Journal: bioRxiv

Article Title: Two independent translocation modes drive neural stem cell dissemination into the human fetal cortex

doi: 10.1101/2025.01.08.631865

Figure Lengend Snippet: (A) Quantification of IST amplitude in bRG cells following treatment with DMSO, nocodazole (1 μ M) or blebbistatin (10 μ M) in cortical organoids (N=3 organoid batches, 329 bRG cells, weeks 9-12). (B) Live imaging of mitotic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (week 8-11). shRNA plasmids co-express GFP. (C) Live imaging of mitotic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (pcw 16-20). (D) Live imaging of in vitro interphasic human bRG cells expressing control or LIS1 shRNA constructs. (E) Quantification of IST amplitude in in vitro interphasic human bRG cells expressing control or LIS1 shRNA constructs (N=3 experiments, 520 bRG cells). (F) Quantification of IST amplitude in in vitro interphasic human bRG cells expressing control or KASH constructs, in the presence of DMSO or blebbistatin (10 μ M) (N=3 experiments, 1198 bRG cells). Yellow arrowheads indicate bRG cell soma, and green and red arrowheads indicate daughter cells. Data are presented as mean values +/− SD. Scale bar = 20 µm. All live imaging montages are in hours:minutes. **p<0,01; ***p<0,001, ns: non-significant by two-tailed unpaired t-tests.

Article Snippet: The following plasmids were used in this study: MSCV-IRES-GFP (Tannishtha Reya, Addgene 20672); VSVG (a gift from P. Benaroch), Human EZR shRNA (TF308420, Origene), Human STK10 shRNA (TF320540, Origene), Human SLK shRNA (TG320620, Origene), Human DYNC1H1 shRNA (TL313335, Origene), Human RDX shRNA (TL309884, Origene), Human MSN shRNA (TL311375, Origene), Human ECT2 shRNA (TL304854, Origene), Human VIM shRNA (TL308419, Origene), Human PAFAH1B1 (LIS1) shRNA (TL310628, Origene), Dominant Negative KASH.

Techniques: Imaging, Expressing, Control, shRNA, Construct, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: Two independent translocation modes drive neural stem cell dissemination into the human fetal cortex

doi: 10.1101/2025.01.08.631865

Figure Lengend Snippet: (A) Live imaging of interphasic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (week 8-11). shRNA plasmids co-express GFP. (B) Live imaging of interphasic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (pcw 16-18). (C) Quantification of IST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (N=3 organoid batches, 899 bRG cells, week 8-11). Two independent shRNA plasmids were used for each knockdown. (D) Quantification of MST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (N=3 organoid batches, 899 bRG cells, week 8-11). (E) Quantification of IST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (N=3 fetal samples, 385 bRG cells, pcw 16-20). (F) Quantification of MST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (N=3 fetal samples, 385 bRG cells, pcw 16-20). (G) Live imaging of interphasic human bRG cells expressing GFP in control cortical organoids and two different patient-derived LIS1-mutated organoids (week 8-11). (H) Quantification of IST amplitude in control cortical organoids and two different patient-derived LIS1-mutated organoids (N=3 organoid batches, 397 bRG cells, week 8-11). (I) Quantification of MST amplitude in control cortical organoids and two different patient-derived LIS1-mutated organoids (N=3 organoid batches, 397 bRG cells, week 8-11). (J) Immunostaining for SOX2 and Nesprin-2 in cortical organoids expressing GFP or the KASH dominant negative together with GFP (week 9). Red arrows indicate nuclear envelope of construct-expressing cells. (K) Live imaging of interphasic human bRG cells expressing control or KASH constructs in human cortical organoids (week 8). KASH plasmid co-expresses GFP. (L) Live imaging of interphasic human bRG cells expressing control or KASH constructs in human fetal tissue (pcw 16). KASH plasmid co-expresses GFP. (M) Quantification of IST amplitude in human bRG cells expressing control or KASH constructs in human cortical organoids (N=3 organoid batches, weeks 8-11, 201 bRG cells). (N) Quantification of MST amplitude in human bRG cells expressing control or KASH constructs in human cortical organoids (N=3 organoid batches, weeks 8-11, 201 bRG cells). (O) Quantification of IST amplitude in human bRG cells expressing control or KASH constructs in human fetal tissue (N=2 fetal samples, pcw 16-18, 40 bRG cells). (P) Quantification of MST amplitude in human bRG cells expressing control or KASH constructs in human fetal tissue (N=2 fetal samples, pcw 16-18, 40 bRG cells). Yellow arrowheads indicate bRG cell soma, and green and red arrowheads indicate daughter cells. Data are presented as mean values +/− SD. Scale bar = 20 µm. All live imaging montages are in hours:minutes. **p<0,01; ****p<0,0001, ns: non-significant by two-tailed unpaired t-tests.

Article Snippet: The following plasmids were used in this study: MSCV-IRES-GFP (Tannishtha Reya, Addgene 20672); VSVG (a gift from P. Benaroch), Human EZR shRNA (TF308420, Origene), Human STK10 shRNA (TF320540, Origene), Human SLK shRNA (TG320620, Origene), Human DYNC1H1 shRNA (TL313335, Origene), Human RDX shRNA (TL309884, Origene), Human MSN shRNA (TL311375, Origene), Human ECT2 shRNA (TL304854, Origene), Human VIM shRNA (TL308419, Origene), Human PAFAH1B1 (LIS1) shRNA (TL310628, Origene), Dominant Negative KASH.

Techniques: Imaging, Expressing, Control, shRNA, Construct, Knockdown, Derivative Assay, Immunostaining, Dominant Negative Mutation, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Two independent translocation modes drive neural stem cell dissemination into the human fetal cortex

doi: 10.1101/2025.01.08.631865

Figure Lengend Snippet: (A) Immunostaining for SOX2 and p-ERM in mitotic bRG cell from week 8 cortical organoid. (B) Live imaging of in vitro mitotic human bRG cells expressing control, Moesin, Radixin, Ezrin or Vimentin shRNA constructs. (C) Quantification of IST amplitude in in vitro interphasic human bRG cells expressing control, Moesin, Radixin, Ezrin or Vimentin shRNA constructs. Two independent shRNA plasmids were used for each knockdown (N=7 control samples and N=3 sample per shRNA condition, 522 bRG cells). (D) Live imaging of mitotic human bRG cells expressing control, Moesin or Vimentin shRNA constructs in human cortical organoids (week 9). (E) Live imaging of mitotic human bRG cells expressing control, Moesin or Vimentin shRNA constructs in human fetal tissue (pcw 20). (F) Quantification of MST amplitude in human bRG cells expressing control, Moesin or Vimentin shRNA constructs in human cortical organoids (N=3 organoid batches, 453 bRG cells, weeks 8-12). (G) Quantification of IST amplitude in human bRG cells expressing control, Moesin or Vimentin shRNA constructs in human cortical organoids (N=3 organoid batches, 453 bRG cells, weeks 8-12). (H) Quantification of MST amplitude in human bRG cells expressing expressing control, Moesin or Vimentin shRNA constructs in human fetal tissue (N=3 fetal samples, 529 bRG cells, pcw 14-20). (I) Quantification of IST amplitude in human bRG cells expressing expressing control, Moesin or Vimentin shRNA constructs in human fetal tissue N=3 fetal samples, 529 bRG cells, pcw 14-20). (J) Schematic representation of the molecular mechanisms driving IST and MST. Yellow arrowheads indicate bRG cell soma, and green and red arrowheads indicate daughter cells. Data are presented as mean values +/− SD. Scale bar = 20 µm. All live imaging montages are in hours:minutes. *p<0,05; **p<0,01, ns: non-significant by two-tailed unpaired t-tests.

