Journal: The Journal of Biological Chemistry

Article Title: Adaptor Protein LRAP25 Mediates Myotonic Dystrophy Kinase-related Cdc42-binding Kinase (MRCK) Regulation of LIMK1 Protein in Lamellipodial F-actin Dynamics *

doi: 10.1074/jbc.M114.588079

Figure Lengend Snippet: MRCK and LRAP25 are required for AlF4−-mediated LIMK1 activation and lamellipodial localization. A, B16-F1 cells with or without a prior 20-min AlF4− treatment were lysed and subjected to immunoprecipitations (IP) using anti-IgG or anti-MRCK antibody as indicated. Immunoprecipitated complexes were assayed for MRCK kinase activity using GST-RMLC as substrate. The 32P autoradiograph is shown. The immunoprecipitates were probed for MRCK (left top panel). Mean values derived from three independent experiments are presented (±S.E.) (right panel). Unpaired Student's t test indicates a significant difference between samples (* denotes p < 0.01). B, B16-F1 cells with or without prior 20 min AlF4− treatment were lysed and subjected to immunoprecipitations using anti-IgG or anti-LIMK1 antibody as indicated. Immunoprecipitated complexes were assayed for LIMK1 kinase activity using cofilin as substrate. The 32P autoradiograph is shown. The immunoprecipitates were probed for LIMK1 (left top panel). Mean values from three independent experiments are presented (±S.E.; n = 3) (right panel). Unpaired Student's t test indicates a significant difference between samples (* denotes p < 0.01). C, B16-F1 cells were treated with AlF4− for 10 min, followed by addition of either DMSO, 5 μm chelerythrine chloride (Che), 5 μm Y-27632 or a combination of Che and Y-27632 for 30 min. Treated cells were lysed and subjected to immunoprecipitations using anti-LIMK1 antibody and subsequently assayed for LIMK1 kinase activity using cofilin as substrate. The 32P autoradiograph is shown. The immunoprecipitates were probed for LIMK1 (left top panel). Mean values from three independent experiments are presented (±S.E.; n = 3) (right panel). Dunnett's multiple comparison test was used after one-way analysis of variance to generate p values (* denotes p < 0.05). D, B16-F1 cells transfected with control (Ctrl), MRCK, siLRAP25-1, or siLRAP25-2 siRNAs were treated with AlF4− and later immunostained for LIMK1-GFP, endogenous MRCKα, and LRAP25 as indicated. Boxed regions are magnified. Scale bar, 10 μm. Characterization of B16-F1 cells stably expressing LIK1-GFP is shown. Parental wild-type and the stable LIMK1-GFP cells were subjected to pull down (PD) with GFP affinity beads. Pulldown products were probed with either anti-GFP or anti-LIMK1 antibodies as indicated. Note that the GFP antibody recognized LIMK1-GFP present in both the total lysate and the pull down, although the LIMK1 antibody only detected LIMK-GFP present at a higher concentration in the pulldown fraction due to its low level in the lysate (bottom panel). E, B16-F1 cells with or without 10 min AlF4− treatment were added with either DMSO, 5 μm Che, 5 μm Y-27632 or a combination of Che and Y-27632 for 30 min. Lysates of treated cells were probed for endogenous Ser(P)-3-cofilin and cofilin. Quantification of relative Ser(P)-3-cofilin levels is shown (±S.E.; n = 3) (bottom panel). Newman-Keuls multiple comparison test was used after one-way analysis of variance to generate p values. * denotes p < 0.05; **, denotes p < 0.01. F, B16-F1 cells transfected with control or combined MRCKα and β siRNAs were treated with AlF4− before lysis. Lysates were probed with anti-MRCKα, anti-MRCKβ, anti-β-actin, anti-Ser(P)-3-cofilin, or anti-cofilin antibodies.

