Journal: Journal of Biological Chemistry

Article Title: The Neurofibromatosis Type 1 Gene Product Neurofibromin Enhances Cell Motility by Regulating Actin Filament Dynamics via the Rho-ROCK-LIMK2-Cofilin Pathway

doi: 10.1074/jbc.m503707200

Figure Lengend Snippet: FIGURE 4. Promotion of cofilin phosphorylation in neurofibromin-depleted cells. A, effect of neurofibromin depletion on the levels of phos- phocofilin. Either control or NF1 siRNA were trans- fected into HeLa cells. After 24 and 48 h, cells were harvested and examined for the levels of Ser(P)3- cofilin by immunoblotting analysis. B, neurofibro- min-depleted cells exhibited a high level of phos- phorylated cofilin. After 24 h of siRNA transfection inaserum-freecondition,HeLacellswerestimulated with 10% serum or 10 M LPA for the indicated peri- odsandthenexaminedforlevelsofSer(P)3-cofilinby immunoblotanalysis.C,enhancementofactinstress fiber formation in neurofibromin-depleted cells. After 24 h of siRNA transfection under a serum-free condition, HeLa cells were stimulated with serum or LPAattheindicatedtimesandthenstainedwithrho- damin phalloidin to visualize the actin cytoskeleton. D, the nonphosphorylatable cofilin S3A mutant sup- presses excessive stress fiber formation in neurofi- bromin-depleted cells. HeLa cells co-transfected with FLAG-cofilin S3A and either control or NF1 siRNA were stained with Alexa 488 phalloidin and anti-FLAG antibody at 24 h after transfection.

Article Snippet: Polyclonal antibodies against Akt, phospho-Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2, LIM kinase 1 (LIMK1), cofilin, and phospho-cofilin (Ser3) were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Control, Western Blot, Transfection, Mutagenesis, Staining

Journal: Journal of Biological Chemistry

Article Title: The Neurofibromatosis Type 1 Gene Product Neurofibromin Enhances Cell Motility by Regulating Actin Filament Dynamics via the Rho-ROCK-LIMK2-Cofilin Pathway

doi: 10.1074/jbc.m503707200

Figure Lengend Snippet: FIGURE 5. Cofilin phosphorylation in neurofi- bromin-depleted cells is induced via a LIMK2 pathway. A, reduction of LIMK1 and LIMK2 expression by siRNA. Immunoblot analysis of whole cell lysates extracted from cells transfected with control siRNA (lanes 1 and 2), LIMK1 siRNA (lanes 3 and 4), or LIMK2 siRNA (lanes 5 and 6). The asterisks indicate nonspecific bands. B, depletion of LIMK2 suppresses cofilin phosphorylation in neurofibromin-depleted cells. NF1 (lanes 3, 5, and 7) or control (lanes 2, 4, and 6) siRNA was co-trans- fected with LIMK1 (lanes 4 and 5) or LIMK2 (lanes 6 and 7) siRNA. The levels of Ser(P)3-cofilin were ana- lyzed by immunoblot after 24 h of siRNA transfec- tion. C, LIMK2 siRNA suppresses the NF1 siRNA-in- duced stress fiber formation. After 24 h of siRNA transfection, cells were stained with Alexa 488 phalloidin to visualize actin cytoskeleton. Exces- sive stress fiber formation in the cell body of neu- rofibromin-depleted cells was significantly sup- pressed by LIMK2 siRNA transfection (bottom panels). D, number of colonies that formed on Matrigel matrix. HeLa cells were transfected with indicated siRNA, and the number of colonies formed at 96 h after the replating was counted under the phase-contrast microscope. Each datum represents the average of three independ- ent experiments. Error bars represent S.D. Statisti- caldifferencesweredeterminedwithattest;*,p 0.001.

