Journal: Tissue Engineering. Part A

Article Title: Opposite Spectrum of Activity of Canonical Wnt Signaling in the Osteogenic Context of Undifferentiated and Differentiated Mesenchymal Cells: Implications for Tissue Engineering

doi: 10.1089/ten.tea.2010.0133

Figure Lengend Snippet: (A) QRT-PCR showing the expression profiles of three downstream targets of canonical Wnt signaling, axin2, myc, and cyclin D1. Quantified mRNA values were normalized by the amounts of glyceraldehyde 3-phophate dehydrogenase mRNA, and results are given as fold induction (*p < 0.05). (B) Immunoblotting analysis of β-catenin performed on nuclear fractions revealed higher levels of nuclear β-catenin in ASCs, E16, and FpN1 cells. Membranes were stripped and reprobed with anti-Lamin B1 antibody to assess for equal loading and transfer of nuclear proteins fraction. Histogram represents the densitometric analysis of electrophoresis bands, and the relative intensities of bands were normalized to their respective loading control and set as 100%. The results are presented as the mean ± standard deviation of three independent experiments. (C) Indirect immunofluorescence staining detected the most intense nuclear staining in ASCs, E16, and FpN1. As negative control normal primary (irrelevant) mouse immunoglobulin G was used. Nuclear counterstaining was performed with DAPI. QRT-PCR, quantitative reverse transcription–polymerase chain reaction; mASCs, mouse adipose-derived stem cells; E16, embryonic-stage day 16 calvarial mesenchymal cells; FpN1, postnatal day 1 frontal bones-derived osteoblast; PpN1, postnatal day 1 parietal bone-derived osteoblast; FpN60, postnatal day 60 frontal bone-derived osteoblast; PpN60, postnatal day 60 parietal bone-derived osteoblast; DAPI, 4′,6-diamidino-2-phenylindole; Wnt, wingless. Color images available online at www.liebertonline.com/ten.

Article Snippet: Nuclear counterstaining was performed using Vectashield H-1200 mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories), and a Zeiss Axioplan microscope equipped with an Axiocam HRc digital camera was used for imaging.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Electrophoresis, Standard Deviation, Immunofluorescence, Staining, Negative Control, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: bioRxiv

Article Title: An Atlas of Phosphorylation and Proteolytic Processing Events During Excitotoxic Neuronal Death Reveals New Therapeutic Opportunities

doi: 10.1101/2020.06.15.151456

Figure Lengend Snippet: (A) CRMP2 is cleaved at sites near T509 in its C-terminal tail. Left inset: the abundance (M/L) ratios of the neo-N-terminal peptides at 30 and 240 min after glutamate treatment. Right inset: the abundance ratios of the identified phosphosites in the C-terminal tails of CRMP2 at 30 and 240 min after glutamate treatment. N.D.: not detected. Red scissors: cleavage sites. P in red sphere: phosphorylation. (B) A model depicting the new mechanism of dysregulation of neuronal CRMP2 during excitotoxicity uncovered by our proteomic findings. In control neurons, CRMP2 undergoes hierarchical phosphorylation by Cdk5 and GSK3 at sites in the C-terminal tail. Cdk5 phosphorylates the priming site S522. Upon phosphorylation, pS522 binds GSK3, which catalyses processive phosphorylation of CRMP2 at three other sites in the order of S518, T514 and T509. In excitotoxic neurons, cleavage of CRMP2 generates a long truncated CRMP2 fragment that lacks the priming site S522, abolishing S522 phosphorylation by Cdk5 and in turn suppressing processive phosphorylation of S518, T514 and T509 by GSK3. The truncation and lack of phosphorylation at T509, T514 and S518 may contribute to the accumulation of the immunoreactive CRMP2 signals at the dendritic blebs shown in panel E. (C) Structure of a phosphomimetic mutant of CRMP2 (PDB accession: 5yz5). Dotted line shows the disordered C-terminal tail region. (D) Western blots of lysates from control and glutamate-treated neurons probed with anti-CRMP2, anti-pT509 CRMP2 and tubulin antibodies. Asterisks: potential hyper-phosphorylated forms of intact CRMP2 detected by the anti-CRMP2 and anti-pT509 CRMP2 antibodies. (E) Fluorescence microscopy images showing actin (phalloidin), CRMP2 and nuclei (DAPI) in control and glutamate-treated neurons. White arrows indicate dendritic blebs. The close-up views of the images in the rectangles marked by white dotted lines are shown. Inset: The number of dendritic blebs per mm 2 in control and the glutamate treated neurons in three biological replicates. **: p < 0,01; ***: p <0.001.

Article Snippet: For all experiments, DNA counterstain DAPI (Molecular Probes, Thermo Fisher Scientific) was applied before coverslipping with ProLong gold anti-fade reagent (Invitrogen).

Techniques: Phospho-proteomics, Control, Mutagenesis, Western Blot, Fluorescence, Microscopy

Journal: International journal of andrology

Article Title: Gper and ESRs are expressed in rat round spermatids and mediate oestrogen-dependent rapid pathways modulating expression of cyclin B1 and Bax.

doi: 10.1111/j.1365-2605.2010.01100.x

Figure Lengend Snippet: Figure 1 Expression of G-protein-coupled oestrogen receptor 1 (Gper) in round spermatids (RS). (a) Reverse transcription-polymerase chain reac- tion analysis of Gper in RS. Mouse spermatogonia GC1 (GC1) cells were used as positive control; negative control ()) contained water instead of cDNA. L19 was used as housekeeping gene. Size in base pair of amplified fragments is indicated. (b) Western blot analysis of Gper was performed on 50 lg of total proteins extracted from RS or from GC1 cells. Blots are representative of three independent experiments with similar results. GAPDH was used as loading control. (c) RS were fixed and incubated with polyclonal anti-Gper antibody (1 : 50) followed by incubation with goat anti-rabbit streptavidine-fluoresceine-conjugated secondary antibody as described in ‘Materials and methods’ (magnification, ·40). Strong immu- nofluorescence was observed in the cytoplasm of RS (part 5). Negative control slides showed immunonegative reaction for Gper (antibody) in RS (part 2). Nuclei of RS were visualized by DAPI staining (parts 1 and 4). Bright field (BF) contrast is shown (parts 3 and 6). The three-dimensional slice view helps to locate spatially the fluorescent signals in the three-dimensional view (3D).

Article Snippet: After being washed with PBS, all slides were cover-slipped with mounting medium containing 4¢-6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnology).

Techniques: Expressing, Reverse Transcription, Positive Control, Negative Control, Western Blot, Control, Incubation, Staining

Journal: International journal of andrology

Article Title: Gper and ESRs are expressed in rat round spermatids and mediate oestrogen-dependent rapid pathways modulating expression of cyclin B1 and Bax.

doi: 10.1111/j.1365-2605.2010.01100.x

Figure Lengend Snippet: Figure 2 Expression of oestrogen receptor (ESR)1 and ESR2 in round spermatids (RS). Reverse-transcription-polymerase chain reac- tion (RT-PCR) analysis of ESR1 (a) and ESR2 (b). R2C cells were used as positive control; negative control ()) contained water instead of cDNA. L19 was used as housekeeping gene. Size in base pair of amplified fragments is indicated. Western blot analysis of ESR1 (c) and ESR2 (d) was performed on 50 lg of total proteins extracted from RS or from R2C cells. Blots are representative of three inde- pendent experiments with similar results. GAPDH was used as loading control. (e) ESR1 and ESR2 optical densities were normalized to GAPDH optical densities in the same lanes. Normalized values were graphed as ESR1 ⁄ ESR2 ratio. (f) RS were fixed and incu- bated with polyclonal anti-ESR1 antibody (1 : 50) followed by incubation with goat anti-rabbit Alexa-F488-conjugated secondary antibody as described in the ‘Materials and methods’ (magnification, ·60). Strong immu- nofluorescence was observed mainly in the cytoplasm of RS. Nuclei of RS were visualized by DAPI staining (red line = 10 lm).

Article Snippet: After being washed with PBS, all slides were cover-slipped with mounting medium containing 4¢-6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnology).

Techniques: Expressing, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Western Blot, Control, Incubation, Staining

Journal: bioRxiv

Article Title: Tandem Requirement for Full Renal D 1 R and D 5 R Activity

doi: 10.1101/736611

Figure Lengend Snippet: A & B : HEK-293 cells were transiently transfected with D 1 R::RFP and D 5 R::GFP and then grown on a cover slip or in a polarized state in a Transwell ™ insert; the colocalization of these receptors was evaluated via laser scanning confocal microscopy. Wheat germ agglutinin (WGA) tagged with Alexa Fluor□ 647 was used to visualized the plasma membrane, while DAPI (#H-1500, Vector Laboratories, Burlingame, CA) was used to visualized the nuclei. Colocalization was observed as discrete areas of white (red+green+blue) or yellow (red+green). Serial images along the X, Y, and Z axes were obtained in cells grown in Transwell ® and stitched together to show 3D colocalization of the receptors. 630x magnification, scale bar = 10 μm, n=3 independent experiments. C : hRPTCs were double-transfected with D 1 R and D 5 R tagged with the C and N termini of the fluorescent protein EYFP, respectively. The cells were grown on cover slips for 48 hours post-transfection, serum-starved for 2 hrs, and then prepared for confocal microscopy. The interaction of the tagged receptors results in the reconstitution and fluorescence of EYFP (pseudocolored green). An overlay of the BiFC signal and the nucleus (pseudocolored blue) is shown to indicate the distribution of the D 1 R-D 5 R complexes, 630x magnification, scale bar =10 μm, n=3 independent experiments. Transfection of D 1 R-EYFP alone was used as negative control.

Article Snippet: The tissues were counterstained with wheat germ agglutinin (WGA) tagged with Alexa Fluor ™ 647 (#A-31573, ThermoFisher Scientific, Waltham, MA) and DAPI (#H-1500, Vector Laboratories, Burlingame, CA) to visualize the plasma membrane and nuclei, respectively.

Techniques: Transfection, Confocal Microscopy, Plasmid Preparation, Fluorescence, Negative Control

Journal: bioRxiv

Article Title: Tandem Requirement for Full Renal D 1 R and D 5 R Activity

doi: 10.1101/736611

Figure Lengend Snippet: A section of the human kidney was double immunostained for endogenous D 1 R (red) and D 5 R (green) ( A ). WGA (magenta) was used to visualize the plasma membrane, e.g., apical brush border of proximal tubules, while DAPI was used to visualize the nuclei. Colocalization in yellow is indicated by the arrows. DIC = differential interference microscopy. Sections of a mouse kidney infused with either vehicle (Basal) or fenoldopam was double immunostained for endogenous D 1 R (green) and D 5 R (red) ( B ). The RPT marker CD15 and DAPI were used to visualize the brush border and nuclei, respectively. Colocalization is indicated by the yellow or white areas in merged images. 630x magnification, scale bar=10 μm, n=3 independent experiments.

Article Snippet: The tissues were counterstained with wheat germ agglutinin (WGA) tagged with Alexa Fluor ™ 647 (#A-31573, ThermoFisher Scientific, Waltham, MA) and DAPI (#H-1500, Vector Laboratories, Burlingame, CA) to visualize the plasma membrane and nuclei, respectively.

Techniques: Microscopy, Marker

Journal: Experimental and Therapeutic Medicine

Article Title: miR-186 promotes tumor growth in cutaneous squamous cell carcinoma by inhibiting apoptotic protease activating factor-1

doi: 10.3892/etm.2018.6679

Figure Lengend Snippet: Effect of miR-186 overexpression or knockdown on cell proliferation, apoptosis, invasion and cell cycle progression in A-431 cells. (A) EdU staining was performed in the NC, miR-186 mimics and miR-186 inhibitors groups. Red indicates EdU staining and blue indicates DAPI. Magnification, ×100. (B) A colony formation assay was performed to assess the effect of miR-186 on cell proliferation in the NC, mimics and inhibitor groups. Magnification, ×100. (C) A Transwell assay was performed to detect the invasive ability of A-431 cells transfected with NC, mimics or inhibitor. Magnification, ×200. (D) A wound-healing assay was used to assess the migratory ability of A-431 cells treated with NC, mimics or inhibitor. Magnification, ×100. (E and F) Annexin V-FITC/PI staining and flow cytometry analysis were performed to evaluate the effects of miR-186 on (E) cell apoptosis and (F) cell cycle distribution in miR-186 overexpressed or inhibited A-431 cells, respectively. *P<0.05, **P<0.01 vs. NC group. ## P<0.01 vs. mimics group. NC, negative control; miR, microRNA; APAF1, apoptotic protease activating factor 1; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Article Snippet: After washing three times with PBS containing 0.1% Triton X-100, cells were blocked with 10% bovine serum albumin (cat. no. FA016-50G; Amresco, LLC, Solon, OH, USA) for 2 h at RT followed by incubation with primary antibodies against APAF1 (1:1,000; cat. no. ab2001; Abcam) and DAPI (1:2,000; cat. no. ab104139; Abcam) at 4°C overnight.

Techniques: Over Expression, Staining, Colony Assay, Transwell Assay, Transfection, Wound Healing Assay, Flow Cytometry, Negative Control

Journal: Investigative ophthalmology & visual science

Article Title: Epigenetic ALYREF/UHRF1/RHOB Axis in Corneal Wound Healing and Implications for Epithelial Tumorigenesis.

doi: 10.1167/iovs.66.3.54

Figure Lengend Snippet: FIGURE 2. UHRF1 promotes cell proliferation and migration in vitro. (A) Immunofluorescence images of DAPI (blue) and EdU (red) staining in control (CT) and Hinokitiol-treated HCECs. Statistical analysis of proliferating cells in the CT and Hinokitiol groups (n = 6 per group). (B) Scratch wound assay images of CT and Hinokitiol-treated HCECs at 0 hours and 24 hours. Statistical analysis of migrating cells in the CT and Hinokitiol-treated HCECs (n = 6 per group). (C) Immunofluorescence images of DAPI (blue) and EdU (red) staining in Lv-negative control (NC) and Lv-UHRF1 HCECs, along with statistical analysis of proliferating cells in each group (n = 6 per group). (D) Scratch wound images of Lv-NC and Lv-UHRF1 HCECs at 0 hours and 20 hours, along with statistical analysis of migrating cells in each group (n = 6/group).

Article Snippet: After antibody staining, the sections were counterstained with DAPI (Beyotime; P0131) and mounted.

Techniques: Migration, In Vitro, Immunofluorescence, Staining, Control, Scratch Wound Assay Assay, Negative Control

Journal: Investigative ophthalmology & visual science

Article Title: Epigenetic ALYREF/UHRF1/RHOB Axis in Corneal Wound Healing and Implications for Epithelial Tumorigenesis.

doi: 10.1167/iovs.66.3.54

Figure Lengend Snippet: FIGURE 5. RHOB silencing ameliorates the suppressive effects of UHRF1 knockdown on HCEC proliferation and migration. (A) Immunoflu- orescence images showing DAPI (blue) and EdU (red) staining in the negative control group (NC) and siRHOB HCECs, with statistical analysis of proliferating cells in each group (n = 6/group). (B) Scratch wound assay images of NC and siRHOB HCECs at 0 hours and 20 hours, with statistical analysis of migrating cells in each group (n = 6/group). NC indicates the negative control group. (C) Western blot analysis of RHOB and UHRF1 protein levels in UHRF1-silenced HCECs following RHOB knockdown, with densitometric quantification of RHOB expression levels, normalized to β-actin (n = 3/group). RT-qPCR analysis of RHOB mRNA expression, also normalized to β-actin (n = 3/group). NC indicates the negative control group. (D) EdU assay assessing proliferation in UHRF1-silenced HCECs following RHOB knockdown, with statistical analysis of proliferating cells in each group (n = 6/group). NC indicates the negative control group. (E) Scratch wound assay evaluating migration in UHRF1-silenced HCECs following RHOB knockdown, with statistical analysis of migrating cells in each group (n = 6/group). NC indicates the negative control group.

Article Snippet: After antibody staining, the sections were counterstained with DAPI (Beyotime; P0131) and mounted.

Techniques: Knockdown, Migration, Staining, Negative Control, Scratch Wound Assay Assay, Western Blot, Expressing, Quantitative RT-PCR, EdU Assay

Journal: Haematologica

Article Title: Uncoupling of the Hippo and Rho pathways allows megakaryocytes to escape the tetraploid checkpoint

doi: 10.3324/haematol.2016.149914

Figure Lengend Snippet: Hippo-p53 axis is activated by genotoxic stress. (A) Western blot analysis of Hippo-p53 pathway proteins in CD41+ megakaryocytes treated with/without 10 μM etoposide for five hours. β-actin was used as loading control. (B) Western blot analysis of p53 and its target genes performed after 12 hours of etoposide treatment at different days (D) of culture (D5, D7 and D9). HSC70 was used as loading control. (C) p53 localization in untreated and 12-h etoposide treated CD41+ MK cells. Cells were stained with anti-p53 antibody. TOTO-3 was used to stain the nucleus. Scale bar=20 μm. (D) YAP localization in untreated CD41+ MK cells and CD41+ MK cells treated by 10 μM etoposide for five hours on day 10 of culture. Cells were stained with anti-YAP antibody. DAPI was used to stain the nucleus. Cells (150) were counted and categorized according to the distribution of YAP between nucleus (N) and cytosol (C). Whereas for control cells, nearly 70% had YAP staining the nucleus, none of the cells in etoposide treated samples showed nuclear localization of YAP. Scale bar=30 μm.

Article Snippet: TOTO-3 iodide or DAPI (Molecular Probes, Life Technology) was applied for nuclear staining.

Techniques: Western Blot, Control, Staining