Article Snippet: Sources of primary antibodies used in immunoblotting are as follows: polyclonal rabbit anti-FLAG (Sigma F7425) and monoclonal anti-FLAG M2-peroxidase (HRP) (Sigma A8592); monoclonal anti-HA F7-HRP (Santa Cruz Biotechnology sc-7392; 0.05–0.1 μg/ml); monoclonal anti-GST (Sigma G1160); rabbit anti-MRCKα ( 23 ); rabbit anti-MRCKβ ( 25 ); mouse monoclonal anti-MRCKα ( 25 ); anti-MYO18A ( 25 ); anti-LIMK1 and anti-pLIMK1(Thr(P)-508) (Cell Signaling Technology); anti-cofilin (Cytoskeleton Inc.); anti-Ser(P)-3-cofilin (Cell Signaling Technology 3311) and rabbit anti-phosphomyosin light chain 2 (Ser(P)-19) (Cell Signaling Technology 3675); mouse monoclonal anti-paxillin (ECM Biosciences PM1071); anti-β-actin (C4) (Santa Cruz Biotechnology sc-47778); rabbit polyclonal anti-ARPC2 (Upstate 07-227); and anti-Ser(P)-3-cofilin (4321) for immunostaining was a gift from J. Bamburg from Colorado State University.

Techniques: Activation Assay, Immunoprecipitation, Activity Assay, Autoradiography, Derivative Assay, Transfection, Stable Transfection, Expressing, Concentration Assay, Lysis

Journal: The Journal of Biological Chemistry

Article Title: Adaptor Protein LRAP25 Mediates Myotonic Dystrophy Kinase-related Cdc42-binding Kinase (MRCK) Regulation of LIMK1 Protein in Lamellipodial F-actin Dynamics *

doi: 10.1074/jbc.M114.588079

Figure Lengend Snippet: LRAP25 depletion affects cell polarization and migration. A, B16-F1 cells were transfected with control siRNA or siLRAP25-1. Cells were treated with AlF4− and subjected to cell morphology analysis. Graph shows the percentage of B16-F1 cells exhibiting morphology corresponding to each of the following categories: small and polarized, enlarged and polarized, or irregular and nonpolarized. β-Actin-GFP expression from co-transfected plasmid marks transfectants for cell count (data not shown). Mean values derived from three independent experiments are shown (±S.E.; n ≥250 for each category). Unpaired Student's t test indicates a significant difference between samples (** denotes p < 0.01) (top panel). Representative cells in each category are marked by red asterisks (phalloidin-stained). B, B16-F1 cells were transfected with control siRNA, siLRAP25-1, plus a GST expression vector or siLRAP25-1 plus a FLAG-tagged siRNA-resistant LRAP25 expression vector. Graph shows the percentage of cells exhibiting morphology categorized as in A (n ≥150 for each category). C, B16-F1 cells were transfected with control siRNA or siLRAP25-1 and treated with AlF4−. Cells were later fixed and stained for F-actin (phalloidin) and paxillin. Cells were transfected with β-Actin-GFP to mark transfectants and lamellipodia. Scale bar, 10 μm. Boxed regions are magnified and shown on the right. D, kymograph analyses showing cell edge advancement dynamics taken from B16-F1 cells transfected with control (Ctrl) siRNA or siLRAP25-1. Dotted yellow lines mark cell edges. Arrowhead indicates the transition point between retraction and protrusion captured during image recording of LRAP25-depleted cells undergoing intermittent retractions (bottom panel). LRAP25-depleted cells displaying both polarized and nonpolarized irregular morphology were analyzed. Red lines indicate locations used to generate kymographs. Red dots indicate line origin (top panel). D and T denote distance and time, respectively. Scale bar, 10 μm. E, average cell edge advancement speed derived from B16-F1 cells transfected with siRNAs as in D. Mean values derived from two independent experiments are shown (±S.E.; n ≥17). Newman-Keuls multiple comparison test was used after one-way analysis of variance to generate p values (**** denotes p < 0.0001). F, migration of B16-F1 cells transfected with control siRNA or siLRAP25-1 was measured in a transwell assay using filters with pore diameters of 5 μm. Graph shows the percentage of cells migrated to the lower chamber derived from three independent experiments (±S.E.). G, model of MRCK complex regulation of LIMK1 and cofilin. Rac activation in response to extracellular signal targets MRCK complex to the cell membrane. Activation of MRCK by lipid hydrolysis (diacylglycerol) results in phosphorylation/activation of LIMK1, which in turn causes phosphorylation/inactivation of the F-actin depolymerizing/severing factor cofilin. Activation of the cofilin phosphatase SSH-1L in response to Rac activation results in dephosphorylation of cofilin. Coordination of the activities of the kinases and phosphatases are required to maintain the dynamic phosphorylation regulation of cofilin that is important for lamellipodial F-actin regulation. In the process of cell protrusion, MRCK translocates from the lamellipodium to the lamella with a corresponding change in complexity. The translocation is believed to be important for coordinating cytoskeletal regulations taking place in the two connected regions. Lp, lamellipodium; Lm, lamella; DAG, diacylglycerol.

