conditional Clic4 knockout mice (PAR1 peptide-induced lung permeability), RhoA activation assay, ERM phosphorylation assay, endothelial barrier assay |
Arteriosclerosis, thrombosis, and vascular biology |
High |
37317855
|
| 2025 |
CLIC4 is expressed on the plasma membrane of sperm cells and is required for IAA-94-sensitive chloride currents (genetic ablation of CLIC4 eliminates these currents). CLIC4 regulates cell volume during sperm maturation without altering membrane potential, motility, or acrosome reaction. CLIC4 channel activity in sperm is modulated by Protein Kinase C (PKC). |
Patch-clamp electrophysiology, CLIC4-null mouse sperm, IAA-94 pharmacology, PKC modulation assay, sperm morphology and functional assays |
BMC biology |
High |
41715063
|
| 2025 |
CLIC4 C-terminus (not N-terminus) encodes CLIC4-specific functions required for S1P-induced Rac1 activation in endothelial cells. The CLIC4 N-terminus encodes determinants for S1P-induced plasma membrane relocalization, but is dispensable for Rac1 activation when the C-terminus is targeted to the membrane via a heterologous signal. The postulated ion channel and thiol-transferase (GST) activities of CLIC4 are NOT required for Rac1 activation. |
Structure-function mutagenesis (N/C-terminal domain swaps, heterologous membrane targeting), Rac1 activation assay, live-cell imaging of plasma membrane translocation, endothelial cell siRNA rescue experiments |
bioRxivpreprint |
Medium |
|