Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 1. sFRP1 was abundantly produced in nerve ECM following injury and associated with nerve degeneration (A) The isolation of sciatic nerve samples and proteomic analysis process. (B) The clustering distribution of injured and uninjured nerve samples as plotted by PCA analysis. (C) Differentially expressed proteins between injured and uninjured nerve samples are displayed in volcano plot. N = 3 mice. Proteins regulated over 1.5-fold changes (adj. p < 0.05) are highlighted in blue (downregulated) and red (upregulated). (D) GO enrichment analysis indicating the classification of differentially expressed proteins related to the biological process category. (E) Differentially expressed proteins in the GO category of ECM are displayed as a heatmap. (F) Western blotting analysis demonstrating increased production of sFRP1 in the injured nerve tissue. (G) Quantification of sFRP1 protein level in sciatic nerves isolated from uninjured and injured mice as indicated by western blot analysis. N = 3 mice. (H) Representative TEM, HE, and TB images of injured nerves isolated from mice treated with WAY-316606 and PBS at 3 weeks post injury. N = 6 mice. (I and J) Quantification of myelinated axon diameter and g-ratio as indicated in TEM images. (K) Quantification of myelinated axon density as indicated in HE-stained images. (L) Representative IHC images of human nerves stained for sFRP1 at 12 h after injury. Statistical significance was determined using two-tailed unpaired Student’s t tests; **p < 0.01; *p < 0.05; ns, no difference. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Produced, Isolation, Western Blot, Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 3. Mice with deletion of sFRP1 in SCs profoundly reduced macrophage infiltration and improved nerve regeneration (A) Sfrp1flox/flox mice were bred with PlpcreErt1 mice to generate tamoxifen-inducible SC-specific sFRP1 knockout (Sfrp1flox/floxPlpcreErt1) and littermate control (Sfrp1flox/flox) mice. (B and C) Representative SCG10 immunostaining and related quantification of sciatic nerves at 14 days post transection. N = 6 mice. The dashed line indicates the transection site. Scale bar, 500 mm. (D and E) Representative F4/80 immunostaining (red) of sciatic nerves taken from the injury site, 1,000, 2,000, and 3,000 mm distal to the injury site and related quantification of infiltrated macrophages. Scale bar, 100 mm. N = 6 mice. (F and G) Western blot analysis and related quantification of TNF-a level in injured nerves at 24 h post transection. N = 3 mice. (H and I) Triple staining of CCL2 (green), F4/80 (red), and NeuN (pink) on sciatic DRG sections from Sfrp1flox/flox and Sfrp1flox/floxPlpcreErt1 mice and related quantification of CCL expression level in DRGs. N = 6 mice. No significant difference of CCL2 expression is observed between DRGs of Sfrp1flox/flox and Sfrp1flox/floxPlpcreErt1 mice. (J–L) Representative TUBB3 immunostaining (green) of sciatic DRG neurons isolated from Sfrp1flox/flox and Sfrp1flox/floxPlpcreErt1 mice (n = 6 mice) and related quantification. DRG neurons were cultured in vitro for 4 days or 7 days. (M and N) Representative immunostaining and related quantification of ATF3 (red) and the neuronal marker NeuN (green) in sciatic DRGs at 24 h after nerve injury. N = 6 mice. Scale bar, 50 mm. Statistical significance in (C) and (E) was analyzed by two-way ANOVA followed by Sidak’s post hoc analysis. Statistical significance was determined using two- tailed unpaired Student’s t tests; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, no significance. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Knock-Out, Control, Immunostaining, Western Blot, Staining, Expressing, Isolation, Cell Culture, In Vitro, Marker, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 4. SFRP1 induces the F4/80+ CD86+ proinflammatory macrophage phenotype and inhibits oxidative metabolism (A and B) The axon length of sciatic DRG neurons demonstrates no significant difference in response to sFRP1 treatment. N = 6 biological replicates. (C) Representative TEM images reveal that the morphology and structure of mitochondria were well preserved in sFRP1-treated neurons. (D and E) Representative TEM images and related quantification of nerve transections (N = 6 mice). The suppressing effect of sFRP1 on axon regrowth is alleviated in a macrophage-deficient condition. (F) Double staining of IL-1b (red) and TNF-a (green) on sFRP1-treated BMDMs. (G) sFRP1-induced phenotypic switch is revealed by flow cytometric quantification. FITC reflects F4/80-positive cells. PE reflects CD206-positive cells. APC reflects CD86-positive cells. (H and I) Quantification of the percentage of IL-1b and TNF-a-positive cells as reflected by Figure 4F. Biological replicates n = 3 with two technical replicates each. (J) Double staining of Arg-1 (red) and Wnt3a (green) on sFRP1 and PBS-treated BMDMs. (K) The internalizing capacity of BMDMs was measured by incubating with 100 mg/mL pHrodo BioParticles (green). BMDMs were visualized by F4/80 (red) staining.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Double Staining, Staining