Article Snippet: Sources of primary antibodies used in immunoblotting are as follows: polyclonal rabbit anti-FLAG (Sigma F7425) and monoclonal anti-FLAG M2-peroxidase (HRP) (Sigma A8592); monoclonal anti-HA F7-HRP (Santa Cruz Biotechnology sc-7392; 0.05–0.1 μg/ml); monoclonal anti-GST (Sigma G1160); rabbit anti-MRCKα ( 23 ); rabbit anti-MRCKβ ( 25 ); mouse monoclonal anti-MRCKα ( 25 ); anti-MYO18A ( 25 ); anti-LIMK1 and anti-pLIMK1(Thr(P)-508) (Cell Signaling Technology); anti-cofilin (Cytoskeleton Inc.); anti-Ser(P)-3-cofilin (Cell Signaling Technology 3311) and rabbit anti-phosphomyosin light chain 2 (Ser(P)-19) (Cell Signaling Technology 3675); mouse monoclonal anti-paxillin (ECM Biosciences PM1071); anti-β-actin (C4) (Santa Cruz Biotechnology sc-47778); rabbit polyclonal anti-ARPC2 (Upstate 07-227); and anti-Ser(P)-3-cofilin (4321) for immunostaining was a gift from J. Bamburg from Colorado State University.

Techniques: Migration, Transfection, Expressing, Plasmid Preparation, Cell Counting, Derivative Assay, Staining, Transwell Assay, Activation Assay, De-Phosphorylation Assay, Translocation Assay

Journal: Oncotarget

Article Title: Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells

doi: 10.18632/oncotarget.25961

Figure Lengend Snippet: Cells were treated with 1% BSA-DMEM (vehicle) or 10 -5 M ALN for 2-8 hours (A) . PC-3 cells were treated with 10 -11 to 10 -5 M ALN for 8 hours to study the effect on the level of total cofilin (B) . The experiments were repeated 3 times. Cofilin immunostaining intensity was analyzed from control and 10 -10 M ALN-treated cells (C) . * p < 0.05 versus vehicle.

Article Snippet: The sections were stained with polyclonal rabbit anti-phospho-Histone-3 (Cell Signaling Technology, Danvers, MA, USA), monoclonal rat anti-CD34 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and polyclonal rabbit anti-cofilin antibody (Abcam, Cambridge, MA, USA) o/n at +4°C, followed by biotin-labeled anti-rat IgG (DAKO Denmark A/S, Glostrup, Denmark), or anti-rabbit IgG secondary antibodies (Sigma-Aldrich, St. Lois, MO, USA).

Techniques: Immunostaining, Control

Journal: Oncotarget

Article Title: Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells

doi: 10.18632/oncotarget.25961

Figure Lengend Snippet: Nude mice were injected s.c . with 10 6 PC-3 cells and treated with i.p. injections of ALN (1 mg/kg, 5 days/week) for 4 weeks before sacrifice. Cofilin index was decreased in PC-3 s.c. tumors of the nude mice treated with ALN compared with tumors of vehicle-treated mice. Cofilin index was calculated by multiplying staining intensity with percentage of cofilin-positive cells. Bar represents 100 μm, * p < 0.05 versus vehicle (A) . pQCT analysis shows that bone mineral density increased in ALN-treated mice. The measurement was done as an intrinsic control for ALN efficacy and biological activity (B) .

