Journal: Molecular therapy. Nucleic acids

Article Title: Icariin attenuates endothelial-mesenchymal transition via H19/miR-148b-3p/ELF5 in ox-LDL-stimulated HUVECs.

doi: 10.1016/j.omtn.2020.11.021

Figure Lengend Snippet: Figure 1. ICA suppressed ox-LDL-induced EndMT in HUVECs (A–E) Expression levels of CD31, VE-cadherin, a-SMA, and S100A4 in HUVECs exposed to 200 mM ox-LDL or ox-LDL+10 mM ICA, determined using western blotting. ##p < 0.01 versus control group; **p < 0.01 versus ox-LDL group; n = 3. Measurement data are expressed as mean ± SD. ICA, icariin; ox-LDL, oxidized low-density lipoprotein; EndMT, endothelial-mesenchymal transition; HUVEC, human umbilical vein endothelial cell.

Article Snippet: The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with TBST containing 5% BSA for 1 h at 20 C–25 C. The PVDF membranes were probed overnight with CD31 (lot no. 3528S, Cell Signaling Technology, Danvers, MA, USA), VE-cadherin Molecular Therapy: Nucleic Acids Vol.

Techniques: Expressing, Western Blot, Control

Journal: Molecular therapy. Nucleic acids

Article Title: Icariin attenuates endothelial-mesenchymal transition via H19/miR-148b-3p/ELF5 in ox-LDL-stimulated HUVECs.

doi: 10.1016/j.omtn.2020.11.021

Figure Lengend Snippet: Figure 2. ICA promoted ox-LDL-induced H19 expression, and H19 affected EndMT in HUVECs (A) The expression of H19 in HUVECs in control, ox-LDL, or ox-LDL+ICA groups was detected using quantitative real-time PCR. (B) Following transfection with recombinant H19 plasmids or sh-H19, the expression of H19 was detected using quantitative real-time PCR. (C–G) The expression of CD31, VE-cadherin, a-SMA, and S100A4 in ox-LDL- treated HUVECs was determined using western blot analysis. (H) Transwell assays were used to assess the migration capacity of HUVECs incubated for 24 h. (I) HUVECs were incubated for 24 h and subjected to a wound-healing assay to assess cell migration. Images were acquired at 0 and 24 h. *p < 0.05, **p < 0.01 versus pLVX-negative control (NC) group; #p < 0.05, ##p < 0.01 versus sh-NC group; n = 3. Measurement data are expressed as mean ± SD. Scale bars, 100 mm.

Article Snippet: The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with TBST containing 5% BSA for 1 h at 20 C–25 C. The PVDF membranes were probed overnight with CD31 (lot no. 3528S, Cell Signaling Technology, Danvers, MA, USA), VE-cadherin Molecular Therapy: Nucleic Acids Vol.

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Transfection, Recombinant, Western Blot, Migration, Incubation, Wound Healing Assay, Negative Control

Journal: Molecular therapy. Nucleic acids

Article Title: Icariin attenuates endothelial-mesenchymal transition via H19/miR-148b-3p/ELF5 in ox-LDL-stimulated HUVECs.

doi: 10.1016/j.omtn.2020.11.021

Figure Lengend Snippet: Figure 3. H19 knockdown attenuated the inhibitory effect of ICA on EndMT (A–E) Western blot analysis was performed to detect the expression of CD31, VE-cadherin, a-SMA, and S100A4 in HUVECs. (F and G) Transwell (F) and wound-healing assays (G) were used to assess cell migration in HUVECs treated with ox-LDL with or without ICA for 24 h. #p < 0.05, ##p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus ox-LDL group; Dp < 0.05, DDp < 0.01 versus ox-LDL+ICA+sh-NC group; n = 3. Measurement data are expressed as mean ± SD. Scale bars, 100 mm.

Article Snippet: The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with TBST containing 5% BSA for 1 h at 20 C–25 C. The PVDF membranes were probed overnight with CD31 (lot no. 3528S, Cell Signaling Technology, Danvers, MA, USA), VE-cadherin Molecular Therapy: Nucleic Acids Vol.

Techniques: Knockdown, Western Blot, Expressing, Migration, Control

Journal: Molecular therapy. Nucleic acids

Article Title: Icariin attenuates endothelial-mesenchymal transition via H19/miR-148b-3p/ELF5 in ox-LDL-stimulated HUVECs.

doi: 10.1016/j.omtn.2020.11.021

Figure Lengend Snippet: Figure 6. miR-148b-3p overexpression reversed H19-mitigated EndMT in ox-LDL-treated HUVECs Cells were transfected with H19 plasmids or miR-148b-3p mimic and exposed to ox-LDL. (A–E) After that, the expression levels of CD31, VE-cadherin, a-SMA, and S100A4 in HUVECs were determined using western blot analysis. (F and G) Cell migration capacity was measured using wound-healing (F) and transwell assays (G). *p < 0.05, **p < 0.01 versus pLVX-NC+miR-NC group; #p < 0.05, ##p < 0.01 versus pLVX-H19+miR-NC group; n = 3. Measurement data are expressed as mean ± SD. Scale bars, 100 mm.

Article Snippet: The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with TBST containing 5% BSA for 1 h at 20 C–25 C. The PVDF membranes were probed overnight with CD31 (lot no. 3528S, Cell Signaling Technology, Danvers, MA, USA), VE-cadherin Molecular Therapy: Nucleic Acids Vol.

