Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Impaired cell migration and structural defects in myeloid cells overexpressing miR-30b and miR-142-3p.

doi: 10.1016/j.bbagrm.2020.194628

Figure Lengend Snippet: Fig. 5. miR-142-3p directly regulate genes involved in cell migration, phagocytosis and cell polarity. (A–C; Upper panel) Sequence alignment of predicted miR-142- 3p binding sites in the 3′UTR of VCL, Dab2 and Skap2. Only the binding sites with mfe < −20 kcal/mol are shown. (A–C; Lower panel) HEK293 cells were co- transfected with dual luciferase reporter plasmids containing 3′UTR of VCL, Dab2 and Skap2 or control vector and miR-142-3p or control mimic. After 36 h, cell lysates were prepared to measure renilla and firefly luciferase activity. Renilla activity was normalized to firefly activity and the ratios were subsequently normalized to empty vector transfected with miR-142-3p mimic set as 1. Data are expressed as ± SEM of four independent transfections. Student's t-test was conducted to calculate p-values. *p < 0.05, **p < 0.01, ***p < 0.001. (D) Expression of VCL, Dab2 and Skap2 in MΦ by western blot. Day 7 differentiated cells were transfected with miR-142-3p mimic or control mimic. Cell lysates were prepared after 36 h of transfection and VCL, Dab2 and Skap2 levels were detected by immunoblotting. The expression level of GAPDH was included as loading control. (E) The corresponding densitometric analysis by Image Lab software is also shown. Data are means ± SEM of three independent experiments (*p < 0.05, **p < 0.01; compared with the control mimic).

Article Snippet: All transfections were performed in quadruplicate using 0.5 μL Lipofectamine 2000 (Invitrogen), 120 ng dual luciferase reporter control (CmiT000001-MT06 miRNA Target clone control vector for pEZX-MT06) or plasmids containing human VCL (NM_003373.3; HmiT018467-MT06), human DAB2 (NM_001244871.1; HmiT055467-MT06) or human SKAP2 (NM_001303468.1; HmiT067400-MT06; All from GeneCopoeia Incorporated, Rockville, MD, USA) were co-transfected with a final concentration of 1 pmol, 5 pmol and 10 pmol of synthetic miR-142-3p or control mimics (10 pmol) (Qiagen).

Techniques: Migration, Sequencing, Binding Assay, Transfection, Luciferase, Control, Plasmid Preparation, Activity Assay, Expressing, Western Blot, Software

Journal: Biologics : Targets & Therapy

Article Title: Elevated Serum Vinculin in Patients with HBV/HCV-Associated Liver Cirrhosis and Hepatocellular Carcinoma: A Pilot Study

doi: 10.2147/BTT.S405500

Figure Lengend Snippet: Serum vinculin in the studied groups.

Article Snippet: Serum VCL level was assayed using Elabscience Human VCL (Vinculin) ELISA Kit, Cta No: E-EL-H1795, Elabscience Biotechnology Inc. USA.

Techniques:

Journal: Biologics : Targets & Therapy

Article Title: Elevated Serum Vinculin in Patients with HBV/HCV-Associated Liver Cirrhosis and Hepatocellular Carcinoma: A Pilot Study

doi: 10.2147/BTT.S405500

Figure Lengend Snippet: Correlation Between Serum Vinculin (VCL) Level with the Laboratory Parameters in the HCC Group (n = 51)

Article Snippet: Serum VCL level was assayed using Elabscience Human VCL (Vinculin) ELISA Kit, Cta No: E-EL-H1795, Elabscience Biotechnology Inc. USA.