Article Snippet: The sections were stained with polyclonal rabbit anti-phospho-Histone-3 (Cell Signaling Technology, Danvers, MA, USA), monoclonal rat anti-CD34 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and polyclonal rabbit anti-cofilin antibody (Abcam, Cambridge, MA, USA) o/n at +4°C, followed by biotin-labeled anti-rat IgG (DAKO Denmark A/S, Glostrup, Denmark), or anti-rabbit IgG secondary antibodies (Sigma-Aldrich, St. Lois, MO, USA).

Techniques: Injection, Staining, Control, Activity Assay

Journal: Oncotarget

Article Title: Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells

doi: 10.18632/oncotarget.25961

Figure Lengend Snippet: PC-3 cells were seeded on 6-well plates or glass slides and transfected with 50, 100 and 200 nM cofilin 1 siRNA or with control siRNA as described in Materials and Methods. After 60 hours, the cells were lysed for Western blotting to detect the levels of total and p-cofilin (A) , fixed on glass slides and immunostained for cofilin (B) , or detached and incubated for 48 hours in an invasion assay (C) . After invasion assay, cells were fixed, stained and counted as described in Materials and Methods. * p < 0.05, ** p < 0.01 versus control. The experiments were repeated 3 times.

Article Snippet: The sections were stained with polyclonal rabbit anti-phospho-Histone-3 (Cell Signaling Technology, Danvers, MA, USA), monoclonal rat anti-CD34 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and polyclonal rabbit anti-cofilin antibody (Abcam, Cambridge, MA, USA) o/n at +4°C, followed by biotin-labeled anti-rat IgG (DAKO Denmark A/S, Glostrup, Denmark), or anti-rabbit IgG secondary antibodies (Sigma-Aldrich, St. Lois, MO, USA).

Techniques: Transfection, Control, Western Blot, Incubation, Invasion Assay, Staining

Journal: Oncotarget

Article Title: Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells

doi: 10.18632/oncotarget.25961

Figure Lengend Snippet: PC-3 cells were transiently transfected with a cofilin expression vector including human myc as a tag gene (PC-3/cofilin cells) or with a control vector (PC-3/mock cells). The cells were treated with 10 -4 -10 -6 M ALN for 24 h and analyzed with Western blots for levels of total cofilin (A) . Success of transfection and loading of protein were assessed on stripped blots by immunodetection of myc (as a transfection control) and tubulin (as a loading control). Results are shown as relative cofilin level (normalized to tubulin and myc). PC-3/mock and PC-3/cofilin cells were pretreated with 10 -4 M ALN for 24 h. Then, medium was changed to 1%BSA-DMEM and migration was studied in a wound density assay up to 92 h (B) . ALN inhibited migration of PC-3/mock cells but did not affect the migration of PC-3/cofilin cells. Results are expressed as mean values together with standard deviation. Mean changes over time were compared with hierarchical linear mixed models suitable for repeated measures. In addition, differences of model based means were compared at each time point and they are shown in four time points (24h, 48h, 72h and 92h).

Article Snippet: The sections were stained with polyclonal rabbit anti-phospho-Histone-3 (Cell Signaling Technology, Danvers, MA, USA), monoclonal rat anti-CD34 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and polyclonal rabbit anti-cofilin antibody (Abcam, Cambridge, MA, USA) o/n at +4°C, followed by biotin-labeled anti-rat IgG (DAKO Denmark A/S, Glostrup, Denmark), or anti-rabbit IgG secondary antibodies (Sigma-Aldrich, St. Lois, MO, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Immunodetection, Migration, Standard Deviation