Techniques: Over Expression, Transfection, Expressing, Western Blot, Migration

Journal: Molecular therapy. Nucleic acids

Article Title: Icariin attenuates endothelial-mesenchymal transition via H19/miR-148b-3p/ELF5 in ox-LDL-stimulated HUVECs.

doi: 10.1016/j.omtn.2020.11.021

Figure Lengend Snippet: Figure 8. ICA inhibited the expression of TGF-b through H19, and galunisertib reversed the promoting effect of sh-H19 on EndMT (A) Cells were transfected with sh-H19 and exposed to ox-LDL (with or without ICA); western blot analysis was performed to detect the expression of transforming growth factor-b (TGF-b) in HUVECs. (B–G) After adding galunisertib, the expression of TGF-b, CD31, VE-cadherin, a-SMA, and S100A4 in HUVECs was also determined using western blot analysis. #p < 0.05, ##p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus ox-LDL group; Dp < 0.05, DDp < 0.01 versus ox-LDL+ICA group; Vp < 0.05, VVp < 0.01 versus ox-LDL+ICA+sh-H19 group; &p < 0.05, &&p < 0.01 versus ox-LDL+ICA+sh-NC group; n = 3. Measurement data are expressed as mean ± SD.

Article Snippet: The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with TBST containing 5% BSA for 1 h at 20 C–25 C. The PVDF membranes were probed overnight with CD31 (lot no. 3528S, Cell Signaling Technology, Danvers, MA, USA), VE-cadherin Molecular Therapy: Nucleic Acids Vol.

Techniques: Expressing, Transfection, Western Blot, Control

Journal: Molecular therapy. Nucleic acids

Article Title: Nuclear-Encoded lncRNA MALAT1 Epigenetically Controls Metabolic Reprogramming in HCC Cells through the Mitophagy Pathway.

doi: 10.1016/j.omtn.2020.09.040

Figure Lengend Snippet: Figure 5. MALAT1 Knockdown Impairs Mitophagy (A) Mitophagy biomarkers were detected by western blot. b-Actin was used as the control. (B) Quantitation of mitophagy biomarker western blot results. ****p < 0.0001, **p < 0.01 compared with the shCT control group. (C) The LC3B II/I ratio. Autophagy was assessed by LC3B-II/I ratio. After knockdown of MALAT1, the LC3B-II/I ratio was significantly decreased. ****p < 0.0001 compared with the shCT control group. (D) Acidic lysosome by Lysotracker staining. The intensity of the fluorescence stained by lysotracker was measured. ****p < 0.0001 compared with the shCT control group.

Article Snippet: Antibodies used for western blot were: Anti-LAMP1 antibody-Lysosome Marker (ab24170), Anti-Cyclophilin F antibody (ab110324), Mitophagy Antibody Sampler Kit (Cell Signaling Technology [CST], #43110), Bcl-2 (124) Mouse monoclonal antibody (mAb) (CST, #15071), Cytochrome c (D18C7) Rabbit mAb (CST, #11940), Caspase-3 antibody (CST, #9662), Bax (D2E11) Rabbit mAb (CST, #5023), b-Actin (8H10D10) Mouse mAb (CST, #3700), and BID antibody (CST, #2002).

Techniques: Knockdown, Western Blot, Control, Quantitation Assay, Biomarker Discovery, Staining

Journal: Molecular therapy. Nucleic acids

Article Title: Nuclear-Encoded lncRNA MALAT1 Epigenetically Controls Metabolic Reprogramming in HCC Cells through the Mitophagy Pathway.

doi: 10.1016/j.omtn.2020.09.040

Figure Lengend Snippet: Figure 6. MALAT1 Affects Cancer Biology Behavior (A) MALAT1 knockdown cells showed decreased cell in- vasion ability. Ordinary one-way ANOVA, followed by Stu- dent’s t test. ****p < 0.0001 compared with the shCT control group. (B) Cell proliferation assay. Total cells were counted after 24 h, 48 h, 72 h, and 96 h. Ordinary one-way ANOVA, followed by Student’s t test. ****p < 0.0001 compared with the shCT control group. (C) Wound healing assay. Scale bar = 50 mm. MALAT1 knockdown cells showed slower wound healing capability indicating slower migration ability. (D) The putative model of MALAT1 as an epigenetic messenger in controlling cancer metabolism. MALAT1 is aberrantly expressed in HCC mitochondria, where it binds to mtDNA and alters mtDNA methylation and mitochondrial function, including mitochondria synthesis, metabolism, mitophagy, ROS regulation, and apoptosis.

Article Snippet: Antibodies used for western blot were: Anti-LAMP1 antibody-Lysosome Marker (ab24170), Anti-Cyclophilin F antibody (ab110324), Mitophagy Antibody Sampler Kit (Cell Signaling Technology [CST], #43110), Bcl-2 (124) Mouse monoclonal antibody (mAb) (CST, #15071), Cytochrome c (D18C7) Rabbit mAb (CST, #11940), Caspase-3 antibody (CST, #9662), Bax (D2E11) Rabbit mAb (CST, #5023), b-Actin (8H10D10) Mouse mAb (CST, #3700), and BID antibody (CST, #2002).