Article Snippet: Polyclonal antibodies against Akt, phospho-Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2, LIM kinase 1 (LIMK1), cofilin, and phospho-cofilin (Ser3) were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Expressing, Western Blot, Transfection, Control, Staining, Microscopy

Journal: Journal of Biological Chemistry

Article Title: The Neurofibromatosis Type 1 Gene Product Neurofibromin Enhances Cell Motility by Regulating Actin Filament Dynamics via the Rho-ROCK-LIMK2-Cofilin Pathway

doi: 10.1074/jbc.m503707200

Figure Lengend Snippet: FIGURE 6. Neurofibromin regulates cofilin-induced actin reorganization via Rho-ROCK pathway. A, ROCK inhibitor Y27632 suppresses NF1 siRNA-induced cofilin phosphoryl- ation. After 24 h of siRNA transfection, HeLa cells were treated with (lanes 3 and 4) or without 10 M Y27632 (lanes 1 and 2) for 1 h and then examined for levels of Ser(P)3-cofilin by immunoblot analysis. B, Y27632 suppresses NF1 siRNA-induced stress fiber formation. After 24 h of siRNA transfection, cells were treated with or without 10 M Y27632 and stained with Alexa 488 phalloidin. C, number of colonies that formed on Matrigel matrix. After transfecting HeLa cells with the indicated siRNA in the presence or absence of Y27632, the number of colonies formed in Matrigel at 96 h was counted under a phase-contrast microscope. Each datum represents the average of three independent experiments. Error bars represent S.D. Statistical differences were determined with a t test; *, p 0.001. D, N19RhoA suppresses NF1 siRNA-induced cofilin phosphorylation. NF1 or control siRNA was co-transfected with mock (lanes 1 and 2) or N19RhoA (a dominant negative form of RhoA) plasmid (lanes 3 and 4). The levels of Ser(P)3-cofilin were analyzed by immunoblot after 24 h. E, N19RhoA suppresses the NF1 siRNA-induced stress fiber formation. Cells treated as described in D were stained with Alexa 488 phalloidin (actin; green) and anti-hemagglutinin antibody (N19RhoA; red) after 24 h of siRNA transfection.

Article Snippet: Polyclonal antibodies against Akt, phospho-Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2, LIM kinase 1 (LIMK1), cofilin, and phospho-cofilin (Ser3) were purchased from Cell Signaling Technology.

Techniques: Transfection, Western Blot, Staining, Microscopy, Phospho-proteomics, Control, Dominant Negative Mutation, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: The Neurofibromatosis Type 1 Gene Product Neurofibromin Enhances Cell Motility by Regulating Actin Filament Dynamics via the Rho-ROCK-LIMK2-Cofilin Pathway

doi: 10.1074/jbc.m503707200

Figure Lengend Snippet: FIGURE 7. NF1-mediated regulation of Rho sig- naling requires Ras activation but is not trans- duced through three major Ras-mediated downstream pathways. A, expression of DN-Ras(S17N) partially suppressed the cofilin phosphorylationinducedbyNF1depletion.NF1or control siRNA was co-transfected with mock (lanes 1 and 2) or DN-Ras (a dominant negative form of Ras) plasmid (lanes 3 and 4). At 24 h after transfec- tion, the levels of Ser(P)3-cofilin, cofilin, and Ras were analyzed by immunoblot. It is noted that cofilin phosphorylation levels in NF1 siRNA- treated cells were partially suppressed when cells were transfected with DN-Ras (lane 4). B, DN-Ras(S17N) partially suppresses the NF1 siRNA-induced stress fiber formation. Cells treated asdescribedinAwerestainedwithAlexa488phal- loidin and anti-Ras antibody after 24 h of siRNA transfection. C, neurofibromin-depleted cells exhibit elevated activation of Ras and its down- stream signaling. After 24 h of siRNA transfection under a serum-free condition, cells were stimu- lated with 10% serum (left panels) or 10 M LPA (right panels) for the indicated periods. Ras activi- ties were analyzed by a pull-down assay using the Ras-binding domain of Raf-1 bound to agarose. ERK and Akt activities were analyzed by immuno- blot using anti-Thr(P)202/Tyr204-ERK1/2 and anti- Ser(P)473-Akt antibodies, respectively. D, the effects of inhibitors for MAPK and PI3K pathways on NF1 siRNA-induced cofilin phosphorylation. After 24 h of siRNA transfection, cells were treated with Me2SO (lanes 1 and 2), 10 M U0126 (lanes 3 and 4), or 20 M PD98059 (lanes 5 and 6) (left pan- els). The transfected cells were also treated with Me2SO (lane 1 and 2), 10 M LY294002 (lanes 3 and 4) or 100 nM wortmannin (lanes 5 and 6) (right pan- els) for 5 h. Cells were then examined for levels of Ser(P)3-cofilin by immunoblot analysis. The levels of phospho-Erk and Akt were also examined to confirm the effects of inhibitors.