Techniques:

Journal: PLoS ONE

Article Title: Estrogen-Dependent Uterine Secretion of Osteopontin Activates Blastocyst Adhesion Competence

doi: 10.1371/journal.pone.0048933

Figure Lengend Snippet: Transcripts of Vcl, Tln1 and Ptk2 that encode vinculin, talin and focal adhesion kinase, respectively, were expressed in blastocysts, however expression levels of these transcripts were not affected by the uterine receptivity. Total RNAs from three pools of 30 blastocysts for each groups were analyzed and representative results presented. ( B–D ) Immunofluorescence staining for VCL in E3.5 Ctrl, E4.0 Ctrl and E4.0 TAM blastocysts. VCL was labeled with Alexa 568 (red). F-actin and nuclei were stained with phalloidin-Atto 488 (green) and DAPI (blue), respectively. ( E–G ) Dual-immunofluorescence staining for integrin β1 and VCL was performed on E4.0 Ctrl blastocysts. Integrin β1 ( E ) and VCL ( F ) were labeled with Alexa 568 and FITC, respectively. Yellow signals in merged image ( G ) indicate co-localization of integrin β1 and VCL. ( H and I ) Immunolocalization of VCL in dormant or activated blastocysts. VCL proteins were labeled with Alexa 568 (red). F-actin and nuclei were stained with phalloidin-Atto 488 (green) and DAPI (blue), respectively. The signals of VCL were found at the surface of activated blastocyst ( I ), whereas that was hardly detectable in the dormant blastocysts ( H ). ( B–I ) Z-stack projection images of confocal laser-scanning microscopy are presented. Multiple samples were analyzed on at least three separate occasions. Representative results are presented.

Article Snippet: Blastocysts were incubated with rat anti-mouse integrin β1 (1∶50 dilution; catalog no. 550531; BD Pharmingen, Franklin Lakes, NJ), rabbit anti-human VCL (1∶100 dilution; catalog no. V4139; Sigma-Aldrich), rabbit anti-human OPN (1∶200 dilution; catalog no. ab8448; Abcam) or rabbit anti-mouse FN (1∶200 dilution; ab23750; Abcam) antibodies, then labeled with Allexa 568 or FITC.

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Confocal Laser Scanning Microscopy

Journal: PLoS ONE

Article Title: Estrogen-Dependent Uterine Secretion of Osteopontin Activates Blastocyst Adhesion Competence

doi: 10.1371/journal.pone.0048933

Figure Lengend Snippet: ( A and B ) Blastocysts collected from E4.0 TAM mice were incubated for 90 min with or without RetroNectin in DMEM/F12 medium. The effect of RetroNectin treatment on expression and distribution of VCL were then evaluated by immunofluorescence. Immunolocalization of VCL was visualized with Alexa 588 (red). F-actin and nuclei were stained with phalloidin-Atto 488 (green) and DAPI (blue), respectively. Z-stack projection images of confocal laser-scanning microscopy are presented. Multiple samples were analyzed on at least three separate occasions. ( C ) Expression of Spp1 , Fn1 and Vtn mRNAs in E3.5 Ctrl, E4.0 Ctrl and E4.0 TAM uteri were evaluated by RT-PCR. Total RNAs from three uteri for each group were analyzed. P.C., positive control. ( D–J ) Immunohistochemistry for OPN ( D–F ) and FN ( G–I ) was performed on uterine tissues from E3.5 Ctrl, E4.0 Ctrl or E4.0 TAM mice. Normal rabbit IgG served as a negative control ( J ). Uterine tissues from at least three animals for each group were analyzed. Representative results are presented. Black arrowheads: luminal epithelium, open arrowheads: glandular epithelium, st: stroma. ( K ) Western blotting for OPN in protein extracts from E3.5 Ctrl, E4.0 Ctrl and E4.0 TAM uteri. Uterine tissues from at least three animals for each group were analyzed. Representative blot is presented. β-actin was used as loading control. Four species of immunoreactive OPN were found at molecular weights of approximately 66 kDa, 62 kDa, 34 kDa and 31 kDa. ( L–P ) In situ hybridization for Spp1 transcripts on E4.0 uterine tissues. Antisense ( L and M ) and sense ( N ) probes for Spp1 mRNA were hybridized on uterine tissues from E4.0 Ctrl ( L and N ) and E4.0 TAM ( M ) mice. ( O and P ) Higher magnification images of L highlighting luminal epithelia ( O ) and glandular epithelia ( P ) of E4.0 Ctrl uterus. Note that the expression of Spp1 mRNA on E4.0 uterus ( L ) was more prominent in glandular epithelia ( P ) than that in luminal epithelia ( O ) and disrupted by TAM treatment ( M ). Sense probe did not show any signals on E4.0 uterus ( N ). Uterine tissues from at least three animals for each group were analyzed. Representative results are presented. Tissue sections were counter-stained with methyl green. Black arrowheads: luminal epithelium, open arrowheads: glandular epithelium, st: stroma.