Techniques: Knockdown, Control, Proliferation Assay, Wound Healing Assay, Migration, Methylation

Journal: Stem cell research

Article Title: CCL5/CCR1 axis regulates multipotency of human adipose tissue derived stromal cells.

doi: 10.1016/j.scr.2012.11.004

Figure Lengend Snippet: Fig. 4 ADSCs respond to CCL5. (A) ADSCs were stimulated with 50 ng/ml of CCL5 for 3 h (right panel) and subjected to immunostaining using CCR1 antibody (red) and DAPI (blue). Non-stimulated cells are shown in the left panel. (B) ADSCs were treated with different concentrations of CCL5 for 20 min. The levels of phospho-AKT, phospho-ERK, AKT, ERK and GAPDH proteins were detected by WB. (C) CCR1, CCR3 and CCR5 expression levels in two ADSC samples were measured in triplicates by qRT-PCR, normalized with GAPDH mRNA expression levels and set as 1 in sample V (left panel). ADSCs were stimulated with 50 ng/ml of CCL5 for 10 min or 1 h, the cells were lysed and subjected to WB analysis using phospho-ERK, phospho-AKT, ERK, AKT and GAPDH antibodies. (D) Migration capacity of ADSCs towards 50 ng/ml of CCL5 (left panel) was examined using a modified Boyden chamber. As a control, CCL5-free media were used (right panel). The migratory cells were stained and observed under a light microscope using 20× objective.

Article Snippet: The antibodies used in the current study were: CCR1 (CKR-1 (H-52), sc-7934, 1:1500) (Santa Cruz Biotechnology, CA, USA), AKT (9272, 1:1000) (Cell Signaling Technology, Beverly, MA, USA), phospho-AKT (Ser473) (9271, 1:1000) (Cell Signaling Technology), ERK (sc-154-G, 1:1500) (Santa Cruz Biotechnology), phospho-ERK (sc-7383, 1:1500) (Santa Cruz Biotechnology), SOX2 (1:750) (CeMines, Evergreen, CO, USA), and GAPDH (G8795, 1:8000) (Sigma).

Techniques: Immunostaining, Expressing, Quantitative RT-PCR, Migration, Modification, Control, Staining, Light Microscopy

Journal: PloS one

Article Title: Differential Inhibition of the TGF-β Signaling Pathway in HCC Cells Using the Small Molecule Inhibitor LY2157299 and the D10 Monoclonal Antibody against TGF-β Receptor Type II.

doi: 10.1371/journal.pone.0067109

Figure Lengend Snippet: Figure 2. Effect of LY2109761 and LY2157299 on SMAD2 phosphorylation upon TGF-b1. (A) Two different HCC cell lines HLE and HLF were pretreated for 16 hours with 100 nM of LY2109761 or LY2157299 and then stimulated with 2 ng of TGF-b1 for 30 min. Phosphorylation of SMAD2 was detected in immunoblots using a rabbit polyclonal antibody directed against Phospho-Smad2 (Ser465/467). (B) Effect of LY2109761 and LY2157299 on E-cadherin expression in HLE and HLF. Increasing expression of E-cadherin is observed in HLE and HLF cells after treatment with both inhibitors for 48 hours. doi:10.1371/journal.pone.0067109.g002

Article Snippet: Rabbit polyclonal anti-Phospho-SMAD2, rabbit polyclonal anti-total SMAD2, mouse monoclonal antiPhospho p44/42, rabbit polyclonal anti p44/p42, rabbit polyclonal anti-Phospho-Akt, and rabbit polyclonal anti-total Akt antibodies were purchased from Cell Signalling technology, Inc. (Beverly, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: PloS one

Article Title: Differential Inhibition of the TGF-β Signaling Pathway in HCC Cells Using the Small Molecule Inhibitor LY2157299 and the D10 Monoclonal Antibody against TGF-β Receptor Type II.

doi: 10.1371/journal.pone.0067109

Figure Lengend Snippet: Figure 6. Effect of LY2157299 and D10 on SMAD2 activity in HCC cells. (A) HepG2 and HLE cells were preincubated with LY2157299 (10 mM) or D10 (25 ng/mL) and then stimulated with TGF-b1 (5 ng/mL) in the presence or absence of LY2157299 or D10 for 30 min or 24 hours. Proteins were extracted and western blotting analysis was performed. Phosphorylation of SMAD2 was detected using a rabbit polyclonal antibody directed against phospho-Smad2 (Ser465/467). (B) HepG2 and HLE cells were silenced for SMAD2, or non-silencing control and cell migration assay was performed. Migrated cells were indicated as migratory index versus non TGF-b1-stimulated control. ***P,0.001 versus TGF-b1-stimulated control-siRNA. doi:10.1371/journal.pone.0067109.g006

Article Snippet: Rabbit polyclonal anti-Phospho-SMAD2, rabbit polyclonal anti-total SMAD2, mouse monoclonal antiPhospho p44/42, rabbit polyclonal anti p44/p42, rabbit polyclonal anti-Phospho-Akt, and rabbit polyclonal anti-total Akt antibodies were purchased from Cell Signalling technology, Inc. (Beverly, MA, USA).