Article Snippet: The following plasmids were used in this study: MSCV-IRES-GFP (Tannishtha Reya, Addgene 20672); VSVG (a gift from P. Benaroch), Human EZR shRNA (TF308420, Origene), Human STK10 shRNA (TF320540, Origene), Human SLK shRNA (TG320620, Origene), Human DYNC1H1 shRNA (TL313335, Origene), Human RDX shRNA (TL309884, Origene), Human MSN shRNA (TL311375, Origene), Human ECT2 shRNA (TL304854, Origene), Human VIM shRNA (TL308419, Origene), Human PAFAH1B1 (LIS1) shRNA (TL310628, Origene), Dominant Negative KASH.

Techniques: Immunostaining, Imaging, In Vitro, Expressing, Control, shRNA, Construct, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: Two independent translocation modes drive neural stem cell dissemination into the human fetal cortex

doi: 10.1101/2025.01.08.631865

Figure Lengend Snippet: (A) Live imaging of an in vitro GBM cell (line U3123) performing IST. (B) Live imaging of an in vitro GBM cell (line U3123) performing MST. (C) Quantification of the fraction of cells performing IST, in 9 GBM lines and compared to in vitro bRG cells (N=3 replicates per line, 1130 cells). (D) Quantification of the fraction of cells performing MST, in 9 GBM lines and compared to in vitro bRG cells (N=3 replicates per line, 1130 cells). (E) Immunostaining for bRG markers SOX2 and HOPX in U3123 GBM line. (F) Live imaging of U3123 GBM line during interphase, treated with DMSO, nocodazole (1 μ M) or blebbistatin (10 μ M). (G) Quantification of IST amplitude following treatment with DMSO, nocodazole (1 μ M) or blebbistatin (10 μ M) in U3123 GBM line (N=3 experiments, 274 GBM cells). (H) Live imaging of U3123 GBM line during mitosis, treated with DMSO, nocodazole (1 μ M) or blebbistatin (10 μ M). (I) Quantification of MST amplitude following treatment with DMSO, nocodazole (1 μ M) or blebbistatin (10 μ M) in U3123 GBM line (N=3 experiments, 274 GBM cells). (J) Live imaging of interphasic U3123 GBM cells expressing control, LIS1 shRNA or KASH dominant negative constructs. (K) Quantification of IST amplitude in U3123 GBM cells expressing control or LIS1 shRNA constructs. (N=3 experiments, 359 GBM cells). (L) Quantification of IST amplitude in U3123 GBM cells expressing control or KASH dominant negative constructs (N=3 experiments, 300 GBM cells). (M) Live imaging of mitotic U3123 GBM cells expressing control, Moesin or Vimentin shRNA constructs. (N) Quantification of MST amplitude U3123 GBM cells expressing control, Moesin or Vimentin shRNA (N=3 experiments, 149 GBM cells). Yellow arrowheads indicate bRG cell soma, and green and red arrowheads indicate daughter cells. Data are presented as mean values +/− SD. Scale bar = 20 µm. All live imaging montages are in hours:minutes. **p<0,01; ***p<0,001; ****p<0,0001, ns: non-significant by two-tailed unpaired t-tests.

Article Snippet: The following plasmids were used in this study: MSCV-IRES-GFP (Tannishtha Reya, Addgene 20672); VSVG (a gift from P. Benaroch), Human EZR shRNA (TF308420, Origene), Human STK10 shRNA (TF320540, Origene), Human SLK shRNA (TG320620, Origene), Human DYNC1H1 shRNA (TL313335, Origene), Human RDX shRNA (TL309884, Origene), Human MSN shRNA (TL311375, Origene), Human ECT2 shRNA (TL304854, Origene), Human VIM shRNA (TL308419, Origene), Human PAFAH1B1 (LIS1) shRNA (TL310628, Origene), Dominant Negative KASH.

Techniques: Imaging, In Vitro, Immunostaining, Expressing, Control, shRNA, Dominant Negative Mutation, Construct, Two Tailed Test

Journal: PLoS ONE

Article Title: Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma

doi: 10.1371/journal.pone.0244069

Figure Lengend Snippet: Using the SEQC patients’ datasets in the R2 Genomics Analysis and Visualization Platform ( http://r2.amc.nl ), event-free survival (top) and overall survival (bottom) curves were generated for (A) EZRIN and (B) MAP2 (Microtubule-associated protein-2) expression in all NB stages patients. Patients’ numbers (n) are shown in parentheses.

Article Snippet: To knock-down ezrin expression, cells were transfected with the silencing Ezrin shRNA lentiviral plasmid (shEzr) (a pool of three target-specific lentiviral vector plasmids by Santa Cruz Biotechnology, Dallas, TX, USA) or with the non-silencing control shRNA plasmid A (shNS) (Santa Cruz Biotechnology).

Techniques: Generated, Expressing

Journal: PLoS ONE

Article Title: Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma

doi: 10.1371/journal.pone.0244069

Figure Lengend Snippet: Immunofluorescence analysis of CHL1 and ezrin plasma membrane localization on (A) HTLA-230 and (B) IMR-32 cells using anti-CHL1 (green) (1) or anti-ezrin (red) (2) antibodies. HTLA-230 in photo A3 were double stained for CHL1 and ezrin (yellow, merge). IMR-32 in photo B3 expressed the transfected pCEFL-CHL1 molecule. Cells were counterstained with DAPI to visualize nuclei (blue) (Scale bars: 10 μm) (C) IMR-32 and HTLA-230 cells were lysed and subjected to Western blot (WB) analysis with anti-CHL1, anti-ezrin (Ezr), anti-radixin (Rdx), and anti-moesin (Moe) antibodies. Only HTLA-230 cell line displayed expression of endogenous CHL1 protein, whereas the three ERM proteins (Ezr, Rdx, and Moe) were equally expressed in both cell lines, and each of them was used as a loading control. (D) Cells lysates were subjected to each anti-ERM protein immunoprecipitation (IP) followed by anti-CHL1 Western blot or to anti-CHL1 immunoprecipitation followed by each anti-ERM protein Western blot. (E-F) Cell lysates from IMR-32 (as CHL1 negative control), and from HTLA-230 transfected with shNS (as non-silencing control) or shEzr, underwent Western blot analysis with anti-CHL1 and each anti-ERM protein antibody. HTLA-230 cells were lysed 7 days after transfection. 85–90% reduction in total ezrin levels was achieved compared to the non-silenced control, considered as 100%, whereas radixin and moesin levels did not change and they were used as loading controls. Data are representative of three independent experiments ± SD (*** p < 0.001). (G) Cell lysates from HTLA-230 transfected with shNS or shEzr were subjected to each anti-ERM protein immunoprecipitation followed by anti-CHL1 Western blot. All the immunoprecipitation results are representative of three independent experiments.