Article Snippet: Polyclonal antibodies against Akt, phospho-Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2, LIM kinase 1 (LIMK1), cofilin, and phospho-cofilin (Ser3) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Expressing, Control, Transfection, Dominant Negative Mutation, Plasmid Preparation, Western Blot, Phospho-proteomics, Pull Down Assay, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: The Neurofibromatosis Type 1 Gene Product Neurofibromin Enhances Cell Motility by Regulating Actin Filament Dynamics via the Rho-ROCK-LIMK2-Cofilin Pathway

doi: 10.1074/jbc.m503707200

Figure Lengend Snippet: FIGURE 8. NF1-GRD type 2 suppresses NF1 siRNA-induced effects. A, NF1-GRD type 2 sup- presses NF1 siRNA-induced cofilin phosphoryla- tion.NF1(lanes2,4,and6)orcontrolsiRNA(lanes1, 3, and 5) was co-transfected with mock (lanes 1 and 2), FLAG-NF1-GRD1 (lanes 3 and 4), or FLAG- NF1-GRD2 (lanes 5 and 6) plasmid. The levels of Ser(P)3-cofilin, cofilin, and FLAG-NF1-GDR were analyzed by immunoblot after 24 h. B, NF1-GRD type 2 suppresses NF1 siRNA-induced stress fiber formation. Cells treated as described in A were stained with Alexa 488 phalloidin (actin; green) and anti-FLAG antibody (red) after 24 h of siRNA transfection. C, the effect of NF1-GRD on the spherical mass formation of neurofibromin-de- pleted cells. NF1 or control siRNA were co-trans- fected with NF1-GRD1 or NF1-GRD2, and then 5000 cells were replated onto a Matrigel-pre- coated dish 24 h after transfection. The number of colonies formed in Matrigel at 120 h was counted under a phase-contrast microscope. Each datum represents the average of three independent experiments. Error bars represent S.D. Statistical differences were determined with a t test; *, p 0.001. D, NF1-GRD1 and NF1-GRD2 suppress the spherical mass formation of neurofibromin-de- pleted cells. Cells treated as described in C were replated onto the Matrigel matrix, and the spheri- cal masses formed at 96 h after the replating were observed by a phase-contrast microscope.

Article Snippet: Polyclonal antibodies against Akt, phospho-Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2, LIM kinase 1 (LIMK1), cofilin, and phospho-cofilin (Ser3) were purchased from Cell Signaling Technology.

Techniques: Transfection, Plasmid Preparation, Western Blot, Staining, Control, Microscopy

Journal: Stem cell research

Article Title: Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

doi: 10.1016/j.scr.2018.03.010

Figure Lengend Snippet: Fig. 1. Actin cytoskeleton is dissembled during adipocyte differentiation of hMSCs via RhoA/LIMK1/CFL1 axis. hMSCs were treated with adipogenic induction medium for the indicated days. Representative data were shown from three independent experiments. A. The expression of adipogenic markers were analyzed by qRT-PCR and normalized against B2M. The expression level at day 13 was set to 1. Data were presented as mean + S.D., n = 3 (upper). Mature adipocytes with lipid droplets were stained by Oil-red O staining (lower). B. G- actin and F-actin level were analyzed by G-actin/F-actin assay followed by quantitation of band density by ImageJ software. C. The expression and phosphorylation status of CFL1, LIMK1, RhoA and DSTN was analyzed by western blot. α-tubulin was used as loading control. Band density in the Western blot was semi-quantitated by ImageJ software.

Article Snippet: Antibodies (total or phosphor) specific for CFL1, LIMK1, Smad2, RhoA, Akt, JNK, p38, Smad1/5/8 were obtained from Cell Signaling Technology; antibodies for total and phosphor-ERKwere purchased from Santa Cruz; antibodies for DSTN, α-tubulin and actin were bought from Sigma.

Techniques: Expressing, Quantitative RT-PCR, Staining, Quantitation Assay, Software, Phospho-proteomics, Western Blot, Control

Journal: Stem cell research

Article Title: Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

doi: 10.1016/j.scr.2018.03.010

Figure Lengend Snippet: Fig. 3. Stabilization of actin cytoskeleton by knocking down CFL1 and DSTN inhibits adipogenesis of hMSCs. hMSCs were transfected with control non-targeting siRNA (siR-Ctrl) or siRNA targeting CFL1 (siR-CFL1), DSTN (siR-DSTN) or both and then treated with adipogenic induction medium for up to 13 days. Representative data were shown from three independent experiments. *P b .05, **P b .01. A. Knocking down efficiency was determined by qRT-PCR and normalized against B2M at indicated day. The gene expression with siR-Ctrl was set to 1. Data were presented as mean ± S.D., n = 3. B. Cell viability was measured by CellTiter-Blue reagent. Data were presented as mean ± S.D., n = 8. C. The expression of adipogenic markers including ADIPOQ, LPL, PPARG and FABP4 was determined by qRT-PCR and normalized against B2M at day 10 (upper). The expression level with siR-Ctrl was set to 1. Data were presented as mean + S.D., n = 3. The accumulation of lipid droplets in mature adipocytes was visualized by Oil-red O staining at day 10 (Lower).

Article Snippet: Antibodies (total or phosphor) specific for CFL1, LIMK1, Smad2, RhoA, Akt, JNK, p38, Smad1/5/8 were obtained from Cell Signaling Technology; antibodies for total and phosphor-ERKwere purchased from Santa Cruz; antibodies for DSTN, α-tubulin and actin were bought from Sigma.

Techniques: Transfection, Control, Quantitative RT-PCR, Gene Expression, Expressing, Staining

Journal: Stem cell research

Article Title: Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

doi: 10.1016/j.scr.2018.03.010

Figure Lengend Snippet: Fig. 4. Inhibition of LIMK1 increased adipocyte differentiation of hMSCs by activation of CFL1. hMSCs were treated with adipogenic induction medium supplemented with DMSO as control (Ctrl) or 1 μM LIMK inhibitor (LIMKi) for 7 or 10 days continuously. Representative data was shown from three independent experiments. **P b .01. A. The total and phosphor-CFL1 were detected by western blot. Actin was used as loading control. B. The accumulation of lipid droplets in mature adipocytes was visualized by Oil-red O staining at day 10. C. The expression of adipogenic markers including CEBPA, ADIPOQ, LPL, PPARG and FABP4 was determined by qRT-PCR and normalized against B2M. Data were presented as mean ± S.D., n = 3.

Article Snippet: Antibodies (total or phosphor) specific for CFL1, LIMK1, Smad2, RhoA, Akt, JNK, p38, Smad1/5/8 were obtained from Cell Signaling Technology; antibodies for total and phosphor-ERKwere purchased from Santa Cruz; antibodies for DSTN, α-tubulin and actin were bought from Sigma.

Techniques: Inhibition, Activation Assay, Control, Western Blot, Staining, Expressing, Quantitative RT-PCR