Techniques: Activity Assay, Western Blot, Phospho-proteomics, Control, Cell Migration Assay

Journal: PloS one

Article Title: Differential Inhibition of the TGF-β Signaling Pathway in HCC Cells Using the Small Molecule Inhibitor LY2157299 and the D10 Monoclonal Antibody against TGF-β Receptor Type II.

doi: 10.1371/journal.pone.0067109

Figure Lengend Snippet: Figure 10. Immunofluorescence staining of p-Smad2 in frozen HCC tissues. Tissues from 30 patients with HCC were stained using an anti-p- Smad2 polyclonal antibody (red) and alpha smooth muscle actin with the monoclonal antibody clone 1A4 (green). In 20 out of 30 HCC tissues (69.0%) positivity for p-Smad2 was obtained; representative cases of p-Smad2-positive (patient #1 and #3) or -negative (patients #7 and #9) tissues are shown. doi:10.1371/journal.pone.0067109.g010

Article Snippet: Rabbit polyclonal anti-Phospho-SMAD2, rabbit polyclonal anti-total SMAD2, mouse monoclonal antiPhospho p44/42, rabbit polyclonal anti p44/p42, rabbit polyclonal anti-Phospho-Akt, and rabbit polyclonal anti-total Akt antibodies were purchased from Cell Signalling technology, Inc. (Beverly, MA, USA).

Techniques: Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes.

doi: 10.1242/jcs.097600

Figure Lengend Snippet: Fig. 7. p90RSK is required for EGF-induced cell migration and HD remodelling. (A) HaCaT cells were pretreated for 1 h, before a migration assay, without (2BI) or with 10 mM BID-1870 (+BI), a pharmacological inhibitor of p90RSK. Cells were either left unstimulated (Ctrl; black bars) or treated with 10 nM EGF (grey bars) and migration assay was performed for 120 min. Cell velocity was quantified (mm/min) as described in Materials and Methods. Values are means 6 s.e.m., n53 (.100 cells per experiment); *P,0.001. (B,C) HaCaT cells were co-transfected with a myr-p90RSK plasmid (myr-p90RSK) and with a plasmid coding for GFP protein (GFP). Immunostaining of co-transfected cells using p90RSK antibody is presented in B (red) and shows that 95% of GFP-positive cells have high p90RSK expression. Forty-eight hours after transfection, cell migration analysis was performed on GFP-positive or -negative cells, unstimulated (Ctrl) or treated with EGF (EGF, 10 nM), UTP (100 mM UTP) or both (EGF/UTP). Data are presented in C and expressed as the mean cell velocity obtained from several independent experiments. Values are means 6 s.e.m., n53; *P,0.001. (D) HaCaT cells were stimulated for the indicated times with 100 mM UTP (UTP), 10 nM EGF (EGF) or both (EGF/UTP). Cell lysates were analysed by western blotting using an anti-phospho-p90RSK (p-p90RSK) antibody as indicated. Immunoblots were then stripped and reprobed with an anti-p90RSK antibody (p90RSK total; upper panel). Histograms show the fold increase of p90RSK phosphorylation normalised to control, untreated cells (n53; *P,0.05). (E) HaCaT cells were transfected with a myr-p90RSK plasmid (myr-p90RSK) or with an empty plasmid (vector) as described in Materials and Methods. Forty-eight hours after transfection, cells were left unstimulated (–, NT) or stimulated with 10 nM EGF (E) or 100 mM UTP (U) or both (E+U) for 5 min. Cell lysates were analysed by western blotting using anti-p90RSK (p90RSK), anti-b4 integrin (b4) or anti- phospho-p90RSK (p-p90RSK) antibodies. After densitometric analysis, the level of p90RSK phosphorylation was calculated as the ratio of p-p90RSK to b4 integrin (p-p90RSK/b4). The values obtained (lower panel) were expressed as fold increase of control (NT, cells untreated and transfected with vector; n53; *P,0.05. (F) HaCaT cells were then either left untreated (2BI) or preincubated for 1 h with 10 mM BID-1870 (+BI). As indicated, cells were then stimulated for 45 min with or without 10 nM EGF. HDs were visualised by confocal microscopy using an anti-b4 integrin antibody. Scale bar: 20 mm. Quantification (right) of HD-positive cells was performed as described in Materials and Methods. Values are means 6 s.e.m. of three independent experiments (.100 cells per experimental conditions); *P,0.001. (G) HaCaT cells were transfected to express the active mutant myr-p90RSK (myr-p90RSK) as described in Materials and Methods. Twenty-four hours after transfection, cells were seeded on collagen-I-coated coverslips for 24 h. Cells were then either left unstimulated (Ctrl) or treated for 45 min with EGF (10 nM), UTP (100 mM) or both. HD were visualised by confocal microscopy using an anti-b4 integrin antibody. The level of p90RSK expression was revealed using an anti-p90RSK antibody. Quantification of HD-positive cells was performed as described in Materials and Methods on cells expressing high levels of myr-p90RSK and cells expressing low levels of myr-p90RSK. Values are means 6 s.e.m. of three independent experiments (.100 cells per experimental conditions); *P,0.001.

Article Snippet: For immunoblotting, rabbit polyclonal antibodies against EGF receptor (EGF-R), phospho-EGF receptor (Tyr845, Tyr992, Tyr1045 or Tyr1068), Elk-1, phospho-Elk1, RSK1/RSK2/RSK3 and rabbit monoclonal antibodies against phospho-MEK1/2, phospho-p44/42 MAPK (p-ERK1/2), p44/42 MAPK (ERK1/2), phospho-p90RSK, p90RSK, phospho-c-Raf, were purchased from Cell Signaling Technology.