Article Snippet: To knock-down ezrin expression, cells were transfected with the silencing Ezrin shRNA lentiviral plasmid (shEzr) (a pool of three target-specific lentiviral vector plasmids by Santa Cruz Biotechnology, Dallas, TX, USA) or with the non-silencing control shRNA plasmid A (shNS) (Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Clinical Proteomics, Membrane, Staining, Transfection, Western Blot, Expressing, Control, Immunoprecipitation, Negative Control

Journal: PLoS ONE

Article Title: Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma

doi: 10.1371/journal.pone.0244069

Figure Lengend Snippet: (A-C) Proteins from HTLA-230 cells transfected with shNS (control) or shEzr and from IMR-32 cells transfected with the empty vector pCEFL (control) or cotransfected with pCEFL-CHL1 and shNS or shEzr, were lysed 7 days after transfection, subjected to Western blot, and probed with anti-CHL1, anti-ezrin, anti-MAP2, anti-p53, anti-phospho-p38 (P-p38), and anti-phospho-ERK 1/2 (P-ERK 1/2) antibodies. (D) 75–80% ezrin expression reduction was achieved in shEzr-IMR-32 cells compared to the non-silenced controls (pCEFL was considered as 100%) with actin used as the loading control. (B-E) Protein levels were quantified by densitometry, normalized to the control values (fold induction = 1) and to the contents of the loading control proteins (actin or total p38 and ERK 1/2), and then visualized by histograms. Data are representative of three independent experiments ± SD. (*** p < 0.001).

Article Snippet: To knock-down ezrin expression, cells were transfected with the silencing Ezrin shRNA lentiviral plasmid (shEzr) (a pool of three target-specific lentiviral vector plasmids by Santa Cruz Biotechnology, Dallas, TX, USA) or with the non-silencing control shRNA plasmid A (shNS) (Santa Cruz Biotechnology).

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma

doi: 10.1371/journal.pone.0244069

Figure Lengend Snippet: Morphological characteristics of cells plated 48 hours after transfection and photographed three days later. (A) HTLA-230 cells transfected with shNS (1–2) or shEzr (3–4): shEzr-HTLA-230 grew faster and lost the neurite-like extensions detectable in control cells (see arrows in photo 2). (D) IMR-32 cells transfected with the negative control pCEFL (1–2) or cotransfected with pCEFL-CHL1 and shNS (3–4) or shEzr (5–6): over-expression of CHL1 in IMR-32 cells strongly reduced growth rate and induced neural differentiation with protrusion of neurite-like extensions (see arrows in photo 4), whereas the co-silencing of ezrin clearly reduced CHL1 effects. (Magnification 4x and 40x). (B-E) Histograms report the percentages of cells extending neurite-like protrusions. Data are representative of three independent experiments ± SD (*** p <0.001). (C-F) HTLA-230 or IMR-32 cells were plated 48 hours after transfection or cotransfection and cultured for 4 days. Every day, cells were trypsinized and counted. Data are representative of three independent experiments ± S.D. (* p < 0.05; *** p < 0.001).

Article Snippet: To knock-down ezrin expression, cells were transfected with the silencing Ezrin shRNA lentiviral plasmid (shEzr) (a pool of three target-specific lentiviral vector plasmids by Santa Cruz Biotechnology, Dallas, TX, USA) or with the non-silencing control shRNA plasmid A (shNS) (Santa Cruz Biotechnology).

Techniques: Transfection, Control, Negative Control, Over Expression, Cotransfection, Cell Culture

Journal: PLoS ONE

Article Title: Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma

doi: 10.1371/journal.pone.0244069

Figure Lengend Snippet: A migration assay was conducted for (A) HTLA-230 cells transfected with shNS or shEzr and for (B) IMR-32 cells transfected with pCEFL or cotransfected with pCEFL-CHL1 and shNS or shEzr. Cells were seeded 48 hours after transfection in the upper chamber of a Transwell insert: the ones which migrated to the lower side 24 hours later were detached and counted. (Three independent experiments ± S.D). (C) Colonies formed by HTLA-230 cells transfected with shNS or shEzr and by (D) IMR-32 cells transfected with pCEFL or cotransfected with pCEFL-CHL1 and shNS or shEzr. Cells were collected 48 hours after transfection and cultured in soft agar for 10 days. (Magnification 4x). Histograms show the mean colony number from three independent experiments ± S.D. (*** p < 0.001).

Article Snippet: To knock-down ezrin expression, cells were transfected with the silencing Ezrin shRNA lentiviral plasmid (shEzr) (a pool of three target-specific lentiviral vector plasmids by Santa Cruz Biotechnology, Dallas, TX, USA) or with the non-silencing control shRNA plasmid A (shNS) (Santa Cruz Biotechnology).

Techniques: Migration, Transfection, Cell Culture

Journal: PLoS ONE

Article Title: Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma

doi: 10.1371/journal.pone.0244069

Figure Lengend Snippet: The intra-cytoplasmic CHL1 C-terminal domain interacts with the N-terminal domain of ezrin, which links cytoskeletal actin through its C-terminal domain. This molecular binding activates MAP2 and p53 pathways and inhibits MAPK phosphorylation, inducing neuronal differentiation rather than cell proliferation and migration, thus conferring less aggressive features on NB (The CHL1 extracellular portion contains six Immunoglobulin (Ig)-like domains and five Fibronectin (FN) type III domains).

Article Snippet: To knock-down ezrin expression, cells were transfected with the silencing Ezrin shRNA lentiviral plasmid (shEzr) (a pool of three target-specific lentiviral vector plasmids by Santa Cruz Biotechnology, Dallas, TX, USA) or with the non-silencing control shRNA plasmid A (shNS) (Santa Cruz Biotechnology).