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 6. Depletion of HSP90 in macrophages attenuated neuroinflammation and nerve degenerative changes exerted by sFRP1 (A) Hsp90aaflox/+ mice were bred with Lyz2-cre mice to generate macrophage-specific HSP90-deficient (Hsp90aaflox/+Lyz2-cre) and littermate control (Hsp90aaflox/+) mice. (B and C) Representative IF images of SCG10 staining and related quantification of sciatic nerves at 2 weeks post injury. The dashed line indicates the transection site. Scale bar, 500 mm. N = 6 mice. (D and E) Representative IF images of F4/80 staining (red) of sciatic nerves and related quantification of macrophages at 2 weeks post injury. Scale bar, 100 mm. N = 6 mice. (F–I) Double staining of TNF-a (red) and IL-1b (green) on nerve longitudinal sections and related quantification. (J–L) Representative TUBB3 staining (green) and related quantification of sciatic DRG neurons isolated from Hsp90aaflox/+ and Hsp90aaflox/+Lyz2-cre mice after 4 days and 7 days of culture. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using two-way ANOVA followed by Sidak’s post hoc analysis in (C) and (E), and using two-tailed unpaired Student’s t tests in (F), (G), (K), and (L); **p < 0.01; ***p < 0.001; *p < 0.05; ns, no significance. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Control, Staining, Double Staining, Isolation, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 7. SFRP1-neutralizing antibody treatment improved axon regeneration in vivo and in vitro (A and B) Representative SCG10 immunostaining and related quantification of murine injured nerves at 2 weeks after nerve transection. The dashed line indicates the transection site. Scale bar, 500 mm. N = 6 mice. (C) Schematic diagram of DRG neuron and macrophage microfluidic coculture chamber assay. (D) Representative optical images of macrophages in the neuron-macrophage coculture chambers. (E and F) Representative TUBB3 immunofluorescent images of neurons in the neuron-macrophage co-culture chambers and related quantification of average axon length in microfluidic channels. Biological replicates n = 3 with two technical replicates each. (G) Schematic diagram of DRG neuron and macrophage direct coculture assay. (H and I) Representative IF images stained for TUBB3 (green) on sciatic DRG neurons, and quantification of average axon length per cell in the direct coculture dishes. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using two-way ANOVA followed by Sidak’s post hoc analysis in (B) and (I) and using two-tailed unpaired Student’s t tests in (F); ***p < 0.001; **p < 0.01; *p < 0.05. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: In Vivo, In Vitro, Immunostaining, Boyden Chamber Assay, Co-Culture Assay, Co-culture Assay, Staining, Two Tailed Test

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 1. DDB1 interacts with and cleaved by NS3/4A. (A) DDB1 interacts with NS3/4A in overexpression system. The 293 cells were transfected with the indicated plasmids. Coimmunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-Flag anti-HA. The lysates were analyzed by immunoblots with anti-DDB1 or anti-HA. (B) Endogenous DDB1 interacts with NS3/4A in JFH-1 infected cells. Huh-7 cells (5 107) were mock-infected or infected with JFH-1 (Multiplicity of Infection, MOI: 0.3) for 3 days. Coimmunoprecipitation was performed with anti-DDB1 or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-DDB1 and anti-NS3. The lysates were analyzed by immunoblots with anti-DDB1 or anti-NS3.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Control, Western Blot, Infection