Techniques: Migration, Transfection, Plasmid Preparation, Immunostaining, Expressing, Western Blot, Phospho-proteomics, Control, Confocal Microscopy, Mutagenesis

Journal: Journal of cell science

Article Title: P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes.

doi: 10.1242/jcs.097600

Figure Lengend Snippet: Fig. 8. Proposed model of UTP involvement in HD formation. During wound healing, inflammatory cells, macrophages, endothelial cells and fibroblasts release numerous growth factors, cytokines, chemokines and extracellular nucleotides in the microenvironment. Together, these molecules form a complex signalling network that governs keratinocyte behaviour. This schematic model depicts the crosstalk signalling pathways in HaCat keratinocytes co-stimulated by EGF and UTP. EGF activates EGF receptor (EGFR), Raf–MEK1/2–ERK1/2 signalling pathway and its effector p90RSK to induce b4 integrin phosphorylation (Frijns et al., 2010), and thus HD disruption. HD dismantling participates to the promotion of cell migration. P2Y2 receptor (P2Y2R) and Gaq activation by UTP transduces an unidentified pathway leading to the inhibition of the MAPK signalling cascade downstream of Raf. The resultant inhibition of p90RSK stabilises b4 integrin in HD, a crucial event for HaCat keratinocyte migration inhibition by UTP.

Article Snippet: For immunoblotting, rabbit polyclonal antibodies against EGF receptor (EGF-R), phospho-EGF receptor (Tyr845, Tyr992, Tyr1045 or Tyr1068), Elk-1, phospho-Elk1, RSK1/RSK2/RSK3 and rabbit monoclonal antibodies against phospho-MEK1/2, phospho-p44/42 MAPK (p-ERK1/2), p44/42 MAPK (ERK1/2), phospho-p90RSK, p90RSK, phospho-c-Raf, were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Disruption, Migration, Activation Assay, Inhibition

Journal: The Journal of cell biology

Article Title: Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment.

doi: 10.1083/jcb.201005140

Figure Lengend Snippet: Figure 2. Neutrophil migration requires flotillin micro domains. (a) fMLP (1 µg/mouse) was injected into subcutaneous air pouches. At the times indicated, mice were culled and the air pouch lavaged with PBS. Leukocyte counts were per- formed based on cell morphology after staining with Wright- Giemsa; a total of 200 cells were counted per slide. +/+, wild type; /, flotillin 1 knockout. Results are mean ± SEM (n = 3–7); these data are representative of three individual ex- periments. *, P < 0.05. (b) Cellular infiltration of the air pouch was monitored by flow cytometry using CD11b and Ly6G expression as markers of neutrophils (Ly6GhiCD11b+ve) and monocytes (Ly6GloCD11b+ve), as shown by the blue gating regions. Numbers in blue are the percentages of total cells in each gate. (c) Absolute cell numbers calculated from FACS analysis of infiltrating cells as in panel b. Results are mean ± SEM (n = 3–7); data are representative of three individual experiments. *, P < 0.05.

Article Snippet: D ow nloaded from http://rupress.org/jcb/article-pdf/191/4/771/1564314/jcb_201005140.pdf by guest on 01 January 2025 JCB • VOLUME 191 • NUMBER 4 • 2010 778 779Function of flotillins in neutrophil recruitment • Ludwig et al. Phospho-myosin light chain 2 (Ser19; 3671) and phospho-myosin light chain 2 (Thr18/Ser19; 3674) antibodies were from Cell Signaling Technology.

Techniques: Migration, Injection, Staining, Knock-Out, Flow Cytometry, Expressing

Journal: The Journal of cell biology

Article Title: Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment.

doi: 10.1083/jcb.201005140

Figure Lengend Snippet: Figure 5. Flotillin microdomains are required for uropod formation and activation of myosin IIa. (a) Migration of control and flotillin 1/ neutrophils into Matrigel in a gradient of MIP2. The dashed white line indicates the top of the Matrigel layer. Triangles represent the direction of the MIP2 concentration gradient; vertical bar, 50 µm. Images are maximum intensity projections derived from 20–25 confocal Z-slices. (b) Quantification of neutrophil migration into Matrigel. The proportion of cells entering the Matrigel was derived by quantifying the fluorescence above and below the dashed white line in panel a. Combined data from four separate experiments are shown. (c) Indirect immunofluorescence with antibodies against flotillin 1 and -spectrin to show colocalization in the uropod of fMLP-stimulated neutrophils. Bar, 20 µm. (d) Comparison of uropod formation in control (+/+) and flotillin 1/ neutrophils. Cells were fixed 15 min after stimulation with fMLP and labeled with antibodies against CD44. Arrows indicate structures defined as uropods. Bar, 20 µm.

Article Snippet: D ow nloaded from http://rupress.org/jcb/article-pdf/191/4/771/1564314/jcb_201005140.pdf by guest on 01 January 2025 JCB • VOLUME 191 • NUMBER 4 • 2010 778 779Function of flotillins in neutrophil recruitment • Ludwig et al. Phospho-myosin light chain 2 (Ser19; 3671) and phospho-myosin light chain 2 (Thr18/Ser19; 3674) antibodies were from Cell Signaling Technology.