Techniques: Binding Assay, Phospho-proteomics, Migration

Journal: Journal of Cancer

Article Title: Nudifloside, a Secoiridoid Glucoside Derived from Callicarpa nudiflora, Inhibits Endothelial-to-Mesenchymal Transition and Angiogenesis in Endothelial Cells by Suppressing Ezrin Phosphorylation

doi: 10.7150/jca.91566

Figure Lengend Snippet: Effects of NDF on the Ezrin in TGF-β1-treated EA.hy 926 cells. A: the effects of NDF on expression of Ezrin and p-Ezrin; B: Effects of NDF on PODXL-Ezrin interaction by immunoprecipitation while anti-Ezrin antibody was used as sedimental protein; C: Effects of NDF on PODXL-Ezrin axis activities by immunoprecipitation while anti-PODXL antibody was used as sedimental protein. *p<0.05 and **p<0.01 compared with control; #p<0.05 and ##p<0.01 compared with TGF-β1; &p<0.05 and &&p<0.01 compared with NDF(5μM). Lane 1: control; lane 2: TGF-β1; lane 3: NDF (5μM); lane 4: NDF (20μM).

Article Snippet: When 70-80% confluency was reached, Ezrin siRNA (SignalSilence® Ezrin siRNA I, CellSignaling Technology) was added at a final concentration of 100 nM by siRNA Transfection Reagent (sc-29528; Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, Control

Journal: Journal of Cancer

Article Title: Nudifloside, a Secoiridoid Glucoside Derived from Callicarpa nudiflora, Inhibits Endothelial-to-Mesenchymal Transition and Angiogenesis in Endothelial Cells by Suppressing Ezrin Phosphorylation

doi: 10.7150/jca.91566

Figure Lengend Snippet: Effects of NDF on Ezrin in VEGF-treated EA.hy 926 cells. A: Effects of NDF on expression of Ezrin and p-Ezrin; B: Effects of NDF on PODXL-Ezrin interaction by immunoprecipitation while anti-Ezrin antibody was used as sedimental protein; C: Effects of NDF on PODXL-Ezrin interaction by immunoprecipitation while anti-PODXL antibody was used as sedimental protein. *p<0.05 and **p<0.01 compared with control; #p<0.05 and ##p<0.01 compared with VEGF. Lane 1: control; lane 2: VEGF; lane 3: NDF (5μM); lane 4: NDF (20μM).

Article Snippet: When 70-80% confluency was reached, Ezrin siRNA (SignalSilence® Ezrin siRNA I, CellSignaling Technology) was added at a final concentration of 100 nM by siRNA Transfection Reagent (sc-29528; Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, Control

Journal: Journal of Cancer

Article Title: Nudifloside, a Secoiridoid Glucoside Derived from Callicarpa nudiflora, Inhibits Endothelial-to-Mesenchymal Transition and Angiogenesis in Endothelial Cells by Suppressing Ezrin Phosphorylation

doi: 10.7150/jca.91566

Figure Lengend Snippet: Effects of NDF on TGF-β1-induced EndoMT and VEGF-induced angiogenesis when Ezrin was knocked-down in EA.hy 926 cells. Effects of NDF on invasion (A) and expression of EndoMT markers (B, C and D) in Ezrin-knocking-down EA.hy 926 cells at presence of TGF-β1; Effects of NDF on tube formation (E and F) , expression of angiogenic markers (G, H and I) in Ezrin-knocking-down EA.hy 926 cells at presence of VEGF. ##p<0.01 compared with CTRL; *p<0.05 and **p<0.01 compared with siRNA.

Article Snippet: When 70-80% confluency was reached, Ezrin siRNA (SignalSilence® Ezrin siRNA I, CellSignaling Technology) was added at a final concentration of 100 nM by siRNA Transfection Reagent (sc-29528; Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Autophagy

Article Title: Hypoxia-induced autophagy drives colorectal cancer initiation and progression by activating the PRKC/PKC-EZR (ezrin) pathway

doi: 10.1080/15548627.2019.1687213

Figure Lengend Snippet: Hypoxia induces EZR phosphorylation in an ATG5 -dependent manner. ( A-B ) Phosphorylation of EZR at Thr567 under normoxic (N) and hypoxic (H) culturing conditions in ( A ) T18 and ( B ) T6 TICs. Data are representative of at least four independent experiments. ( C-D ) Activation of EZR after ATG5 knockdown in ( C ) T18 and ( D ) T6 TIC cultures after 16 h of hypoxia. Quantification was performed based on four independent experiments (mean ± SD) and is shown on the right side of the respective graph. *p < 0.05. ( E-G ) Activation of EZR following silencing of ( E ) BNIP3 ( F ) BNIP3L and ( G ) BECN1 after 16 h of hypoxia in T18 TIC cultures. Data are representative of three independent experiments and similar results were obtained for T6 TICs. ( H-I ) Effect of PRKCA siRNA on EZR phosphorylation under normoxia and hypoxia (16 h) in ( H ) T6 and ( I ) T18 TICs. Data are representative of two independent experiments per TIC culture. ( J-K ) Effect of NSC305787 and NSC668394, two chemical inhibitors of PKC-mediated EZR phosphorylation in hypoxic (16 h) T6 TICs. Data are representative of at least two independent experiments. ( L ) Effect of Go6976, a chemical inhibitor of PRKC, on the phosphorylation of EZR in hypoxic (16 h) T6 TICs. Data are representative of three independent experiments.

Article Snippet: Ready-to-use lentiviral particles were used to generate spheroids with stable knockdowns of ATG5 (sc-41445-V), BECN1 (sc-29797-V), BNIP3 (sc-37451-V), BNIP3L (sc-37453-V), HIF1A (sc-35561-V), and EZR (sc-35349-V) via short hairpin RNA or respective control vectors (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Activation Assay, Knockdown

Journal: Autophagy

Article Title: Hypoxia-induced autophagy drives colorectal cancer initiation and progression by activating the PRKC/PKC-EZR (ezrin) pathway

doi: 10.1080/15548627.2019.1687213

Figure Lengend Snippet: Inhibition of autophagy reverses hypoxia-mediated phenotype in patient-derived TICs. ( A ) Sphere-forming capacity (%) was determined by carrying out single cell assays in ATG5 -defective (sh ATG5 ) and control (scr) T18 TICs under 10 d of normoxia (N) and hypoxia (H). Representative figure of 4 independent experiments. Data are presented as mean with a confidence interval of 0.95, p-value calculated using a chi-square test. ( B ) Colony numbers under hypoxia (H) for control (scr) and ATG5 -defective (sh ATG5 ) TICs derived from patients T6, T18, and T20. Data are presented as a representative figure of at least 2 independent experiments per TIC culture, mean ± SD, ***p < 0.001. ( C-E ) Colony numbers after 10–14 d under normoxia (N) and hypoxia (H) for control (scr) and BECN1 -defective (sh BECN1 ) TICs derived from patients ( C ) T6, ( D ) T18 and ( E ) T20. ( F-G ) Colony numbers after 10–14 d of hypoxia for small inhibitor of EZR (NSC668394) treated ( F ) T6 and ( G ) T18 TICs. ( H-I ) Colony numbers after 10–14 d of hypoxia and Go6976 treatment for ( H ) T6 and ( I ) T18 TICs. For 3D clonogenic assays of C-I, data are representative of at least 3 independent experiments, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Ready-to-use lentiviral particles were used to generate spheroids with stable knockdowns of ATG5 (sc-41445-V), BECN1 (sc-29797-V), BNIP3 (sc-37451-V), BNIP3L (sc-37453-V), HIF1A (sc-35561-V), and EZR (sc-35349-V) via short hairpin RNA or respective control vectors (Santa Cruz Biotechnology).