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 2. NS3/4A cleaves DDB1 at C378. (A) Cleavage of DDB1 by NS3/4A is inhibited by the NS3/4A inhibitor VX-950. The 293 cells were transfected with N-terminal or C-terminal Flag-tagged DDB1 (N-Flag-DDB1 or DDB1-C-Flag respectively) and HA-NS3/4A. The transfected cells were treated with VX-950 (0.2 mM) or left untreated for 1 day before immunoblot analysis with anti-Flag or anti-HA. (B) Alignment of the junction sequences of NS proteins of HCV and the potential NS3/4A cleavage sites in TC-PTP, VISA, TRIF and DDB1. (C) NS3/4A cleaves DDB1 at C378. The 293 cells were transfected with the indicated plasmids and cells lysates were analyzed by immunoblots with anti-Flag or anti-HA. (D) DDB1 N-terminal cleavage product migrated similarly to overexpressed DDB1(1–378) mutant. The 293 cells were transfected with the indicated plasmids, treated with VX-950 or left untreated for 1 day before immunoblot analysis with with anti-Flag or anti-HA.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Transfection, Western Blot, Mutagenesis

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 3. DDB1 plays a critical role in HCV replication. (A) Overexpression of DDB1 potentiates HCV RNA replication. Huh-7 cells (1 106) were transfected with the indicated amounts of Flag-DDB1 plasmid for 24 h and then cells were split and mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-Flag or anti-b-actin. Graphs show mean7SD, n¼3. (B) Knockdown of DDB1 inhibits HCV RNA replication. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (C) Knockdown of DDB1 inhibits HCV protein expression. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 3 days, and the cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (D) Knockdown of DDB1 inhibits production of infectious HCV particles. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show mean7SD, n¼3. (E) Knockdown of DDB1 inhibits RNA replication of HCV subgenomic replicon. Control or DDB1-RNAi knockdown Huh-7 cells and Huh-7 Con1 subgenomic replicon cells were cultured for 3 days. The cells (2 106) were collected and intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. Cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (F) DDB1 has no effects on HCV entry. Control or DDB1-RNAi knockdown Huh-7 cells were infected with HCVpp for 3 days (NC: Negative Control, HCVpp pakaging without HCV E1E2). The lysates of infected cells were assayed by luciferase reporter assays and immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Western Blot, Knockdown, Control, Expressing, Staining, Cell Culture, Negative Control, Luciferase

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 4. DDB1 cleavage is required for HCV replication. (A) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) The indicated stable cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh medium was added for 48 h. The JFH-1-containing medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D) Control or DDB1-RNAi knockdown Huh-7 cells were stably transduced with empty vector, DDB1, DDB1(C378R), off-target nonsense mutants of DDB1 or DDB1(C378R) respectively. Two days later, cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-actin. Graphs show meanþSD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Stable Transfection, Infection, Quantitative RT-PCR, Western Blot, Staining, Control, Knockdown, Transduction, Plasmid Preparation

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 5. DDB1 cleavage products do not affect the HCV infection. (A) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D–F) Control or DDB1-RNAi-#1 (targeted sequence is within the cDNA fragment encoding aa379–1140) transduced Huh-7 cells were further transfected with empty vector, Flag-DDB1(1–378), Flag-DDB1(379–1140*) (*, a RNAi off-target mutant),or a combination of Flag-DDB1(1–378) and Flag-DDB1(379–1140*) by Lipofectamine 2000. One day post transfection, the cells were split and mock infected or infected with JFH-1(MOI: 0.3) for 3 (D, E) or 1 (F) day. Intracellular HCV RNA levels (D), intracellular viral particles (E), or viral titers in the medium (F) were then determined as described in (A–C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Infection, Stable Transfection, Transduction, Quantitative RT-PCR, Western Blot, Staining, Control, Sequencing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Oncology reports

Article Title: Overexpression of LASP1 is associated with proliferation, migration and invasion in esophageal squamous cell carcinoma.

doi: 10.3892/or.2012.2199

Figure Lengend Snippet: Figure 1. mRNA expression of LASP1. (A) Relative expression level of LASP1 was measured by qRT-PCR in 56 pairs of ESCC and adjacent normal esopha geal tissues. The expression level of LASP1 was higher in ESCC tissues than that in the adjacent normal tissues; *P<0.05. (B) Fold-change of LASP1 expression level in ESCC tissues compared with the adjacent normal tissues. (C) Fold-change of LASP1 expression level in two ESCC cell lines (ECA109 and KYSE510) compare with the normal esophageal tissues. LASP1 expression data were presented as the means ± SD and were normalized to β-actin expression. Relative quantification of LASP1 expression was calculated using the 2-ΔΔCt method.