Techniques: Activation Assay, Migration, Control, Concentration Assay, Derivative Assay, Fluorescence, Immunofluorescence, Comparison, Labeling

Journal: Translational Oncology

Article Title: Epithelial Ovarian Cancer-Induced Angiogenic Phenotype of Human Omental Microvascular Endothelial Cells May Occur Independently of VEGF Signaling 1 2

doi:

Figure Lengend Snippet: Ovarian cancer cell secretome increases HOMEC cellular responses and contains a range of potential proangiogenic proteins. (A) Proliferation of HOMECs treated with CM from SKOV3 and A2780 cells for 24, 48, or 72 hours assessed using the WST-1 assay. Controls received basal medium only. **P ≤ .01 and ***P ≤ .001 versus control levels (100%). (B) Migration of HOMECs in co-culture with ovarian cancer cells and HDFs (co-culture control) assessed by the ThinCert migration assay. After 24 hours, fluorescence of migrated cells was quantified. ***P ≤ .001 versus control (100%) and ###P ≤ .001 versus HDFs. (C) HOMEC Oris cell migration after 48 hours in the presence of VEGFA165 (20 ng/ml) ± SU5416 (10 µM). Controls received medium alone. ***P ≤ .001 versus control (100%) and ###P ≤ .001 versus VEGFA165. (D) Migration of HOMECs in co-culture with ovarian cancer cells ± anti-VEGFA antibody (500 ng/ml) or SU5416 (10 µM) assessed by the ThinCert migration assay. ***P ≤ .001 versus control (100%), **P ≤ .01 versus control (100%), and NS versus A2780 or SKOV3. (E) VEGF- and EOC cell-induced tube-like structure formation of HOMECs and the effects of the SU5416 inhibitor. HOMECs were plated onto fibrin matrices and exposed to either VEGFA165 or EOC cells in co-culture ± SU5416 (10 µM). Negative controls received medium alone. Tube-like structure formation was quantified. *P ≤ .05, **P ≤ .01, and ***P ≤ .001 versus control (100%), ###P ≤ .001 versus VEGF, and NS versus SKOV3/A2780. For A to E, data are presented as means ± SD. P values were calculated by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. (F) Shotgun proteomics of SKOV3 CM by the 4800 MALDI TOF/TOF analyzer. Significant results were determined by selecting proteins that matched with two or more peptides with total ion CIs of >95%, followed by a literature search to determine proteins with a possible role in angiogenesis. (G) Analysis of CL, CD, IGFBP-7, and VEGF concentration in CM from SKOV3 and A2870. Commercially available ELISAs were used for determination of VEGF, CD, and CL concentrations, whereas the IGFBP-7 ELISA protocol was developed in-house. All experiments were carried out in duplicate on three separate samples. B/D, below detection limit.

Article Snippet: Supplementary Figures and Tables: Click here to view. (416K, pdf) Reagents and Equipment The following reagents and equipment were used: ELISA-grade BSA, human thrombin, EC growth supplement from bovine neural tissue, CD, CL, MMP9, thrombin, and human plasma (Cat. No. T7009) from Sigma; DMEM-BS from Gibco (Paisley, United Kingdom); WST-1 from Roche (Burgess Hill, United Kingdom); PF04217903 and PlGF-1 from R&D Systems; VEGFA 165 , Proteome Profiler human angiogenesis array, CD DuoSet ELISA, and mouse anti-human monoclonal IGFBP-7 antibody (clone 192520; capture antibody); streptavidin-HRP conjugate and ELISA substrate reagent; HDFs, fibroblast growth medium 2, MV2 endothelial medium kit, and HGF from PromoCell/PromoKine (Heidelberg, Germany); calcein AM from eBioscience (Hatfield, United Kingdom); MaxiSorp microplates (Nunc); IGFBP-7, rabbit polyclonal anti-human IGFBP-7 (biotinylated, detection antibody), and CD ELISA from Abcam (Cambridge, United Kingdom); SKOV3 and A2780 cell lines from the European Collection of Cell Cultures; ThinCerts (8.0-µm pore diameter) and 24-well plates (Cat. No. 662638) from Greiner Bio One (Stonehouse, United Kingdom); Oris Cell migration assay (Cat. No. cma1.101) from Platypus Technologies (Fitchburg, WI); fibrinogen, human plasma (Cat. No. 341578), and Amicon centrifugal filters (Cat. No. UFC800324) from Merck Millipore; anti-VEGFA antibody (Cat. No. 07-1419; recognizes isoforms 121, 165, and 189 of VEGFA); VectaSpin Micro (Cat. No. 6835-3001) from GE Healthcare/Whatman.

Techniques: WST-1 Assay, Migration, Co-Culture Assay, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Translational Oncology

Article Title: Epithelial Ovarian Cancer-Induced Angiogenic Phenotype of Human Omental Microvascular Endothelial Cells May Occur Independently of VEGF Signaling 1 2

doi:

Figure Lengend Snippet: Bioactive effects of fractionated A2780 and SKOV3 CM on HOMEC migration. (A and C) Relative protein content of fractionated CM from SKOV3 (A) and A2780 (C) assessed by absorbance at 280 nm. A 100 ≤ 10 kDa prefraction of both tumor CM was separated by size exclusion gel filtration, and 1-ml fractions were collected. Distinct protein peaks were observed in both profiles. (B and D) Fractions 11 and 12 of CM from SKOV3 (B) and A2780 (D) obtained by FPLC increase HOMEC migration. The bioactivity of protein-rich fractions (11 and 12) produced by FPLC was assessed in HOMECs using the Oris Cell migration assay. The experiment was performed on three separate cell isolations in heptaplicate. Data are presented as means ± SD. *P ≤ .05, **P ≤ .05, and ***P < .001 versus control (100%); P values were calculated by the Mann-Whitney U test.

Article Snippet: Supplementary Figures and Tables: Click here to view. (416K, pdf) Reagents and Equipment The following reagents and equipment were used: ELISA-grade BSA, human thrombin, EC growth supplement from bovine neural tissue, CD, CL, MMP9, thrombin, and human plasma (Cat. No. T7009) from Sigma; DMEM-BS from Gibco (Paisley, United Kingdom); WST-1 from Roche (Burgess Hill, United Kingdom); PF04217903 and PlGF-1 from R&D Systems; VEGFA 165 , Proteome Profiler human angiogenesis array, CD DuoSet ELISA, and mouse anti-human monoclonal IGFBP-7 antibody (clone 192520; capture antibody); streptavidin-HRP conjugate and ELISA substrate reagent; HDFs, fibroblast growth medium 2, MV2 endothelial medium kit, and HGF from PromoCell/PromoKine (Heidelberg, Germany); calcein AM from eBioscience (Hatfield, United Kingdom); MaxiSorp microplates (Nunc); IGFBP-7, rabbit polyclonal anti-human IGFBP-7 (biotinylated, detection antibody), and CD ELISA from Abcam (Cambridge, United Kingdom); SKOV3 and A2780 cell lines from the European Collection of Cell Cultures; ThinCerts (8.0-µm pore diameter) and 24-well plates (Cat. No. 662638) from Greiner Bio One (Stonehouse, United Kingdom); Oris Cell migration assay (Cat. No. cma1.101) from Platypus Technologies (Fitchburg, WI); fibrinogen, human plasma (Cat. No. 341578), and Amicon centrifugal filters (Cat. No. UFC800324) from Merck Millipore; anti-VEGFA antibody (Cat. No. 07-1419; recognizes isoforms 121, 165, and 189 of VEGFA); VectaSpin Micro (Cat. No. 6835-3001) from GE Healthcare/Whatman.

Techniques: Migration, Filtration, Produced, Cell Migration Assay, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Weak Power Frequency Magnetic Field Acting Similarly to EGF Stimulation, Induces Acute Activations of the EGFR Sensitive Actin Cytoskeleton Motility in Human Amniotic Cells

doi: 10.1371/journal.pone.0087626

Figure Lengend Snippet: Vinculin stained: A–D; fascin stained: E–H; Arp3 stained: I–L. A: negative control (N-con); B: sham-exposed; C: exposed to 0.4 mT power frequency MF (MF); D: pre-treated with 100 nM EGF (EGF); E: negative control (N-con); F: sham-exposed; G: exposed to 0.4 mT power frequency MF (MF); H: pre-treated with 100 nM EGF (EGF); I: negative control (N-con); J: sham-exposed; K: exposed to 0.4 mT power frequency MF; L: pre-treated with 100 nM EGF (EGF)); M: summary of the gray value analysis of the proportion of vinculin-associated adhesion spots to the total cell area, analyzed by ImageJ; compared with N-con and Sham P<0.05 (*); N: summary of the gray value analysis of fascin and Arp3 by ImageJ analysis, compared with N-con and Sham P<0.05 (*). Bar in A–L: 10 µm. The detailed information of experimental conditions and repeating numbers of samples is seen in .

Article Snippet: The mouse monoclonal antibody to fascin (Cat.No: ab78487), anti-myosin light chain antibody [MLC] (Cat.No: ab97891), and anti-Arp3 antibody (Cat.No: ab56817) were from Abcam, and the vinculin antibody (Cat.No: 4650) was from Cell Signaling Technology.

Techniques: Staining, Negative Control

Journal: PLoS ONE

Article Title: Weak Power Frequency Magnetic Field Acting Similarly to EGF Stimulation, Induces Acute Activations of the EGFR Sensitive Actin Cytoskeleton Motility in Human Amniotic Cells

doi: 10.1371/journal.pone.0087626

Figure Lengend Snippet: A: F-actin/G-actin content in supernatant or precipitate; NC: negative control, 1 µM phalloidin was added when cracking the cells; PC: positive control, 1 µM cytochalasin-D was added when cracking the cells. B: gray value summary of actin content by software ImageJ, compared with N-con and Sham P<0.05 (*). C: focal adhesion-associated signal protein vinculin, filopodia-associated signal protein fascin, lamellipodia-associated signal protein Arp3, and stress fiber-associated signal protein MLC content. D: gray value summary of vinculin, fascin, and Arp3 by software ImageJ, compared with N-con and Sham P<0.05 (*). E: gray value summary of stress fiber-associated MLC by software ImageJ, compared with N-con and Sham P<0.05 (*). F: total content of actin and MLC in FL cells, compared with N-con and Sham. G: gray value of total content of actin and MLC protein in FL cells; compared with Sham P>0.05. The detailed information of experimental conditions and repeating numbers of samples is seen in .

Article Snippet: The mouse monoclonal antibody to fascin (Cat.No: ab78487), anti-myosin light chain antibody [MLC] (Cat.No: ab97891), and anti-Arp3 antibody (Cat.No: ab56817) were from Abcam, and the vinculin antibody (Cat.No: 4650) was from Cell Signaling Technology.

Techniques: Negative Control, Positive Control, Software

Journal: PLoS ONE

Article Title: Weak Power Frequency Magnetic Field Acting Similarly to EGF Stimulation, Induces Acute Activations of the EGFR Sensitive Actin Cytoskeleton Motility in Human Amniotic Cells

doi: 10.1371/journal.pone.0087626

Figure Lengend Snippet: A: Time-dependent recovery of F-actin content after withdrawing power frequency MFs. Sham: sham-exposed to power frequency MF for 0.5 hours; 0–2 h: exposed to power frequency MF for 0.5hours,field withdrawn and then sham-exposed for 0hour to 2hours; the summary of the gray value analysis of F-actin and cell area was analyzed by software ImageJ; compared with Sham, 0–1.5 h P<0.05 (*), 2 h P>0.05. B: content of vinculin, fascin, MLC and Arp3 proteins in FL cells 2 hours after withdrawing power frequency MF; C: gray value summary of vinculin, fascin, MLC and Arp3 content in FL cells 2 hours after exposing to MFs by software ImageJ, compared with Sham, P>0.05. The detailed information of experimental conditions and repeating numbers of samples is seen in .

Article Snippet: The mouse monoclonal antibody to fascin (Cat.No: ab78487), anti-myosin light chain antibody [MLC] (Cat.No: ab97891), and anti-Arp3 antibody (Cat.No: ab56817) were from Abcam, and the vinculin antibody (Cat.No: 4650) was from Cell Signaling Technology.

Techniques: Software

Journal: PLoS ONE

Article Title: Weak Power Frequency Magnetic Field Acting Similarly to EGF Stimulation, Induces Acute Activations of the EGFR Sensitive Actin Cytoskeleton Motility in Human Amniotic Cells

doi: 10.1371/journal.pone.0087626

Figure Lengend Snippet: The information of experimental conditions and repeating numbers of samples for each target.

Article Snippet: The mouse monoclonal antibody to fascin (Cat.No: ab78487), anti-myosin light chain antibody [MLC] (Cat.No: ab97891), and anti-Arp3 antibody (Cat.No: ab56817) were from Abcam, and the vinculin antibody (Cat.No: 4650) was from Cell Signaling Technology.

Techniques: Flow Cytometry, Western Blot, Control, Migration

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Fold changes in the microarray for all 35 genes were found to be differentially regulated by C. pneumoniae infection and satisfied the comparison filtering criteria

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Microarray, Infection, Comparison, Binding Assay, Coagulation

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: C. pneumoniae stimulates TLR2 expression in rVSMCs. (A) Validation of TLR2 gene expression profiling using quantitative real-time RT-PCR normalized to GAPDH expression. Data shown are mean values of PCR replicates from individual groups. (B) TLR2 mRNA expression at the indicated time points after C. pneumoniae infection. rVSMCs infected with C. pneumoniae (5 × 105 IFU) for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h or control cells were lysed to prepare the total RNA. cDNA was amplified for 30 cycles, and PCR products were separated by agarose gel electrophoresis. Relative TLR2 mRNA expression levels determined by standard PCR (n = 3 replicates per group). (C) C. pneumoniae infection stimulates TLR2 protein expression in rVSMCs. Cells were infected with C. pneumoniae for 0 h, 12 h, or 24 h. Cell lysates were separated by SDS-PAGE, and blots were probed with anti-TLR2 and anti-β-actin antibodies, followed by donkey anti-goat IgG-HRP and goat anti-mouse IgG-HRP antibodies, and developed with enhanced chemiluminescence (ECL).

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing, Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Infection, Control, Amplification, Agarose Gel Electrophoresis, SDS Page

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Confocal microscopy analysis of the TLR2 expression pattern in C. pneumoniae-infected rVSMCs. rVSMCs were grown in coverslips and infected with C. pneumoniae for 60 h and then were fixed, permeabilized, and stained using specific antibodies. C. pneumoniae inclusions were observed using a mouse polyclonal antibody to C. pneumoniae. Fluorescence micrographs were stained with TLR2-specific antibody. Cell nucleus was stained with DAPI (4′,6-diamidino-2-phenylindole).

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Confocal Microscopy, Expressing, Infection, Staining, Fluorescence

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Effects of TLR2 on rVSMC migration induced by C. pneumoniae infection. The TLR2-neutralizing antibody was added 1 h before C. pneumoniae infection. (A) Wound healing assay. “Scratch wounds” were created by scraping the confluent cell monolayer with a sterile pipette tip, and then cells were infected with C. pneumoniae at an infectious dose of 5 × 105 IFU. Photographs were taken of the same wounded area of each well at 0 h and 24 h. The scratched regions were photographed under an inverted Nikon microscope (×100 magnification) at 24 h after C. pneumoniae infection. Migration velocity is presented as a ratio of the cellular recoverage area to the whole wound area. *, P < 0.05 versus control; **, P < 0.05 versus C. pneumoniae infection group. (B) Transwell migration assay. Cell morphology was observed by staining with 0.1% crystallin violet. The number of cells that had migrated through the pores was quantified by counting nine independent visual fields using a microscope (×200 magnification). *, P < 0.05 versus control; **, P < 0.05 versus the C. pneumoniae infection group.

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Migration, Infection, Wound Healing Assay, Sterility, Transferring, Microscopy, Control, Transwell Migration Assay, Staining

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: The TLR2-neutralizing antibody suppresses Akt phosphorylation induced by C. pneumoniae infection. rVSMCs cultured for 24 h in 6-well plates were incubated with the TLR2-neutralizing antibody (10 μg/ml) and then infected with C. pneumoniae. Equal amounts of protein lysates were subjected to SDS-PAGE, and blots were probed with anti-Akt, anti-phospho-Akt (Ser 473), and anti-β-actin antibodies, followed by the corresponding horseradish peroxidase-conjugated secondary antibodies, and developed with ECL. P-Akt indicates phosphorylated Akt. *, P < 0.05 versus control; **, P < 0.05 versus the C. pneumoniae infection group.

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Phospho-proteomics, Infection, Cell Culture, Incubation, SDS Page, Control