Techniques: Inhibition, Derivative Assay, Control

Journal: Autophagy

Article Title: Hypoxia-induced autophagy drives colorectal cancer initiation and progression by activating the PRKC/PKC-EZR (ezrin) pathway

doi: 10.1080/15548627.2019.1687213

Figure Lengend Snippet: ATG5 deficiency limits tumor initiation and progression in vivo . ( A ) In vivo tumor growth after subcutaneous injection of 10,000 T18-derived TIC cells and subsequent intraperitoneal treatment with either CQ or PBS (n = 12 tumors per group). Data are presented as means tumor volumes (mm 3 ) ± SEM. ( B ) T18 tumor weight after treatment with CQ or PBS, respectively. Data are presented as mean tumor weights (mg) ± SEM. ( C ) In vivo tumor growth after subcutaneous injection of 10,000 T20-derived TIC cells with/without stable knockdown of ATG5 , n = 5 mice/group. ( D-E ) Tumor ( D ) growth and ( E ) weight in immune-deficient mice (NSG mice), 8 weeks after subcutaneous injection of 10,000 cells derived from primary T18 TICs, following a stable knockdown of ATG5 or respective control vector; n = 6/group. A representative ATG5 protein expression of extracted T18-derived xenografts is shown. All data are shown as mean ± SEM. Two-way ANOVA followed by the Tukey post-hoc test was used to test for statistical significance in A, C and D. Paired t-tests were used to assess significance in B, and E. *p < 0.05, ***p < 0.001 ( F ) p-EZR immunofluorescence staining in extracted xenograft tumors from (D). Scale bar: 100 µm. Representative images are shown (left panel) as well as the quantification of p-EZR-positive area (right panel). Four mice out of the six from (D) were used, as the remaining two were used in (G). Data are presented as mean ± SD, *p < 0.05. ( G ) Serial in vivo limiting dilution experiment with T18 TICs, following stable knockdown of ATG5 or the corresponding control vector. After an initial round of xenotransplantation, two extracted tumors (from mice appearing in [D]) were dissociated and different cell doses (100, 250 and 500 cells) were subcutaneously injected into secondary recipient NSG mice. Secondary tumor incidence was evaluated after 12 weeks. Statistical significance was assessed with a Chi-square test ** p < 0.01. ( H ) Potential mechanism of action. Hypoxia within a solid tumor leads to activation of autophagy, especially in TICs. Kinases, such as PRKCA, are activated and further induce phosphorylation of EZR on Thr567 in TICs. EZR, most likely through MAPK14/p38 activation, leads to increased self-renewal capacity of TICs in vitro and in vivo.

Article Snippet: Ready-to-use lentiviral particles were used to generate spheroids with stable knockdowns of ATG5 (sc-41445-V), BECN1 (sc-29797-V), BNIP3 (sc-37451-V), BNIP3L (sc-37453-V), HIF1A (sc-35561-V), and EZR (sc-35349-V) via short hairpin RNA or respective control vectors (Santa Cruz Biotechnology).

Techniques: In Vivo, Injection, Derivative Assay, Knockdown, Control, Plasmid Preparation, Expressing, Immunofluorescence, Staining, Activation Assay, Phospho-proteomics, In Vitro

Journal: Autophagy

Article Title: Hypoxia-induced autophagy drives colorectal cancer initiation and progression by activating the PRKC/PKC-EZR (ezrin) pathway

doi: 10.1080/15548627.2019.1687213

Figure Lengend Snippet: The relevance of the hypoxia/autophagy/EZR pathway in human TICs. ( A ) Immunofluorescence staining and colocalization in human tumor tissues (from six patients) for CA9 (a downstream target of HIF1A), BNIP3L, MAP1LC3A, p-EZR, and POU5F1 (refer to Fig. S7 for patient characteristics including TNM staging and HE stainings). The specificity of all used antibodies was carefully validated (please see Material and Methods and Supplementary data). Scale bar: 100 µm. ( B ) Staining correlation in human CRC tissues. Measures were standardized (z-score) for each patient. Dot colors indicate different patients. A repeated measure correlation test was performed in order to account for the within-individual association of paired measures (using the rmcorr package in R; see Material and Methods). The rmcorr r coefficient and the Holm adjusted p-values are reported on each plot.