Journal: bioRxiv

Article Title: Vector analysis of steerable mechanical tension across nuclear lamina

doi: 10.1101/462275

Figure Lengend Snippet: (A) MCF-7 cells were treated with ADM, H 2 O 2 , and CHX separately. The samples were stained for both β-actin and α-tubulin. The cells were also stained with DAPI to localize the nuclei. The images were captured through confocal laser microscopy after immunofluorescence staining (n = 10). (B) Intracellular nanoparticle size distribution in MCF-7 cells separately treated with ADM, H 2 O 2 , and CHX (n = 10). (C) MCF-7 cell intracellular osmotic pressure was measured via osmometery after the cells were exposed to ADM, H 2 O 2 , and CHX. (**: 0.001 < p < 0.05, ns: p > 0.05, Tukey-b test, n = 10). (D) P-cofilin, cofilin, actin, p-stathmin, stathmin, and tubulin levels in MCF-7 cells that had been treated with ADM, H 2 O 2 , or CHX (n = 6). (E) In the H 2 O 2 group, the ΔpNOP could be divided into two parts by comparing the total intensity of the protein nanoparticles, generated from MF and MT depolymerization, respectively (versus , line 1, row 2). Scale bar, 10 μm. All error bars represent SEM.

Article Snippet: Antibodies were purchased from commercial sources: rabbit anti-lamin B1 antibody (13435S, Cell Signaling Technology, Temecula, USA), rabbit anti-β-actin antibody (4970P, Cell Signaling Technology, Temecula, USA), mouse anti-tubulin-α antibody (T5168, Boster, Wuhan, China), rabbit anti-vimentin antibody (5741S, Cell Signaling Technology, Temecula, USA), rabbit anti-GFP antibody (2956S, Cell Signaling Technology, Temecula, USA), mouse anti-phospho-cofilin (Ser24) (bs-10252R, Bioss, Woburn, USA), mouse anti-cofilin antibody (PB0035, Boster, Wuhan, China), rabbit anti-phospho-stathmin 1 (Ser38) (bs-3432R, Bioss, Woburn, USA), and mouse anti-stathmin 1 antibody (BA1417, Boster, Wuhan, China).

Techniques: Staining, Microscopy, Immunofluorescence, Generated

Journal: International Journal of Molecular Medicine

Article Title: The marine natural product, dicitrinone B, induces apoptosis through autophagy blockade in breast cancer

doi: 10.3892/ijmm.2022.5186

Figure Lengend Snippet: DB does not affect the pH or the hydrolytic function of lysosomes. (A) Fluorescence images of MCF7 and MDA-MB-231 cells transfected with double fluorescent mRFP-GFP-LC3 lentivirus and treated with DB, CQ alone or in combination for 6 h. Scale bars, 10 µ m. (B) Fluorescence microscopy with AO staining. MCF7 and MDA-MB-231 cells were treated with 20 µ M DB or 60 µ M CQ for 6 h and then stained with AO. The red fluorescence represents the acidic vesicles. Scale bars, 20 µ m. (C) Flow cytometry for LysoTracker Red in MCF7 and MDA-MB-231 cells treated with DB (0, 5, 10 and 20 µ M) for 6 h. (D) Representative western blots and corresponding protein quantification plots of CTSD and CTSB protein expression in MCF7 and MDA-MB-231 cells treated with 20 µ M DB for 6 h. Cofilin was used as a loading control. DB, dicitrinone B; RFP, red fluorescent protein; GFP, green fluorescent protein; LC3, microtubule associated protein 1 light chain 3; AO, acridine orange; CQ, chloroquine; DB, dicitrinone B; CTSD, cathepsin D; CTSB, cathepsin B.

Article Snippet: 7074P2 and 7076P2, respectively) were purchased from Cell Signaling Technology, Inc. Cathepsin D (CTSD; cat. no. BM1577; mouse), β-actin (cat. no. BM3873; rabbit), cofilin (cat. no. PB9033; rabbit) and cathepsin B (CTSB; cat. no. A01456-3, rabbit) antibodies were purchased from Wuhan Boster Biological Technology, Ltd. FITC-linked goat anti-rabbit IgG (cat. no. ZF-0311) was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Puromycin was purchased from InvivoGen (cat. no. ant-pr-1).

Techniques: Fluorescence, Transfection, Microscopy, Staining, Flow Cytometry, Western Blot, Expressing, Control