Article Snippet: Blastocysts were incubated with rat anti-mouse integrin β1 (1∶50 dilution; catalog no. 550531; BD Pharmingen, Franklin Lakes, NJ), rabbit anti-human VCL (1∶100 dilution; catalog no. V4139; Sigma-Aldrich), rabbit anti-human OPN (1∶200 dilution; catalog no. ab8448; Abcam) or rabbit anti-mouse FN (1∶200 dilution; ab23750; Abcam) antibodies, then labeled with Allexa 568 or FITC.

Techniques: Incubation, Expressing, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy, Reverse Transcription Polymerase Chain Reaction, Positive Control, Immunohistochemistry, Negative Control, Western Blot, In Situ Hybridization

Journal: PLoS ONE

Article Title: Estrogen-Dependent Uterine Secretion of Osteopontin Activates Blastocyst Adhesion Competence

doi: 10.1371/journal.pone.0048933

Figure Lengend Snippet: ( A and B ) IVF-derived blastocysts were incubated with OPN in DMEM/F12 media for 10 min then transferred to culture medium without OPN. The effect of OPN-priming on blastocyst adhesiveness was monitored for rhodamine-FN binding at 10 min ( A ) and 60 min ( B ) after OPN treatment. ( C and D ) IVF-derived blastocysts were incubated with or without OPN in DMEM/F12 media for 90 min and examined for VCL expression ( C ) and AKT phosphorylation ( D ) by immunoblotting. Three pools of ten blastocysts for each control group (Control) and OPN treated group (OPN) were analyzed. β-actin (ACTB) and total AKT were used as loading controls for C and D , respectively. ( E and F ) IVF-derived blastocysts were incubated with or without OPN in DMEM/F12 for 90 min and examined for expression and distribution of VCL by immunofluorescence staining. ( G–I ) Effects of small molecule inhibitors for FAK ( G ), PI3K ( H ) or MEK1/2 ( I ) on the expression and distribution of VCL during OPN-induced blastocyst adhesiveness were examined by VCL immunofluorescence staining. ( E–I ) Immunolocalization of VCL was visualized with Allexa 568 (red). F-actin and nuclei were stained with phalloidin-Atto 488 (green) and DAPI (blue), respectively. Z-stack projection images of confocal laser-scanning microscopy are presented. ( A , B and E – I ) Multiple samples were analyzed on at least three separate occasions. Representative results are presented.

Article Snippet: Blastocysts were incubated with rat anti-mouse integrin β1 (1∶50 dilution; catalog no. 550531; BD Pharmingen, Franklin Lakes, NJ), rabbit anti-human VCL (1∶100 dilution; catalog no. V4139; Sigma-Aldrich), rabbit anti-human OPN (1∶200 dilution; catalog no. ab8448; Abcam) or rabbit anti-mouse FN (1∶200 dilution; ab23750; Abcam) antibodies, then labeled with Allexa 568 or FITC.

Techniques: Derivative Assay, Incubation, Binding Assay, Expressing, Western Blot, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy

Journal: Medical sciences (Basel, Switzerland)

Article Title: Plasma Concentrations of Vinculin versus Talin-1 in Coronary Artery Disease.

doi: 10.3390/medsci10030046

Figure Lengend Snippet: Figure 1. The vinculin and talin-1 interaction regulate integrin clustering at focal adhesions (This figure was adapted from Peng X et al. [11]).

Article Snippet: For the measurement of plasma vinculin concentrations, the frozen plasma was thawed, and then an enzyme-linked immunosorbent assay (ELISA) (Human Vinculin ELISA Kit, CUSABIO, Houston, TX, USA) was used.

Techniques:

Journal: Medical sciences (Basel, Switzerland)

Article Title: Plasma Concentrations of Vinculin versus Talin-1 in Coronary Artery Disease.

doi: 10.3390/medsci10030046

Figure Lengend Snippet: Figure 2. Plasma vinculin concentrations and CAD or the number of vessels with >50% stenosis. Vinculin concentrations were not different between the CAD(-) and CAD groups (p = 0.325). Moreover, there was no significant difference in vinculin concentrations among the four groups of CAD(-), 1VD, 2VD, or 3VD (p = 0.202). The central line shows the median, and the box shows the 25th to 75th percentiles.

Article Snippet: For the measurement of plasma vinculin concentrations, the frozen plasma was thawed, and then an enzyme-linked immunosorbent assay (ELISA) (Human Vinculin ELISA Kit, CUSABIO, Houston, TX, USA) was used.

Techniques: Clinical Proteomics

Journal: Medical sciences (Basel, Switzerland)

Article Title: Plasma Concentrations of Vinculin versus Talin-1 in Coronary Artery Disease.

doi: 10.3390/medsci10030046

Figure Lengend Snippet: Figure 4. Correlations of plasma talin-1 concentrations with vinculin or CRP concentrations. No correlation was found between vinculin and talin-1 concentrations (rs = −0.06, p = 0.308) (left). However, talin-1 concentrations correlated with CRP concentrations (rs = 0.31, p < 0.001) (right).

Article Snippet: For the measurement of plasma vinculin concentrations, the frozen plasma was thawed, and then an enzyme-linked immunosorbent assay (ELISA) (Human Vinculin ELISA Kit, CUSABIO, Houston, TX, USA) was used.

Techniques: Clinical Proteomics

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Impaired cell migration and structural defects in myeloid cells overexpressing miR-30b and miR-142–3p

doi: 10.1016/j.bbagrm.2020.194628

Figure Lengend Snippet: (A-C; Upper panel) Sequence alignment of predicted miR-142–3p binding sites in the 3′UTR of VCL, Dab2 and Skap2. Only the binding sites with mfe <−20 kcal/mol are shown. (A-C; Lower panel) HEK293 cells were co-transfected with dual luciferase reporter plasmids containing 3′UTR of VCL, Dab2 and Skap2 or control vector and miR-142–3p or control mimic. After 36 h, cell lysates were prepared to measure renilla and firefly luciferase activity. Renilla activity was normalized to firefly activity and the ratios were subsequently normalized to empty vector transfected with miR-142–3p mimic set as 1. Data are expressed as ± SEM of four independent transfections. Student’s t-test was conducted to calculate p-values. *p<0.05, **p<0.01, ***p<0.001. (D) Expression of VCL, Dab2 and Skap2 in MΦ by Western blot. Day 7 differentiated cells were transfected with miR-142–3p mimic or control mimic. Cell lysates were prepared after 36 h of transfection and VCL, Dab2 and Skap2 levels were detected by immunoblotting. The expression level of GAPDH was included as loading control. (E) The corresponding densitometric analysis by Image Lab software is also shown. Data are means ± SEM of three independent experiments (*p <0.05, **p<0.01; compared with the control mimic).

Article Snippet: All transfections were performed in quadruplicate using 0.5 μL Lipofectamine 2000 (Invitrogen), 120 ng dual luciferase reporter control (CmiT000001-MT06 miRNA Target clone control vector for pEZX-MT06) or plasmids containing human VCL ( {"type":"entrez-nucleotide","attrs":{"text":"NM_003373.3","term_id":"50593538","term_text":"NM_003373.3"}} NM_003373.3 ; HmiT018467-MT06), human DAB2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001244871.1","term_id":"349585059","term_text":"NM_001244871.1"}} NM_001244871.1 ; HmiT055467-MT06) or human SKAP2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001303468.1","term_id":"744066862","term_text":"NM_001303468.1"}} NM_001303468.1 ; HmiT067400-MT06; All from GeneCopoeia Incorporated, Rockville, MD, USA) were co-transfected with a final concentration of 1 pmol, 5 pmol and 10 pmol of synthetic miR-142–3p or control mimics (10 pmol) (Qiagen).

Techniques: Sequencing, Binding Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Expressing, Western Blot, Software