Article Snippet: Ready-to-use lentiviral particles were used to generate spheroids with stable knockdowns of ATG5 (sc-41445-V), BECN1 (sc-29797-V), BNIP3 (sc-37451-V), BNIP3L (sc-37453-V), HIF1A (sc-35561-V), and EZR (sc-35349-V) via short hairpin RNA or respective control vectors (Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: CLIC5A interacts directly with ezrin, radixin, and moesin. A . yeast two-hybrid (Y2H) assay. Plates contain yeast colonies expressing untagged CLIC5A from the “bait” vector and ezrin 432-586 , radixin 432-583 , or moesin 432-577 from the “prey” vector. Colony growth on double-deficient medium (DDO, lacking leucine and tryptophan) indicates both vectors are present. Blue colonies indicate induced α-galactosidase activity in the presence of X-α-gal (DDO/X) due to direct interactions between bait and prey proteins. Growth on plates containing the antibiotic aureobasidin A (DDO/X/A) indicate a direct interaction between bait and prey proteins resulting in Aureobasidin A resistance. The inset for DDO/X/A plates containing CLIC5A/radixin 432-583 or CLIC5A/moesin 432-577 represents a 2× digital magnification (representative of 3 biologically separate experiments). B , WB with anti(α)-ezrin, α-radixin and α-moesin antibodies of total cell lysates (input) and GST or GST-CLIC5A pulldowns (PD) from untransfected COS-7 cells. Each of the three lanes for input, GST pulldown, and GST-CLIC5A pulldown are from three biologically distinct experiments. C , semiquantitative Y2H mapping of interactions between distinct ERM and CLIC5A constructs. Input and GST-CLIC5A pulldown (PD) is shown (representative of three independent experiments). D , microscale thermophoresis (MST) left panel: total protein stain of recombinant, purified GST-His6x, GST-His6x-CLIC5A, and untagged ezrin 432-586 proteins. Right panel: normalized thermophoresis-induced change in fluorescence for fluorescently labeled GST-His6x or GST-His6x-CLIC5A in the presence of increasing concentrations of purified ezrin 482-586 (H549N/T567E). The calculated affinity (Kd) between GST-His6x-CLIC5A and ezrin 432-586 (H549N/T567E) was 29 ± 12 μM (mean ± SD, n = 3 biological replicates). E , WB of HA-ezrin 297-586 , HA-ezrin 432-586 , and HA-ezrin 432-570 produced by in vitro transcription/translation before (input) and after pulldown (PD) by immobilized GST-CLIC5A. F , α-GFP WB of transiently expressed full-length GFP-ezrin 1-586 , GFP-ezrin 432-586 , and GFP-ezrin 432-570 in COS-7 cells and pulled from the cell lysates by immobilized GST or GST-CLIC5A (PD) (representative of three biologically independent experiments). G , coimmunoprecipitation of HA-ezrin 1-586 , HA-ezrin 432-586 , and HA-ezrin 432-570 coexpressed with GFP-CLIC5A in COS-7 cells (representative of three biological replicates). CLIC, chloride intracellular channel; DDO, double dropout; ERM, ezrin, radixin, and moesin; GST, glutathione S-transferase; WB, Western blot.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Y2H Assay, Expressing, Plasmid Preparation, Activity Assay, Construct, Microscale Thermophoresis, Staining, Recombinant, Purification, Fluorescence, Labeling, Produced, In Vitro, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: ERM knockdown partially displaces CLIC5A from its peripheral location. A , localization of transiently expressed GFP-CLIC5A in living HeLa cells transfected with nonspecific siRNA (Scr siRNA) or triple ezrin, moesin, and radixin siRNA (ERM siRNA) (representative of three biologically distinct experiments). B , quantification of the peripheral: cytoplasmic GFP-CLIC5A ratio in living HeLa cells transfected with GFP-CLIC5A and with Scr siRNA or triple ERM siRNA from a single experiment (Student’s t test). C , mean peripheral: cytoplasmic GFP-CLIC5A in living HeLa cells transfected with GFP-CLIC5A and Scr siRNA or triple ERM siRNA (mean ± SD, n = 3 biologically independent experiments, Student’s t test). D : WB with anti-ezrin, anti-radixin, and anti-moesin antibodies showing individual and triple ezrin, radixin, and moesin siRNA-mediated knockdown of endogenous ezrin, radixin and moesin in HeLa cells. E : Quantification of ERM knockdown from three independent experiments. (One-way ANOVA for ezrin abundance: F = 21.17; p < 0.001, for moesin abundance: F = 11.50; p < 0.0009, for radixin abundance: F = 17.68; p < 0.0002, and for CLIC5A abundance: F = 1.564; p = 0.265. p values for post hoc Dunnett’s multiple comparisons are shown). CLIC, chloride intracellular channel.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Knockdown, Transfection

