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: Cancer Research

Article Title: Aurora-A Regulation of Nuclear Factor-κB Signaling by Phosphorylation of IκBα

doi: 10.1158/0008-5472.can-06-2272

Figure Lengend Snippet: Figure 2. Aurora-A phosphorylation of InBa. A, HeLa cells transfected with plasmids expressing Aurora-A and IKKh, and treated with Aurora (VX-680) and IKKh (IKKIV) inhibitors alone or in combination. Cell lysates were analyzed for phosphorylated InBa (S32/36) levels by immunoblotting. Aurora-A, IKKh, and ezrin antibodies were used as controls. B, IKKh-deficient MEFs were transfected with empty vector or plasmids expressing Aurora-A, IKKh, or in combination in the presence or absence of proteasomal inhibitor MG132. Cell lysates were analyzed for phosphorylated InBa (S32/36) levels by immunoblotting. Aurora-A, IKKh, and ezrin antibodies were used as controls.

Article Snippet: Antibodies used were IAK1 (Aurora-A; BD Transduction Laboratories), V5 (PK; Serotec), a-tubulin and Flag (Sigma), PARP, Bcl-xL, and phospho-Ser 32/Ser36 InBa (Cell Signaling Technology), cyclin D1 and p53 (Oncogene), total InBa, ezrin, and MYC (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Phospho-proteomics, Transfection, Expressing, Western Blot, Plasmid Preparation

Journal: Cancer Research

Article Title: Aurora-A Regulation of Nuclear Factor-κB Signaling by Phosphorylation of IκBα

doi: 10.1158/0008-5472.can-06-2272

Figure Lengend Snippet: Figure 3. Aurora-A–induced phosphorylation of InBa at Ser32/Ser36 and its inhibition rescues TNFa-induced InBa degradation. A, HeLa cells cotransfected with plasmids expressing Aurora-A and empty vector, PK-InBa, PK-InBa-S32A, PK-InBa-S36A, or PK-InBa-S32/36A, respectively. Cell lysates were analyzed for phosphorylated InBa (S32/36) levels by immunoblotting (top). Anti-InBa antibody shows the endogenous and exogenous InBa (middle). Tubulin and MYC-Aurora-A used as controls (bottom). B, in MCF7 and MCF7-overexpressing MYC–Aurora-A cells, the levels of S32/S36-phosphorylated InBa were assessed by immunoblotting. Bottom, levels of InBa and Aurora-A, respectively. C, HeLa cells with or without TNFa were treated with VX-680 and IKKIV inhibitors towards Aurora-A and IKKh, respectively, and total InBa levels were assessed by immunoblotting. The relative levels of InBa in Aurora-A and IKKh-inhibited and control cell lysates were quantified by densitometry (bottom). Tubulin provides a loading control. D, HeLa cells cotransfected with plasmids expressing Aurora-A or empty vector and treated with MEK1 (PD98059), PI3K (LY294002), Aurora (VX-680), and IKKh (IKKIV) inhibitors, respectively. Cell lysates were analyzed for phosphorylated InBa (S32/36) and Aurora-A levels by immunoblotting. Ezrin used as a loading control.

Article Snippet: Antibodies used were IAK1 (Aurora-A; BD Transduction Laboratories), V5 (PK; Serotec), a-tubulin and Flag (Sigma), PARP, Bcl-xL, and phospho-Ser 32/Ser36 InBa (Cell Signaling Technology), cyclin D1 and p53 (Oncogene), total InBa, ezrin, and MYC (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Phospho-proteomics, Inhibition, Expressing, Plasmid Preparation, Western Blot, Control

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: eLife

Article Title: G-protein-coupled receptor signaling and polarized actin dynamics drive cell-in-cell invasion

doi: 10.7554/eLife.02786

Figure Lengend Snippet: ( A ) Immunolabeling of endogenous mDia1 (green) and phalloidin staining of F-actin (red) of a MCF10A cell undergoing entosis. Nuclei are labeled by DAPI (blue). Scale bar 5 μm. ( B ) Visualization of mDia1-GFP (green) and mCherry-LifeAct (red) localization at the invading cell rear in fixed and non-permeabilized HEK293 cells co-transfected with LPAR2 to trigger cell-in-cell invasion events. Merged image including bright-field and DAPI (blue) is shown in the right panel. Scale bar 5 μm. ( C ) Immunolabeling of endogenous Ezrin (green) and F-actin (red) of control and mDia1 siRNA-treated MCF10A cells. ( D ) MCF10A cell population after incomplete siRNA treatment against mDia1 showing mDia1 knockdown of the upper two cells (red only) and endogenous mDia1 detection of the lower three cells were labeled for mDia1 (green) and F-actin (red). Note the presence of mDia1 on cellular blebs, while the two upper mDia1-negative cells fail to bleb. 2 frames are shown from a confocal z-scan using a LSM 700 (Zeiss). ( E ) MCF10A cells treated with indicated siRNAs were analyzed for the number of blebbing cells (n = 3 ± SD, p<0.007, t test). ( F ) MCF10A cells pretreated for 40 min with 20 μM of the LPAR inhibitor Ki16425 before analysis of the number of blebbing cells (n = 3 ± SD, p<0.001, t test). ( G ) MCF10A cells expressing LifeAct-GFP (green) or LifeAct-mCherry (red) silenced for control or mDia1 respectively. White arrowheads in the first frame indicate red (siDia1) and green (siMOCK) cell in contact with a host cell. Red arrowhead indicates addition of 100 nM Latrunculin B (LatB) at time frame 104 min. ( H ) MCF10A cells treated with indicated siRNAs for 48 hr were analyzed for entosis (n = 3 ± SD analyzed by one way ANOVA followed by Dunnett's post-tests compared with siMOCK group). ( I ) HEK293 cells expressing Flag-LPAR2 to trigger cell-in-cell invasion events were treated with indicated siRNAs for 48 hr before analyzing entosis rates (n = 3 ± SD analyzed by One way ANOVA followed by Dunnett's post-tests compared with Flag-LPAR2 expressing siMOCK group). DOI: http://dx.doi.org/10.7554/eLife.02786.012

Article Snippet: Poly (2-hydroxyethyl methacrylate) (PolyHEMA) was purchased from Polysciences Inc. Antibodies against EDG4 were from Assay Biotechnology; LPAR receptors, Ezrin and LARG from Santa Cruz Biotechnology; PDZ-RhoGEF from IMGENEX; pMLC2 from Sigma and mDia1 from BD Biosciences. pCMV6-XL5 LPAR2 expression vector were purchased from OriGene (SC117226). pWPXL-based lentiviral expression vectors for H2B, LPAR2, Gα12, and Gα12Q/L were generated using standard PCR-based procedures or in the case of LifeAct-GFP were a kind gift from Oliver Fackler.

Techniques: Immunolabeling, Staining, Labeling, Transfection, Control, Knockdown, Expressing

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: British Journal of Cancer

Article Title: Co-targeting of FAK and MDM2 triggers additive anti-proliferative effects in mesothelioma via a coordinated reactivation of p53

doi: 10.1038/bjc.2016.331

Figure Lengend Snippet: NF2 regulates the interaction of FAK–p53 and MDM2–p53. ( A ) The NF2–FAK complex was evaluated in MESO257 by NF2 and FAK immunoprecipitation followed by FAK and NF2 immunoblotting. ( B ) Nuclear localisation of NF2, FAK, p53, and MDM2 was evaluated in MESO257 by immunoblotting. Poly(ADP-ribose) polymerase (PARP) is a nuclear localisation control, and GAPDH is a cytoplasmic control. ( C ) In MESO257 with stable NF2 shNRA expression, p53, NF2, FAK, MDM2, and p21 expression were evaluated by immunoblotting after FAK shRNA knockdown for 72 h. β -Actin staining is a loading control. p53 expression quantifications are standardised to the empty vector control. ( D ) Expression of p53 and p21 was evaluated in MESO257 cell membrane, cytoplasm, and nucleus with stable NF2 shNRA expression by immunoblotting. ( E ) Cell viability was evaluated by a cell titre Glo ATP-based luminescence assay in MESO257 with stable expressed NF2 shRNA , at 72 h post-infection with FAK shRNA . Data were normalised to empty lentivirus infections, and represent the mean values (±s.d.) from quadruplicate cultures. Statistically significant differences between untreated control and treatments or between vector control and FAK shRNA or NF2 shRNA and NF2+FAK shRNA are presented as * P <0.05, ** P <0.01.

Article Snippet: Human NF2 full-length cDNA expression plasmid (Catalogue: TC124024) was obtained from Origene (Rockville, MD, USA).

Techniques: Immunoprecipitation, Western Blot, Control, Expressing, shRNA, Knockdown, Staining, Plasmid Preparation, Membrane, Luminescence Assay, Infection

Journal: Journal of cell communication and signaling

Article Title: Ezrin regulates skin fibroblast size/mechanical properties and YAP-dependent proliferation.

doi: 10.1007/s12079-017-0406-6

Figure Lengend Snippet: Fig. 1 Ezrin knockdown decreases skin fibroblast size. Primary skin fibroblasts were transfected with non-specific control siRNA or Ezrin siRNAs (20 nM) for 48 h. a Ezrin mRNA levels. N = 4. b Ezrin protein levels. N = 3. c Ezrin immunostaining. Images represent four independent experiments. Bar = 100 μm. Enlargement of the boxed region is shown to the bottom (bars = 50 μm). d Cells were stained with CellTracker® fluorescent dye. Red fluorescence delineates cell cytoplasm; blue fluores- cence delineates nuclei. The relative cell surface areas were quantified by ImageJ. Bars = 50 μm. N = 3. e Cells were cultured in 3D type I collagen

Article Snippet: Ezrin #1 siRNA was purchased from Santa Cruz Biotechnology (SC535349, Santa Cruz, CA, UAS).

Techniques: Knockdown, Transfection, Control, Immunostaining, Staining, Fluorescence, Cell Culture

Journal: Journal of cell communication and signaling

Article Title: Ezrin regulates skin fibroblast size/mechanical properties and YAP-dependent proliferation.

doi: 10.1007/s12079-017-0406-6

Figure Lengend Snippet: Fig. 2 Ezrin knockdown decreases skin fibroblast mechanical properties. Primary skin fibroblasts were transfected with non-specific control siRNA or Ezrin siRNAs (20 nM). Cells were then cultured in 3D type I collagen gels for 48 h as described in Methods. Mechanical proper- ties were determined by atomic force microscopy (AFM) PeakForce Quantitative NanoMechanics mode and ana- lyzed by Nanoscope Analysis software. a Representative bottom-view bright field image of AFM. AFM cantilever positioned on the cells are shown (black circle). Arrow heads indicate cells. b Cell traction force (nN). Representative images of traction force (left). Horizontal bars indi- cate traction forces scales (nN). N = 4. c Tensile strength (Pa). Representative images of traction force (left). Horizontal bars indi- cate tensile strength scales (Pa). N = 4. d Deformation (nm). Representative images of defor- mation (left). Horizontal bars in- dicate deformation scales (nm). N = 4. Data are expressed as mean ±SEM, *p < 0.05 vs control si

Article Snippet: Ezrin #1 siRNA was purchased from Santa Cruz Biotechnology (SC535349, Santa Cruz, CA, UAS).

Techniques: Knockdown, Transfection, Control, Cell Culture, Microscopy, Software

Journal: Journal of cell communication and signaling

Article Title: Ezrin regulates skin fibroblast size/mechanical properties and YAP-dependent proliferation.

doi: 10.1007/s12079-017-0406-6

Figure Lengend Snippet: Fig. 3 Ezrin knockdown impairs YAP nuclear translocation in skin fibroblasts. Primary skin fibroblasts were transfected with non-specific control siRNA or Ezrin siRNAs (20 nM) for 48 h. a YAP immunostaining. Images represent five independent exper- iments. Bar = 100 μm. Enlargement of the boxed region is shown to the bottom (bar = 50 μm). b YAP protein levels in nucleus. c YAP protein levels in cytosols. d Phosphorylated YAP (Ser127) protein levels in nucleus. e Phosphorylated YAP (Ser127) protein levels in cytosols. YAP nuclear protein levels were nor- malized by lamin A/C as a load- ing control. YAP cytosol protein levels were normalized by SOD as a loading control. Protein levels were determined by Western blots. Insets show representative Western blots. N = 3. Data are expressed as mean±SEM, *p < 0.05 vs control si

Article Snippet: Ezrin #1 siRNA was purchased from Santa Cruz Biotechnology (SC535349, Santa Cruz, CA, UAS).

Techniques: Knockdown, Translocation Assay, Transfection, Control, Immunostaining, Western Blot

Journal: Journal of cell communication and signaling

Article Title: Ezrin regulates skin fibroblast size/mechanical properties and YAP-dependent proliferation.

doi: 10.1007/s12079-017-0406-6

Figure Lengend Snippet: Fig. 4 Ezrin knockdown inhibits YAP target gene expression in skin fibroblasts. Primary skin fibroblasts were transfected with non-specific control siRNA or Ezrin siRNAs (20 nM) for 48 h. a CCN1 and CCN2 mRNA levels. N = 3. b CCN1 and CCN2 protein levels. N = 3. (c and d) Cells were transfected with non-specific control siRNA (left bar) or Ezrin siRNAs (middle bar) or Ezrin siRNAs plus constitutively active YAP (right bar) for 48 h. c YAP protein levels. N = 3. d CCN1 and CCN2 protein levels. N = 3. Protein levels were determined by Western blots. Protein levels were normalized by β-actin as a load- ing control. Insets show repre- sentative Western blots. Data are expressed as mean±SEM, *p < 0.05 vs control si

Article Snippet: Ezrin #1 siRNA was purchased from Santa Cruz Biotechnology (SC535349, Santa Cruz, CA, UAS).

Techniques: Knockdown, Targeted Gene Expression, Transfection, Control, Western Blot

Journal: Journal of cell communication and signaling

Article Title: Ezrin regulates skin fibroblast size/mechanical properties and YAP-dependent proliferation.

doi: 10.1007/s12079-017-0406-6

Figure Lengend Snippet: Fig. 5 Ezrin knockdown inhibits fibroblasts proliferation via impaired YAP activity. Cells were transfected with non-specific control siRNA or Ezrin siRNAs or Ezrin siRNAs plus constitu- tively active YAP for two days. a Cells were harvested two days after transfection and 2 × 105 cells were cultured in 60 mm plates for one day (top panel) and 6 days (bottom panel) followed by stain with crystal violet blue dye to vi- sualize cells. N = 4. b To quantify crystal violet, methanol was added to solubilize the dye, followed by reading optical den- sity (O.D.540). Results are expressed as the mean ± SEM, N = 4, *p < 0.05 vs control si. c Cells were harvested two days after transfection and 2.5 × 105

Article Snippet: Ezrin #1 siRNA was purchased from Santa Cruz Biotechnology (SC535349, Santa Cruz, CA, UAS).

Techniques: Knockdown, Activity Assay, Transfection, Control, Cell Culture, Staining