Journal: Stem cell research

Article Title: Generation of a heterozygous knockout human embryonic stem cell line for the OCIAD1 locus using CRISPR/CAS9 mediated targeting: BJNhem20-OCIAD1-CRISPR-39.

doi: 10.1016/j.scr.2015.12.037

Figure Lengend Snippet: Fig. 1. (A) Strategy for targeting OCIAD1 exon3 by CRISPR-CAS9. (B) Genomic targeting of OCIAD1 exon3 of BJNhem20-OCIAD1-CRISPR-39 identified by T7 endonuclease mediated digestion of high fidelity exon 3 amplicon detecting deletions in this clone. (C) Chromatograms showing sequence analysis to confirm heterozygous mutation in the targeted region of OCIAD1. (D) Western blot analysis showing decreased OCIAD1 levels in BJNhem20-OCIAD1-CRISPR-39. Graph represents standard error mean after analysis of three biological replicates using single factor ANOVA (P value 0.001). (E) Karyotype analysis of BJNhem20-OCIAD1-CRISPR-39. (F) Analysis of transcript levels of pluripotency marker genes Oct4, Nanog, TDGF and Sox2 by reverse transcription and polymerase chain reaction (RT-PCR) amplification. GAPDH was used to normalize transcript levels. (G) Immunostaining analysis of BJNhem20-OCIAD1-CRISPR-39 for depletion of OCIAD1 and for pluripotency markers Oct4, SSEA4 and TRA1-81 as indicated, compared to parental hESC line BJNhem20. (H) Differentiation analysis of BJNhem20-OCIAD1-CRIPSR-39: embryoid bodies stained to show differentiation to all the three germ layers by immunostaining for AFP, Brachyury and β-III tubulin marking the endoderm, mesoderm and ectoderm respectively.

Article Snippet: Primary antibodies used were against OCIAD1 (Abcam Ab91574), Oct4 (BD Biosciences BD611203), TRA1-81 and SSEA4 (kind gift from Peter Andrews, University of Sheffield, UK), Brachyury (Santa Cruz Biotech Cat no. SC-17743), β-III tubulin (Santacruz SC-51670), AFP (Sigma Chemical Pvt.

Techniques: CRISPR, Amplification, Sequencing, Mutagenesis, Western Blot, Marker, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Staining

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Effects of oxygen tension on retinal levels of HIF-1α, VEGF and β3-AR from PD7 to PD17. ( A ) Schematic diagram of the OIR model including DMOG administration daily from PD7 to PD12. ( B ) Representative blots showing protein levels of HIF-1α, VEGF and β3-AR as evaluated by Western blot in retinal extracts at different times from normoxic controls or OIR mice without or with DMOG administration. β-actin was used as the loading control. ( C – E ), Relative densitometric analyses of the protein levels of HIF-1α, VEGF and β3-AR. ( F ) Retinal mRNA levels of β3-AR at different times from controls or OIR mice untreated or treated with DMOG. * p < 0.05 vs. normoxic controls. One-way ANOVA followed by Tukey’s multiple comparison post-hoc test. Each histogram represents the mean ± SEM of data from 6 independent samples.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Western Blot, Control, Comparison

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Schematic representation of mouse and human β3-AR genes including their upstream sequences. ( A ) In the mouse gene, 5 exons (E1–E5; solid boxes) and 4 introns (dashed lines) are depicted. They potentially express up to 6 different alternative mRNAs of which 3 codify for the canonical β3-AR protein (yellow mRNAs) while the other 3 for an alternative β3-AR protein with a different C-terminal sequence (purple mRNAs). The putative transcription-start site (TSS) is indicated by the red arrow. The positions of the 6 potential HBSs relative to the TSS are in green. All of them contain the minimal HBS consensus sequence (underlined sequence 5′-ACGTG-3′). ( B ) In the human gene, the positions of the 6 potential HBSs relative to the TSS are in green. All of them contain the minimal consensus sequence (underlined sequence 5′-ACGT-3′). The putative TSS is indicated by the red arrow. The highly conserved nucleotides G −2 and/or C +5 in the mouse and human HBSs sequence are highlighted in red.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Sequencing

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: HIF-1α modeling and HIF-1/DNA docking. ( A ) Root-mean-square (RMS) displacement of protein backbone (black arrow indicates the time at which the stabilization of the protein structure occurs). ( B ) RMS fluctuation of aminoacid displacement relative to the starting structure and the principal domains of the HIF-1α protein, accordingly colored in ( C ). ( D ) HIF-1α protein modelized in its dimeric form showing the correct interaction with the DNA fragment. The two monomers are reported in green and orange respectively, while the DNA fragment is highlighted in blue. The binding site generated by dimerization is better shown in the focus section.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Binding Assay, Generated

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Docking analysis of model 1 and model 3. ( A ) HIF-1/HBS #1 best association complex: full structure and focus on HIF-1-DNA interactions (boxes). ( B ) HIF-1/HBS #3 best association complex: full structure and focus on HIF-1-DNA interactions (boxes).

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques:

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: HIF-1α interaction with HBS #1 and corresponding β3-AR gene expression at PD12 (from 0 to 12 h of hypoxia) or at PD17. ( A ) Schematic diagram of the OIR model pointing to the specific times under analysis. ( B ) Data from HIF-1α chromatin immunoprecipitation and HBS #1-specific qPCR (ChIP-qPCR) represented as fold enrichment relative to IgG input. ( C ) Corresponding levels of β3-AR mRNA. White bars represent data from retinas of normoxic controls while grey bars represent data from hypoxic mice. One-way ANOVA followed by Tukey’s multiple comparison post-hoc test. Each histogram represents the mean ± SEM of data from 6 independent samples. * p < 0.05 vs. normoxic controls ( n = 6 samples).

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Gene Expression, Chromatin Immunoprecipitation, ChIP-qPCR, Comparison

Journal: Oncotarget

Article Title: Crosstalk between integrin αvβ3 and ERα contributes to thyroid hormone-induced proliferation of ovarian cancer cells

doi: 10.18632/oncotarget.10757

Figure Lengend Snippet: ( A ) mRNA levels of ERα , TRβ1 , ITG αv and ITG β3 from MCF-7, SKOV-3 and OVCAR-3 were measured by quantitative real-time PCR and normalized with 18Sr RNA. Results were expressed as folds of increase. ( B ) SKOV-3 cells were treated in the presence or absence of indicated peptides with T 3 or T 4 for 3 days, cells in 96 wells were subjected to the MTT assay. ( C ) scramble or shRNA of integrin αv or β3 were transiently transfected to SKOV-3 cells and treated with thyroid hormone (T 4 ) for 30 min, and whole cell lysates were harvested for western blot analysis. Indicated antibodies were used. Compared to sc: p < 0.05 :* p < 0.01 :** p < 0.001 :*** ; Compared to sc treated with T 4 : p < 0.05: † p < 0.01: †† p < 0.001: †††

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween, and then incubated with selected antibodies overnight: PCNA (GTX100539, GeneTex Inc. CA, USA), Lamin B (GTX103292, GeneTex Inc.), integrin αv (SC9969, Santa Cruz Inc., Santa Cruz, CA, USA ), integrin β3 (SC6627, Santa Cruz), phospho-ERK1/2 (4377S, Cell Signaling Technology, Danvers, MA, USA), total-ERα (8644S, Cell Signaling Technology), ERα-pS118(2511p, Cell Signaling Technology), ERα-pS167 (5587p, Cell Signaling Technology).

Techniques: Real-time Polymerase Chain Reaction, MTT Assay, shRNA, Transfection, Western Blot

Journal: Oncotarget

Article Title: Crosstalk between integrin αvβ3 and ERα contributes to thyroid hormone-induced proliferation of ovarian cancer cells

doi: 10.18632/oncotarget.10757

Figure Lengend Snippet: SKOV- 3 cells were treated with ( A ) thyroid hormone, ( B ) estrogen, for different periods of time as indicated. ( C ) SKOV-3 cells treated with thyroid hormone or estrogen for 10 min in the absence or presence of ICI were fixed and stained with anti-integrin αv and phospho-ERα (S167) antibodies, and subsequently with fluorescent secondary antibodies. Nuclear punctate of phosphorylated ERα-induced by T 4 (shown in green) were increased with time and co-localized with integrin αvβ3 (shown in red) to yield a yellow color. Nuclei were stained with DAPI and showed as blue. The phosphorylation of ERα and nuclear translocation of integrin αv were inhibited by ICI. Quantitative fluorescence intensities are shown as average intensity per cell. p < 0.05: * p < 0.01: **

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween, and then incubated with selected antibodies overnight: PCNA (GTX100539, GeneTex Inc. CA, USA), Lamin B (GTX103292, GeneTex Inc.), integrin αv (SC9969, Santa Cruz Inc., Santa Cruz, CA, USA ), integrin β3 (SC6627, Santa Cruz), phospho-ERK1/2 (4377S, Cell Signaling Technology, Danvers, MA, USA), total-ERα (8644S, Cell Signaling Technology), ERα-pS118(2511p, Cell Signaling Technology), ERα-pS167 (5587p, Cell Signaling Technology).

Techniques: Staining, Phospho-proteomics, Translocation Assay, Fluorescence

Journal: Oncotarget

Article Title: Crosstalk between integrin αvβ3 and ERα contributes to thyroid hormone-induced proliferation of ovarian cancer cells

doi: 10.18632/oncotarget.10757

Figure Lengend Snippet: SKOV-3 were pre-treated in the presence or absence of ICI for 30 min prior to another 30 min of T 4 treatment and harvested for ChIP. Total cell lysate was immunoprecipitated with anti-integrin αv and pulled-down DNA was measured with qPCR. Compared to control: p < 0.01: **; Compared to T 4 alone: p < 0.001: †††

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween, and then incubated with selected antibodies overnight: PCNA (GTX100539, GeneTex Inc. CA, USA), Lamin B (GTX103292, GeneTex Inc.), integrin αv (SC9969, Santa Cruz Inc., Santa Cruz, CA, USA ), integrin β3 (SC6627, Santa Cruz), phospho-ERK1/2 (4377S, Cell Signaling Technology, Danvers, MA, USA), total-ERα (8644S, Cell Signaling Technology), ERα-pS118(2511p, Cell Signaling Technology), ERα-pS167 (5587p, Cell Signaling Technology).

Techniques: Immunoprecipitation, Control