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: Phosphorylation enhances the CLIC5A/ezrin interaction. A , left panel: WB for endogenous ezrin and endogenous phosphorylated ERM proteins (pERM) in COS-7 cell lysates and GST-CLIC5A pulldowns (PD) from cells treated with vehicle or calyculin A (50 nM) for 30 min prior to lysis. Right panel: densitometric quantification (mean ± SD, n = 3 independent experiments, Student’s t test). B , right panel: α-GFP WB of total cell lysate (input) and GST or GST-CLIC5A pulldown (PD) from lysates of COS-7 transiently expressing GFP-ezrin 432-586 (WT), phosphorylation deficient GFP-ezrin 432-586 (T567A; “A”) or phosphomimetic GFP-ezrin 432-586 (T567D; “D”). Right panel: densitometric quantification of GST-CLIC5A pulldown of transiently expressed GFP-ezrin 432-586 (WT, A or D mutants; one-way ANOVA F = 5.42; p = 0.04, p values for post hoc Dunnett’s multiple comparisons are shown). C , confocal fluorescence microscopy of living HeLa cells transfected with RFP-CLC5A ( magenta ) and/or full-length GFP-ezrin 1-586 ( green ) without (WT) or with a phosphomimetic T567D mutation (the scale bar represents 12 μm). Cells were either transfected separately ( top row ) with RFP-CLIC5A or GFP-ezrin and then mixed 1:1 (control conditions) or they were cotransfected ( bottom row ) with RFP-CLIC5A and GFP-ezrin. The white arrows represent line plots analyzed for pixel intensity (representative of three biologically distinct experiments). D , line plot pixel intensity for RFP ( magenta ) and GFP ( green ) for lines in ( C ). E , quantification of the transiently expressed GFP-ezrin 1-586 (WT or T567D mutant) peripheral: cytoplasmic pixel intensity in the presence and absence of transiently expressed RFP-CLIC5A. Top panel: each data point represents a single cell from a single experiment (two-way ANOVA: interaction F = 19.3, p < 0.0001; CLIC5A effect F = 23.6, p < 0.0001; ezrin mutant versus WT effect F = 156.9; p < 0.0001, p values for pos t hoc Tukey’s multiple comparisons are shown). Bottom panel: each data point represents the mean for one of three biologically independent experiments. (Two-way ANOVA: interaction F = 28.8, p < 0.0007; CLIC5A effect F = 32.7, p < 0.0004; ezrin mutant versus WT effect F = 97.3; p < 0.0001, p values for post hoc Tukey’s multiple comparisons are shown). F , quantification of the RFP-CLIC5A peripheral: cytoplasmic pixel intensity in the presence or absence of transiently expressed GFP-ezrin1-586 (WT or T567D mutant). Top panel: each datapoint represents a single cell from a single experiment (one-way ANOVA F = 0.297, p = 0.744). Bottom panel: each data point represents the mean for one of three biologically independent experiments (one-way ANOVA F = 0.796, p = 0.488). G , Pearson correlation coefficient for colocalization of transiently expressed RFP-CLIC5A and GFP-ezrin (WT or T567D mutant). Right panel: each data point represents a separate image (each image containing 4–10 cells) from a single experiment (two-way ANOVA: interaction F = 13.51, p < 0.0007; separate versus cotransfection F = 1175, p < 0.0001; ezrin mutant versus WT effect F = 22.54; p < 0.0001, p values for post hoc Tukey’s multiple comparisons are shown). Right panel: each datapoint represents the mean from one of three biologically distinct experiments (mean ± SD, Student’s t test). CLIC, chloride intracellular channel; ERM, ezrin, radixin, and moesin; GST, glutathione S-transferase; WB, Western blot.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Phospho-proteomics, Lysis, Expressing, Fluorescence, Microscopy, Transfection, Mutagenesis, Control, Cotransfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: Indirect association of CLIC5A and Rac1. A , WB showing endogenous Rac1 and ezrin as well as transiently expressed GFP-CLIC5A in total cell lysates (TLC) and the same proteins captured by immobilized PAK-protein binding domain (PAK-PBD) from HeLa cells transiently expressing GFP or GFP-CLIC5A. The GFP-CLIC5A expressing cells were treated, or not, with the Rac1 inhibitor NSC23766 (100 μM × 10 min) (representative of three biologically distinct experiments). B , α-GFP WB of GST- or GST-CLIC5A pulldowns from lysates of COS-7 cells transiently expressing WT, constitutively active (L61) or dominant negative (N17) GFP-Rac1 constructs (representative of three independent experiments). C , live-cell imaging of HeLa cells transiently expressing RFP-CLIC5A (magenta) and WT GFP-Rac1 ( green ). The scale bar represents 10 μm. The white lines represent line plots analyzed in ( D ). Colocalization ( white ) in the merged image is observed at the dorsal cell periphery and in the perinuclear location (representative of three biologically distinct experiments). D , pixel intensity of line plots in ( C ) for RFP-CLIC5A ( magenta ) and GFP-Rac1 ( green ). E , Pearson correlation coefficients of all images from three biologically distinct experiments. 1, 2, and 3 = cotransfection of GFP-Rac1 with RFP-CLIC5A; 2’ and 3’ = separate transfection of GFP-Rac1 or RFP-CLIC5A followed by 1:1 mixing of cells. F , in vitro loading of purified, recombinant His-Rac1 with GTPγS in vitro . His-Rac1 (500 ng) was incubated with GDP or GTPγS followed by affinity capture of Rac1-GTPγS by immobilized PAK-PBD. Top panel: protein blot. Bottom panel: α-Rac1 WB. G , α-Rac1 WB of purified His-Rac1 and GST- or GST-CLIC5A pulldowns from cell-free solutions containing WT Rac1, Rac1-GTPγS, or Rac1-GDP (representative of three independent experiments). CLIC, chloride intracellular channel; GST, glutathione S-transferase; WB, Western blot.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Protein Binding, Expressing, Dominant Negative Mutation, Construct, Live Cell Imaging, Cotransfection, Transfection, In Vitro, Purification, Recombinant, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: ERM and Rac-1 activation are amplified by the CLIC5A/ezrin interaction. A , endogenous pERM abundance in lysates and detergent resistant pellets of COS-7 cells transfected with GFP-CLIC5A cDNA and increasing concentrations of GFP-ezrin 432-586 (T567D) cDNA . GFP-CLIC5A cDNA was kept constant and the GFP-CLIC5A: GFP-ezrin 432-586 (T567D) cDNA transfection ratio was 1:1, 1:2, 1:4, and 1:8. Left panel , representative WB. Right panel, densitometric quantification of the endogenous pERM: endogenous ezrin ratio (mean ± SD, n = 3 independent experiments, one-way ANOVA: F = 7.90; p = 0.0007. p values shown represent post hoc Dunnett’s multiple comparisons). B , pERM abundance in lysates and detergent resistant pellets of COS-7 cells transiently transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of GFP- e zrin 432-570 cDNA. Left panel , representative WB. Right panel, densitometric quantification of the endogenous pERM: endogenous ezrin ratio (mean ± S.D., n = 3 biologically independent experiments, two-way ANOVA: interaction F = 12.25; p = 0.01; CLIC5A effect: F = 39.63, p = 0.0002; ezrin 432-570 effect F = 22.58. p = 0.0014; p values shown represent post hoc Tukey’s multiple comparisons). C , endogenous Rac1 WB for COS-7 cell lysates and endogenous Rac1-GTP captured by PAK-PBD pulldown (PD) from COS-7 cells transiently transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of GFP-ezrin 432-586 (T567D) cDNA. Left panel, representative WB. Right panel: densitometric quantification of endogenous Rac1-GTP/total endogenous Rac1 (mean ± SD, n = 3 independent experiments, two-way ANOVA: interaction F = 6.32, p = 0.036; CLIC5A effect F = 29.25, p = 0.001; ezrin 432–586 effect F = 5.88; p = 0.042, p values for post hoc Tukey’s multiple comparisons are shown). D , PAK-PBD pulldown (PD) of endogenous Rac1-GTP from lysates of COS-7 cells transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of transiently expressed GFP-ezrin 432 - 570 cDNA. Left panel, representative WB. Right panel, densitometric quantification of endogenous Rac1-GTP/total endogenous Rac1 (mean ± S.D., n = 3 independent experiments, two-way ANOVA: interaction F = 0.029, p = 0.87; CLIC5A effect F = 62.17 p < 0.0001; ezrin 432–570 effect F = 0.012; p = 0.91, p values for post hoc Tukey’s multiple comparisons are shown). E , endogenous Rac1-GTP abundance in human glomerular endothelial cells (hGENs) determined by Rac1-GTP G-LISA. The cells were transduced with control adenoviral-vector (ad-Vector) or untagged CLIC5A cDNA in the same vector (ad- CLIC5A ) at an increasing multiplicity of infection (MOI) (mean ± SD, n = 3 independent experiments). F , representative WB of lysates from hGEN cells transduced with ad-Vector or ad- CLIC5A with or without ezrin-specific siRNA. G , change relative to baseline of Rac1-GTP in hGEN cells transduced with 30 MOI ad-Vector or ad- CLIC5A with or without ezrin-specific siRNA (mean ± SD, n = 4 independent experiments, two-way ANOVA: interaction F = 6.35, p = 0.027; CLIC5A effect F = 34.18, p < 0.0001; ezrin siRNA effect F = 6.86; p = 0.023, p values shown represent post hoc Tukey’s multiple comparisons). H , coimmunoprecipitation of endogenous Rho-GDI with endogenous ezrin in the presence and absence of transiently expressed GFP-CLIC5A. Left panel: representative α-Rho GDI, α-ezrin, and α-CLIC5A WB of lysates (input) and α-ezrin immunoprecipitates (IP). Right panel: Quantification of endogenous Rho-GDI and endogenous ezrin immunoprecipitated with α-ezrin antibodies (n = 3 biologically distinct experiments, mean ± SD, Student’s t test). CLIC, chloride intracellular channel; PBD, protein binding domain; pERM, phosphorylated ezrin, radixin, and moesin proteins; Rho-GDI, Rho guanine nucleotide dissociation inhibitor; WB, Western blot.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Activation Assay, Amplification, Transfection, Transduction, Control, Plasmid Preparation, Infection, Immunoprecipitation, Protein Binding, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: CLIC5A-dependent feed-forward ezrin activation loop. 1 , the soluble (inactive) conformation of ezrin, in which ezrin N and C termini self-associate with high affinity encounters plasma membrane PI(4,5)P 2 . 2 , a conformational change brought about by binding of the ezrin N-terminal FERM domain to membrane PI(4,5)P 2 unmasks the ezrin C-terminal domain which binds filamentous actin producing the open/active conformation of ezrin. 3 , this open ezrin conformation is subject to phosphorylation of T567 by PKC or Rho-dependent kinase (ROCK) enhancing actin binding. 4 , the actin-associated phosphorylated ezrin C terminus directly binds CLIC5A. 5 , CLIC5A binding stabilizes the open/active conformation of ezrin. 6 , open/active ezrin sequesters inhibitory Rho-GDI (GDI), removing it from Rac-GDP and resulting in spatially restricted Rac1-GTP generation. Open/active ezrin also binds NHERF1 (EBP50) and NHERF2. EBP50 can recruit the Rac GEF ARHGEF7 also known as β-Pix ( , ), which would also enhance Rac1-GTP formation in the absence of the GDI. Rac1-GTP is known to stimulate the localized generation of PI(4,5)P 2 by PI4P5 kinases. By stabilizing the active ezrin hub, CLIC5A promotes Rac1-GTP–stimulated accumulation of PI(4,5)P 2 which then serves to further activate ezrin. Thus, the interaction of CLIC5A with the open/active conformation of ezrin results in feed-forward amplification loop of ezrin-stimulated Rac1 activity and PI(4,5)P 2 generation, in turn serving to stabilize the local cortical actin cytoskeleton. CLIC, chloride intracellular channel; PI(4,5)P 2 , phosphatidylinositol 4,5 bisphosphate; Rho-GDI, Rho guanine nucleotide dissociation inhibitor.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Activation Assay, Clinical Proteomics, Membrane, Binding Assay, Phospho-proteomics, Amplification, Activity Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Ezrin/NF-κB Pathway Regulates EGF-induced Epithelial-Mesenchymal Transition (EMT), Metastasis, and Progression of Osteosarcoma