Article Snippet: After transferring the protein to a nitrocellulose membrane and blocking with 3% non-fat dry milk in 10 mM Tris, pH 7.5, 10 mM NaCl, 0.1% (w/v) Tween-20, the membrane was first incubated with the antibody raised against LASP1 (1:4,000) followed by incubation with HRP goat anti-rabbit IgG (Proteintech, Chicago, IL, USA), which were diluted to 1:3,00.

Techniques: Expressing, Quantitative RT-PCR

Journal: Oncology reports

Article Title: Overexpression of LASP1 is associated with proliferation, migration and invasion in esophageal squamous cell carcinoma.

doi: 10.3892/or.2012.2199

Figure Lengend Snippet: Figure 2. The protein expression level of LASP1 by western blot analysis revealed that LASP1 expression was upregulated in 13 pairs of ESCC tis sues (T) compared to that in the adjacent normal esophageal tissues (N). The LASP1 protein was normalized to β-actin.

Article Snippet: After transferring the protein to a nitrocellulose membrane and blocking with 3% non-fat dry milk in 10 mM Tris, pH 7.5, 10 mM NaCl, 0.1% (w/v) Tween-20, the membrane was first incubated with the antibody raised against LASP1 (1:4,000) followed by incubation with HRP goat anti-rabbit IgG (Proteintech, Chicago, IL, USA), which were diluted to 1:3,00.

Techniques: Expressing, Western Blot

Journal: Oncology reports

Article Title: Overexpression of LASP1 is associated with proliferation, migration and invasion in esophageal squamous cell carcinoma.

doi: 10.3892/or.2012.2199

Figure Lengend Snippet: Figure 3. Histological expression pattern of LASP1 by IHC. Immunoreactivity for the positive staining of LASP1 was localized in the cytoplasm and nuclei. (A) Negative control in ESCC (PBS instead of the primary antibody as nega tive control); (B) strong expression of LASP1 in ESCC; (C) weak expression of LASP1 in ESCC (magnification, x400).

Article Snippet: After transferring the protein to a nitrocellulose membrane and blocking with 3% non-fat dry milk in 10 mM Tris, pH 7.5, 10 mM NaCl, 0.1% (w/v) Tween-20, the membrane was first incubated with the antibody raised against LASP1 (1:4,000) followed by incubation with HRP goat anti-rabbit IgG (Proteintech, Chicago, IL, USA), which were diluted to 1:3,00.

Techniques: Expressing, Staining, Negative Control, Control

Journal: Oncology reports

Article Title: Overexpression of LASP1 is associated with proliferation, migration and invasion in esophageal squamous cell carcinoma.

doi: 10.3892/or.2012.2199

Figure Lengend Snippet: Figure 4. Expression of LASP1 in the ECA109 and KYSE510 cells was effectively suppressed by transfection with siR-LASP1. (A and C) LASP1 mRNA level was inhibited in ECA109 cells transfected with siR-LASP1 compared with NT and NC cells. (B and D) LASP1 mRNA level was inhibited in KYSE510 cells transfected with siR-LASP1 compared with NT and NC cells. (E and G) LASP1 protein level was inhibited in ECA109 cells transfected with siR-LASP1 compared with NC cells. (F and H) LASP1 protein level was inhibited in KYSE510 cells transfected with siR-LASP1 compared with NC cells. The LASP1 expression level was normalized to β-actin; *P<0.05. NT, non-transfected; NC, negative-control; siR-LASP1, siRNA-LASP1.

Article Snippet: After transferring the protein to a nitrocellulose membrane and blocking with 3% non-fat dry milk in 10 mM Tris, pH 7.5, 10 mM NaCl, 0.1% (w/v) Tween-20, the membrane was first incubated with the antibody raised against LASP1 (1:4,000) followed by incubation with HRP goat anti-rabbit IgG (Proteintech, Chicago, IL, USA), which were diluted to 1:3,00.

Techniques: Expressing, Transfection, Negative Control

Journal: Oncology reports

Article Title: Overexpression of LASP1 is associated with proliferation, migration and invasion in esophageal squamous cell carcinoma.

doi: 10.3892/or.2012.2199

Figure Lengend Snippet: Figure 5. Silencing of LASP1 in ESCC cells inhibits proliferation in vitro. Two ESCC cell lines (ECA109 and KYSE510) were transfected with 75 ng of siR-LASP1 or negative-control siRNA. Cell proliferation was determined using the MTT assay at 24, 48 and 72 h after transfection in 3 different independent experiments.ve-control-siR and cells. There was significant difference in the OD at 24, 48 and 72 h in (A) ECA109 and (B) KYSE510 cells following transfec tion with siR-LASP compared with NT and NC cells. Marked cell growth inhibition was noted in (C) ECA109 and (D) KYSE510 cell lines after transfection with siR-LASP1 compared to NC and NT cells at 72 h. Data are presented as the means ± SD of the absorbance value [optical density (OD)] of cells. *P<0.05. NT, non-transfected; NC, negative-control; siR-LASP1, siRNA-LASP1.

Article Snippet: After transferring the protein to a nitrocellulose membrane and blocking with 3% non-fat dry milk in 10 mM Tris, pH 7.5, 10 mM NaCl, 0.1% (w/v) Tween-20, the membrane was first incubated with the antibody raised against LASP1 (1:4,000) followed by incubation with HRP goat anti-rabbit IgG (Proteintech, Chicago, IL, USA), which were diluted to 1:3,00.

Techniques: In Vitro, Transfection, Negative Control, MTT Assay, Control, Inhibition

Journal: Oncology reports

Article Title: Overexpression of LASP1 is associated with proliferation, migration and invasion in esophageal squamous cell carcinoma.

doi: 10.3892/or.2012.2199

Figure Lengend Snippet: Figure 6. Silencing of LASP1 in ESCC cells inhibits migration in vitro. Two ESCC cell lines (ECA109 and KYSE510) were transfected with 75 ng of siR‑LASP1 or control-negative-siRNA. Cell migration was evaluated by the migration assay 6 h after transfection in three different independent experiments. (A) An obvious reduction was noted in cell migration of ECA109 cells after transfection with siR-LASP1 when compared to NC-siRNA-transfected and NT cells. (B) Obvious inhibition of cell migration of KYSE510 cells was noted after transfection with siR-LASP1 when compared to NC-siRNA-transfected and NT cells. Results are expressed as the means ± SD of five randomly selected x200 magnification fields. *P<0.05. NT, non-transfected; NC, negative-control; siR-LASP1, siRNA-LASP1.

Article Snippet: After transferring the protein to a nitrocellulose membrane and blocking with 3% non-fat dry milk in 10 mM Tris, pH 7.5, 10 mM NaCl, 0.1% (w/v) Tween-20, the membrane was first incubated with the antibody raised against LASP1 (1:4,000) followed by incubation with HRP goat anti-rabbit IgG (Proteintech, Chicago, IL, USA), which were diluted to 1:3,00.

Techniques: Migration, In Vitro, Transfection, Control, Inhibition, Negative Control

Journal: Oncology reports

Article Title: Overexpression of LASP1 is associated with proliferation, migration and invasion in esophageal squamous cell carcinoma.

doi: 10.3892/or.2012.2199

Figure Lengend Snippet: Figure 7. Silencing of LASP1 in ESCC cells inhibits invasion in vitro. Two ESCC cell lines (ECA109 and KYSE510) were transfected with 75 ng of siR‑LASP1 or control negative-siRNA. Cell invasion was evaluated by the invasion assay 18 h after transfection in three different independent experiments. (A) An obvious inhibition was noted in the cell invasiveness of ECA109 cells after transfection with siR-LASP1 when compared to NC-siRNA-transfected and NT cells. (B) An obvious inhibition was noted in cell invasiveness of KYSE510 cells after transfection with siR-LASP1 compared to NC-siRNA-transfected and NT cells. Results are expressed as the means ± SD of five randomly selected x200 magnification fields *P<0.05. NT, non-transfected; NC, negative-control; siR-LASP1, siRNA-LASP1.

Article Snippet: After transferring the protein to a nitrocellulose membrane and blocking with 3% non-fat dry milk in 10 mM Tris, pH 7.5, 10 mM NaCl, 0.1% (w/v) Tween-20, the membrane was first incubated with the antibody raised against LASP1 (1:4,000) followed by incubation with HRP goat anti-rabbit IgG (Proteintech, Chicago, IL, USA), which were diluted to 1:3,00.

Techniques: In Vitro, Transfection, Control, Invasion Assay, Inhibition, Negative Control

Journal: Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan

Article Title: Induction of differentiation by panaxydol in human hepatocarcinoma SMMC-7721 cells via cAMP and MAP kinase dependent mechanism.

doi: 10.1248/yakushi.131.993

Figure Lengend Snippet: Fig. 5. Panaxydol Regulates the Expression Levels of p21, p-ERK and Id1 in SMMC-7721 Cells The levels of p21, p-ERK and Id1 were examined by immunoblotting analysis after 5 days incubation with 5, 10 and 20 mM panaxydol. Lane C: control group, Lane 1～3: SMMC-7721 cells treated with 5, 10, 20 mM panaxydol respectively. Data (mean±S.D.) were summarized from three independent experiments (n＝3), p＜0.05, p＜0.01 vs. control group.

Article Snippet: Immunoblotting analysis was carried out as previously described and the following antibodies were used: monoclonal antibody anti-p21 (Protein Tech, 1:1000), poly-anti-p-ERK1 /2 (Cell Signaling, 1:1000) or primary monoclonal antibody anti-Id.

Techniques: Expressing, Western Blot, Incubation, Control

Journal: Journal of cell science

Article Title: Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration.

doi: 10.1242/jcs.155663

Figure Lengend Snippet: Fig. 2. Liprin-a1, ERC1a and LL5 proteins regulate the stability of lamellipodia. (A) Time lapse of MDA- 231 cells on fibronectin. White arrows indicate the appearance of new protrusions in cells transfected with the indicated siRNAs. Scale bar: 10 mm. (B) Liprin-a1, ERC1a or LL5 silencing (Lip, liprin-a1; ERC, ERC1a; LL5s, both LL5a and LL5b) in cells migrating on fibronectin. Lamellipodial stability is reduced compared to transfection with control siRNA (Luc). Results are mean6s.e.m. (n510 cells). (C) Cells overexpressing liprin-a1 (L4) produce less frequent and more stable lamellipodia compared to control (C9) cells. Effects of liprin-a1 overexpression are abolished by silencing liprin-a1, ERC1a or LL5 proteins. Results are mean6s.e.m. (n510 cells). (D) Overexpression of ERC1a or LL5b decreases the frequency of lamellipodia formation and increases lamellipodial persistence (n55–10 cells). (E) ERC1a overexpression rescues the frequency of lamellipodia formation (top) and partially rescues the loss of their persistence (bottom) induced by liprin- a1 silencing. Results are mean6s.e.m. (n59–17 cells). *P,0.05; **P,0.005 compared with control or as indicated.

Article Snippet: The polyclonal antibody (pAb) against liprin-a1 was as described previously (Asperti et al., 2009); the commercial mAb against liprin-a1 was from Proteintech.

Techniques: Transfection, Control, Over Expression

Journal: Journal of cell science

Article Title: Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration.

doi: 10.1242/jcs.155663

Figure Lengend Snippet: Fig. 3. ERC1a colocalizes with liprin-a1 and LL5b at the edge of migrating cells. MDA-231 cells migrating on fibronectin were immunostained for the endogenous proteins: colocalization at protrusions (arrows) of ERC1a, liprin-a1 and LL5a (A); ERC1a with LL5b and LL5a (B); ERC1a, liprin-a1 and LL5b (C). Scale bars: 10 mm. Lower panels in A and C are threefold enlargements of areas indicated by arrowheads. Arrows indicate examples of colocalization. (D) Frames from cells expressing the indicated proteins and migrating on fibronectin. GFP-tagged liprin-a1, ERC1a, LL5a and LL5b are concentrated in clusters near the leading edge of migrating cells. Cells cotransfected with GFP and Mito-RFP (Mitochondria-RFP) were used as controls. Scale bar: 10 mm. (E) Frames from a time- lapse series of a cell co-transfected with mCherry and GFP–ERC1a. Scale bar: 20 mm. (F) Kymograph for mCherry and GFP–ERC1a from the lamellipodium of the cell shown in E (white line) showing intense accumulation of ERC1a near the lamellipodium during the protrusive phase. The signal for ERC1a decreases abruptly once the lamellipodium halts (red arrow) and retracts (blue arrow). Right, diagram of the intensity of fluorescence for ERC1a (green line) and mCherry (red line). The intensity of ERC1a signal increases soon after lamellipodial protrusion is detectable (red line) and abruptly decreases when the protrusive activity stops.

Article Snippet: The polyclonal antibody (pAb) against liprin-a1 was as described previously (Asperti et al., 2009); the commercial mAb against liprin-a1 was from Proteintech.

Techniques: Expressing, Transfection, Fluorescence, Activity Assay

Journal: Journal of cell science

Article Title: Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration.

doi: 10.1242/jcs.155663

Figure Lengend Snippet: Fig. 5. Silencing of liprin-a1, ERC1a or LL5 proteins enhances the intracellular accumulation of total b1 integrins. (A) ERC1a-positive (ERC) clusters at protrusions are contiguous, but do not overlap, with paxillin-positive focal adhesions. Scale bars: 10 mm. (B) Frames from time-lapse of a cell co-transfected with mCherry–zyxin and GFP–ERC1a. ERC1a accumulates behind peripheral focal adhesions at the front of the migrating cell. Scale bar: 10 mm. (C,D) Cells co- transfected with GFP and the indicated siRNAs (Lip, siLip, liprin-a1; siERC1a, ERC1a; LL5s, siLL5a+b, both LL5a and LL5b) were incubated with the mAb TS2/16 before fixation for immunofluorescence. Merge: GFP (green), integrins (red), DAPI (blue). Results are mean6s.e.m. for the normalized density of b1 integrin at the cell surface (n536–47 cells). (E,F) Cells transfected with GFP and siRNAs were incubated overnight at 37˚C with the TS2/16 mAb. Surface labeling of integrins was removed before fixation and detection of the internalized integrins with fluorescently tagged anti-mouse-IgG antibody. Scale bar: 20 mm (C,E). Silencing of any of the three proteins enhanced total b1 internalization (F). Bars, normalized mean6s.e.m. (n563–85 cells); *P,0.05; **P,0.005 compared with control.

Article Snippet: The polyclonal antibody (pAb) against liprin-a1 was as described previously (Asperti et al., 2009); the commercial mAb against liprin-a1 was from Proteintech.

Techniques: Transfection, Incubation, Immunofluorescence, Labeling, Control

Journal: Journal of cell science

Article Title: Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration.

doi: 10.1242/jcs.155663

Figure Lengend Snippet: Fig. 8. The PH domain of LL5b is required for effective migration, and for the localization of endogenous liprin-a1 and ERC1a near the protruding cell edge. (A) siRNAs specific for endogenous LL5 proteins (LL5s) (upper two blots from top) do not interfere with the expression of GFP-tagged mouse wild- type and mutant LL5b (lowest blot). Immunoblotting was performed on lysates (15 mg protein) from transfected cells to detect the proteins indicated on the left of each blot. The anti-LL5b antibody recognizes the endogenous human protein, but not the murine overexpressed protein. Luc, luciferase. (B,C) Subcellular localization of endogenous liprin-a1 in cells co-transfected with siRNAs for either control or LL5 proteins, together with the indicated GFP constructs. Scale bars: 20 mm. (D) Effects of the expression of wild-type LL5b or the mutant LL5bM on the accumulation of endogenous liprin-a1 at the cell edge. Results are mean6s.e.m. (n545–76 cells). *P,0.05; **P,0.005 compared with control. (E,F) Localization of endogenous ERC1a in cells co-transfected with siRNAs for either control or LL5 proteins, together with the indicated GFP constructs. Scale bars: 20 mm. (G) The negative effects of LL5 proteins depletion on the speed of migration on fibronectin are rescued by GFP–LL5b, but not by GFP–LL5bM. Results are mean6s.e.m. (n542–81 cells). The effects of LL5 proteins depletion on the formation (H) and the persistence (I) of lamellipodia during migration on fibronectin are rescued by the wild-type GFP–LL5b. The GFP–LL5bM

Article Snippet: The polyclonal antibody (pAb) against liprin-a1 was as described previously (Asperti et al., 2009); the commercial mAb against liprin-a1 was from Proteintech.

Techniques: Migration, Expressing, Mutagenesis, Western Blot, Transfection, Luciferase, Control, Construct

Journal: Scientific reports

Article Title: MiR-424-5p reversed epithelial-mesenchymal transition of anchorage-independent HCC cells by directly targeting ICAT and suppressed HCC progression.

doi: 10.1038/srep06248

Figure Lengend Snippet: Figure 6 | miR-424-5p disrupted the cell-cell adhesion complex by directly targeting ICAT. (a) HEK293 cells were transfected with 40 nM, 80 nM and 120 nM of miR-424-5p and cultured for 24 h. Cell lysates were extracted and subjected to immunoprecipitation with anti-b-catenin antibody followed by western blot analysis with anti-E-cadherin antibody. Proteins in whole-cell lysates were used as input control for analysis by western blot with anti-E- cadherin antibody and anti-ICAT antibody, respectively. (b) HEK293 cells were transfected with 0.5 mg, 1 mg and 2 mg of HA tagged ICAT recombinant and cultured for 24 h. Cell lysates were extracted and subjected to immunoprecipitation with anti-b-catenin antibody followed by western blot analysis with anti-E-cadherin antibody. Proteins in whole-cell lysates were used as input control. Similar results were got from three independent experiments. (c) HepG2 cells were transfected with 50 nM of 3 synthesized siRNAs targeting ICAT or nonsense control.48 h after transfection, the inhibitory effect of siRNA were analyzed by western blot. (d) The HepG2 cells were transfected with a mixture of Si-1 and Si-3 targeting ICAT, and cultured in anchorage deprived condition for 24 h before analysis of EMT related markers by real-time PCR. (e–f) HepG2 cells were transfected with miR-424-5p or HA-tagged ICAT recombinant and cultured in anchorage deprived condition for 24 h. Expression of E-cadherin, N-cadherin and ICAT was determined by western blot (e) and quantified by band intensity analysis of these proteins normalized to that of b-actin (f). (g–h) BEL7402, SMMC7721 and HepG2 HCC cells transfected with miR-424-5p, miR-424-5p inhibitor or Si-ICAT were cultured as anchorage deprived cells for 24 h before being harvested and analyzed for their invasion capability (g). The invaded cells were quantified and presented histogram were representative one from three independent experiments performed in duplicate (h). *P,0.05, **P , 0.01, ***P , 0.001.

Article Snippet: After centrifugation at 14,000 rpm for 10 min, supernatants were collected and incubated with protein G plus-agrose immunoprecipitation reagent together with anti-b-catenin antibody (proteintech, Chicago, USA).

Techniques: Transfection, Cell Culture, Immunoprecipitation, Western Blot, Control, Recombinant, Synthesized, Real-time Polymerase Chain Reaction, Expressing

Journal: Scientific reports

Article Title: MiR-424-5p reversed epithelial-mesenchymal transition of anchorage-independent HCC cells by directly targeting ICAT and suppressed HCC progression.

doi: 10.1038/srep06248

Figure Lengend Snippet: Figure 7 | miR-424-5p inhibited the tumorigenicity of HCC cells in vivo in BALB/c nude mice. (a) The 5-week BALB/c nude mice were subcutaneously injected with SMMC7721 cells. Images presented were the isolated tumors transfected with miR-424-5p plasmid or mock control on day 12 after transfection. (b–c) The volume (b) and weight(c) of the miR-424-5p plasmid or mock control transfected tumors were assayed on day 18 after transplantation of HCC cells. (d) Expression of miR-424-5p in the miR-424-5p plasmid or mock control transfected HCC tumors was analyzed by real- time PCR and normalized to U6. (e) Expression of ICAT in HCC tumors transfected with the miR-424-5p or mock plasmid was analyzed by real-time PCR and normalized to b-actin. (f) The correlation analysis between the expression levels of miR-424-5p and ICAT. (g–i) The expression of E- cadherin(g), N-cadherin(h) and b-catenin(i) in the HCC tumors transfected with miR-424-5p plasmid or mock control. *P,0.05, **P,0.01.

Article Snippet: After centrifugation at 14,000 rpm for 10 min, supernatants were collected and incubated with protein G plus-agrose immunoprecipitation reagent together with anti-b-catenin antibody (proteintech, Chicago, USA).

Techniques: In Vivo, Injection, Isolation, Transfection, Plasmid Preparation, Control, Transplantation Assay, Expressing, Real-time Polymerase Chain Reaction