doi: 10.12659/MSM.906945

Figure Lengend Snippet: EGF promoted Ezrin phosphorylation and EMT, and silencing Ezrin suppressed EGF-induced EMT in osteosarcoma cells. GAPDH was used as internal control (* compared to the Con., P <0.05; # compared to EGF, P <0.05).

Article Snippet: Small interfering RNA (siRNA) silencing Ezrin (5′-GUG GGA UGC UCA AAG AUA ATT-3′) was designed and synthesized by GenePharma (Shanghai, China).

Techniques: Phospho-proteomics, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Ezrin/NF-κB Pathway Regulates EGF-induced Epithelial-Mesenchymal Transition (EMT), Metastasis, and Progression of Osteosarcoma

doi: 10.12659/MSM.906945

Figure Lengend Snippet: Ezrin was responsible for NF-κB activity in osteosarcoma cells. GAPDH was used as internal control (* comparing to the Con., P <0.05; # compared to EGF, P <0.05; & compared to siRNA+EGF, P <0.05).

Article Snippet: Small interfering RNA (siRNA) silencing Ezrin (5′-GUG GGA UGC UCA AAG AUA ATT-3′) was designed and synthesized by GenePharma (Shanghai, China).

Techniques: Activity Assay, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Ezrin/NF-κB Pathway Regulates EGF-induced Epithelial-Mesenchymal Transition (EMT), Metastasis, and Progression of Osteosarcoma

doi: 10.12659/MSM.906945

Figure Lengend Snippet: EGF induced EMT by Ezrin/NF-κB in osteosarcoma cells (* compared to the Con., P <0.05).

Article Snippet: Small interfering RNA (siRNA) silencing Ezrin (5′-GUG GGA UGC UCA AAG AUA ATT-3′) was designed and synthesized by GenePharma (Shanghai, China).

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Ezrin/NF-κB Pathway Regulates EGF-induced Epithelial-Mesenchymal Transition (EMT), Metastasis, and Progression of Osteosarcoma

doi: 10.12659/MSM.906945

Figure Lengend Snippet: Proliferation, migration and motility of MG63 and U20S with NF-κB inhibitor, silencing Ezrin and EGF treatments (X±SDs).

Article Snippet: Small interfering RNA (siRNA) silencing Ezrin (5′-GUG GGA UGC UCA AAG AUA ATT-3′) was designed and synthesized by GenePharma (Shanghai, China).

Techniques: Migration

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Ezrin/NF-κB Pathway Regulates EGF-induced Epithelial-Mesenchymal Transition (EMT), Metastasis, and Progression of Osteosarcoma

doi: 10.12659/MSM.906945

Figure Lengend Snippet: Suppressing Ezrin/NF-κB reversed EGF-induced morphological change and promotion of proliferation, migration, and motility of osteosarcoma cells (* compared to the Con., P <0.05).

Article Snippet: Small interfering RNA (siRNA) silencing Ezrin (5′-GUG GGA UGC UCA AAG AUA ATT-3′) was designed and synthesized by GenePharma (Shanghai, China).

Techniques: Migration

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Ezrin/NF-κB Pathway Regulates EGF-induced Epithelial-Mesenchymal Transition (EMT), Metastasis, and Progression of Osteosarcoma

doi: 10.12659/MSM.906945

Figure Lengend Snippet: Reducing Ezrin suppressed metastasis of EGF-injected MG63 and U20S xenografts of BALB/c-nu mice. ( A ) BALB/c-mu mouse with osteosarcoma xenografts. ( B ) Liver and lung weights of tumorigenic mice. ( C ) HPRT mRNA level in livers and lungs of tumorigenic mice. ( D ) E-cadherin, vimentin, and p65 protein levels in livers and lungs of tumorigenic mice. (HPRT, hypoxanthine guanine phosphoribosyl transferase; Con.+PBS, tumor xenografts and PBS injection; siRNA+PBS, tumor xenografts with silencing Ezrin and PBS injection; Con.+EGF, tumor xenografts and EGF injection; siRNA+EGF, tumor xenografts with silencing Ezrin and EGF injection; * compared to Con.+PBS, P <0.05; # compared to siRNA+PBS, P <0.05; & compared to Con.+EGF, P <0.05).

Article Snippet: Small interfering RNA (siRNA) silencing Ezrin (5′-GUG GGA UGC UCA AAG AUA ATT-3′) was designed and synthesized by GenePharma (Shanghai, China).

Techniques: Injection

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Ezrin/NF-κB Pathway Regulates EGF-induced Epithelial-Mesenchymal Transition (EMT), Metastasis, and Progression of Osteosarcoma

doi: 10.12659/MSM.906945

Figure Lengend Snippet: Liver and lung weight of mice with osteosarcoma (X±SDs) (g).

Article Snippet: Small interfering RNA (siRNA) silencing Ezrin (5′-GUG GGA UGC UCA AAG AUA ATT-3′) was designed and synthesized by GenePharma (Shanghai, China).

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Ezrin/NF-κB Pathway Regulates EGF-induced Epithelial-Mesenchymal Transition (EMT), Metastasis, and Progression of Osteosarcoma

doi: 10.12659/MSM.906945

Figure Lengend Snippet: Liver and lung HPRT of mice with osteosarcoma (X±SDs).

Article Snippet: Small interfering RNA (siRNA) silencing Ezrin (5′-GUG GGA UGC UCA AAG AUA ATT-3′) was designed and synthesized by GenePharma (Shanghai, China).

Techniques: