Journal: Oncogene

Article Title: Control of PD-L1 expression by miR-140/142/340/383 and oncogenic activation of the OCT4–miR-18a pathway in cervical cancer

doi: 10.1038/s41388-018-0347-4

Figure Lengend Snippet: miR-18a increases PD-L1 levels by repressing SOX6 to activate the Wnt/β-catenin pathway and inactivate p53 signaling. a Heat map of qRT-PCR data showing the expression of Wnt signaling pathway-related genes in CaSki cells transfected with the miR-18a mimic or its negative control. Red: upregulation of mRNA; green: downregulation of mRNA. b Western blotting analysis of active β-catenin, cyclin D1, and PD-L1 in CaSki and SiHa cells transfected with the miR-18a mimic or its negative control with or without the SOX6 expression vector ( n = 3). c , d TOP/FOP-Flash reporter assays of CaSki ( c ) and SiHa ( d ) cells co-transfected with the miR-18a mimic or its negative control and the SOX6 expression vector or control ( n = 4). e Co-immunoprecipitation assays reveal associations between endogenous SOX6 and β-catenin in CC cells. Cell lysates are labeled as input. IgG was used as the control ( n = 3). f , g qRT-PCR ( f ) and western blotting ( g ) of WISP3 expression in CaSki and SiHa cells transfected with the SOX6 expression vector or control vector ( n = 3). h Schematic representation of the human WISP3 gene promoter. The predicted SOX6 binding site is shown in red, and the primer sequences for ChIP-qPCR assays are underlined. i SOX6 occupancy at its potential binding site in the WISP3 gene promoter was analyzed using ChIP-qPCR assays. c-Myc, a known SOX6 target, was used as a positive control ( n = 3). j CaSki and SiHa cells were transfected with the wild-type (WT) or mutant (MU) WISP3 -promoter luciferase plasmid with or without the SOX6 expression vector. Relative luciferase activity was assessed ( n = 4). k Western blot analysis of SOX6, WISP3, active β-catenin, and PD-L1 in CaSki and SiHa cells transfected with SOX6 expression vector or control vector and WISP3 expression vector or the appropriate control ( n = 3). l Western blot analysis of p53, p21, and PD-L1 in CaSki and SiHa cells transfected with the SOX6 expression vector or control vector in the presence or absence of p53 siRNA ( n = 3). m qRT-PCR analysis of miR-34a expression in CaSki and SiHa cells transfected with the SOX6 expression vector in the presence or absence of p53 siRNA ( n = 3). n Western blot analysis of PD-L1 in CaSki and SiHa cells transfected with the miR-34a mimic or its negative control ( n = 3). ** P < 0.01, *** P < 0.001

Article Snippet: The following primary antibodies were used: PD-L1 (17952-1-AP, ProteinTech), SOX6 (NBP1-85811, Novus), WNK2 (07-2261, Millipore), BTG3 (ab112938, Abcam), RBSP3 (ab106973, Abcam), active β-catenin (05-665, Millipore), OCT4 (ab19857, Abcam). p-AKT (sc-293125), p-ERK1/2 (sc-16982), total β-catenin (sc-7963), ERK1/2 (sc-514302), PTEN (sc-7974), cyclin D1 (sc-8396), p21 (sc-6246), and GAPDH (sc-47724) were obtained from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Western Blot, Plasmid Preparation, Control, Immunoprecipitation, Labeling, Binding Assay, ChIP-qPCR, Positive Control, Mutagenesis, Luciferase, Activity Assay

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a FGF4 is a key signal that promotes differentiation of pluripotent mESCs. b Workflow for phosphoproteomic analysis of FGF4 signalling in Fgf4 −/− mESCs. Volcano plot showing significantly modified phosphosites after stimulation of Fgf4 −/− mESCs with FGF4 for 5 min ( c ) and 20 min ( d ). Phosphosites on known FGF4 pathway components are highlighted. e Protein kinase phosphopeptides that are significantly upregulated (>2-fold) on at least one time point (5 or 20 min) compared with control. Data are presented as mean ± SD ( n = 3). f Fgf4 −/− mESCs were stimulated with FGF4 for the indicated time, and EPHA2 pS898 and EPHA2 levels determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Modification, Control, Western Blot

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Average protein copy number per cell determined for receptor kinases in mESCs, using quantitative whole-cell proteomics. Data are presented as mean ± SD ( n = 3). b Workflow for quantification of EPH–EFN interactions in mESCs by EFN ligand affinity purification mass spectrometry. c Proof-of-principle identification of EPH–EFN interactions by EFN ligand affinity purification. EPHA2 levels were determined by immunoblotting. d Coomassie staining of EFNA1/B1 affinity purification from mESCs. EFNA1, EFNB1 and EPHA2 proteins are indicated. (*) = non-specific band. e Mass-spectrometry analysis of 75–130-kDa region of the Coomassie stained EFNA1/EFNB1 affinity purification shown in ( d ). Total spectral counts recovered for each EPH receptor family member are indicated. f EFNA1/B1 affinity purification from intact Epha2 +/+ and pooled Epha2 −/− mESCs. Phosphotyrosine (pTyr), EPHA2 and ERK1/2 levels were determined by immunoblotting. Note that the pTyr signal is specific for EPHA2, and is not detected in the absence of EFN ligand. g EPHA2 was immunoprecipitated from Epha2 +/+ and Epha2 −/− mESCs, and pTyr, EPHA2, EFNA1 and ERK1/2 levels determined by immunoblotting. h Epha2 +/+ mESCs were stimulated with EFNA1-expressing Epha2 −/− mESCs for 15 or 40 min. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Affinity Purification, Mass Spectrometry, Western Blot, Staining, Immunoprecipitation, Expressing

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Epha2 +/+ , Epha2 −/− or Epha2 −/− mESCs (clone C4) stably expressing EPHA2 were cultured in the absence of LIF for 48 h. EPHA2, KLF4, DNMT3B, NANOG and OCT4 levels were determined by immunoblotting. b Epha2 +/+ or Epha2 −/− (clone C4) mESCs were maintained in 2i or differentiated in N2B27 media for 72 or 96 h, respectively, whereupon 10% of cells were replated in 2i. Total alkaline phosphatase staining is represented relative to Epha2 +/+ mESCs. The total number of alkaline phosphatase-positive colonies for Epha2 +/+ and Epha2 −/− mESCs is shown, and also represented relative to Epha2 +/+ mESCs. Data show mean ± SEM ( n = 3); statistical significance was determined using unpaired two-sided Student’s t test comparing Epha2 −/− with the Epha2 +/+ control (**** P < 0.0001, ** P = 0.0016). c Epha2 +/+ or Epha2 −/− (clone C4) mESCs stably expressing EFNA1, along with the respective parental controls, were grown in LIF/FBS, and KLF4, NANOG, EPHA2, EFNA1 and ERK1/2 levels determined by immunoblotting, or EPHA2 immunoprecipitated and pTyr and EPHA2 levels determined by immunoblotting. d Epha2 +/+ , Epha2 −/− or Epha2 −/− mESCs stably expressing EPHA2 were differentiated as embryoid bodies for 10 days, and the levels of Fgf5 , Brachyury , Mixl and Cer1 mRNA determined by qRT-PCR. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values. The results shown are for technical replicates from two independent experiments, including three Epha2 −/− clones ( n = 3); statistical significance at day 4 was determined using unpaired two-sided Student’s t test comparing each group with the Epha2 +/+ control (ns = not significant, * P = 0.0252, ** P = 0.0045, *** P < 0.0001). e Epha2 +/+ mESCs cultured in LIF/FBS were stimulated with 1 μg/ml clustered EFNA1 for the indicated times. ppERK1/2, total ERK1/2, STAT3 pY705 and total STAT3 levels were determined by immunoblotting. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting. ppERK1/2 signal was quantified; data show mean ± SD ( n = 3); statistical significance was determined using one-sample two-sided t test comparing each group with control, theoretical mean = 1 (ns = not significant, 5 min; * P = 0.0467, 30 min; *** P = 0.0005, 45 min; ** P = 0.0014, 60 min; ** P = 0.0018). f Epha2 +/+ mESCs cultured in LIF/FBS were stimulated with 1 μg/ml clustered EFNA1 for the indicated times. SHP2 was immunoprecipitated, and SHP2 and EPHA2 levels detected by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Stable Transfection, Expressing, Cell Culture, Western Blot, Staining, Control, Immunoprecipitation, Quantitative RT-PCR, Whisker Assay, Clone Assay

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Diagram of potential phosphorylation sites within the EPHA2 S898 motif. Mass spectrometry analysis detects phosphorylation of at least three sites in EPHA2 immunoprecipitated from FGF4-stimulated Fgf4 −/− mESCs (see Supplementary Table ). b Fgf4 −/− mESCs were treated with 10 μM of the indicated inhibitors for 1 h, and stimulated with FGF4 for 10 min. EPHA2 pS898, EPHA2 and AKT pS473 levels were determined by immunoblotting. c Epha2 −/− mESCs were transfected with either wild type or 5E EPHA2 constructs, and stimulated with 1 μg/ml clustered EFNA1 for 15 min. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting and quantified. Data show mean ± SD ( n = 4). d EPHA2 was immunoprecipitated from EPHA2 WT knock-in (KI) or 5A KI cell lines and pTyr and EPHA2 levels determined by immunoblotting (upper panel). Relative pTyr/EPHA2 signal was quantified (lower panel). Data show mean ± SD ( n = 4). e Phase-contrast images of EPHA2 WT KI or 5A KI mESC lines; scale bar = 100 µM. f EPHA2 WT KI or 5A KI cell lines were cultured in LIF/FBS medium for 48 h, and KLF4, NANOG, DNMT3B, OCT4 ppERK1/2 and ERK1/2 levels determined by immunoblotting. g Epha2 +/+ mESCs were differentiated as embryoid bodies for 10 days, and EPH receptor expression determined by qRT-PCR at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of four technical replicates ( n = 4). h Epha2 +/+ mESCs were differentiated as embryoid bodies for 10 days, and EFN ligand expression determined by qRT-PCR at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of four technical replicates ( n = 4). Epha2 ( i ) and Efna1 ( j ) mRNA expression in 2i mESCs undergoing differentiation in N2B27 was determined by qRT-PCR analysis at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of two technical and three biological replicates ( n = 3). Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Phospho-proteomics, Mass Spectrometry, Immunoprecipitation, Western Blot, Transfection, Construct, Knock-In, Cell Culture, Expressing, Quantitative RT-PCR, Whisker Assay

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Epha2 +/+ mESCs were transfected with the indicated siRNAs, and EPHA2, OCT4 and SOX2 levels determined by immunoblotting. A non-specific band was used as a loading control. b Epha2 and Oct4 mRNA expression was determined by qRT-PCR following transfection of Epha2 +/+ mESCs with control or OCT4 siRNA. Data show mean ± SEM of four technical replicates (n = 4). c Pluripotency transcription factor-binding sites in Epha2 gene regulatory regions were extracted from CODEX mESC ChIP-SEQ data ( http://codex.stemcells.cam.ac.uk ). d Epha2 +/+ mESCs were transfected with the indicated siRNAs, and stimulated with 1 μg/ml clustered EFNA1 for 15 min. EPHA2 was immunoprecipitated using EPHA2 antibody, and pTyr and EPHA2 levels determined by immunoblotting. e EFNA1/B1 affinity purification from control, dasatinib-treated or siOct4-transfected mESCs. pTyr, EPHA2, OCT4 and ERK1/2 levels were determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Transfection, Western Blot, Control, Expressing, Quantitative RT-PCR, Binding Assay, ChIP-sequencing, Immunoprecipitation, Affinity Purification

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: Expression (RPKM) of EPH ( a , b ) or EFN ( c , d ) mRNA in the inner cell mass (ICM) or trophectoderm (TE) of 64-cell mouse embryos ( a , c ) or 5-day human embryos ( b , d ). a , c n = 33 biologically independent cells for ICM, and n = 28 biologically independent cells for TE. b , d n = 73 biologically independent cells for ICM, and n = 142 biologically independent cells for TE. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values. e EPHA2 regulation and function in mESCs. In the pluripotent state, OCT4 and other pluripotency factors promote EPHA2 receptor expression, enabling activation by EFNA ligands to support pluripotency by restraining ERK1/2. During differentiation, FGF4 drives ERK1/2–RSK activity to phosphorylate and inhibit EPHA2, whilst ERK1/2 suppresses an OCT4–EPHA2 transcriptional module to disable EPHA2 receptor expression. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Expressing, Whisker Assay, Activation Assay, Activity Assay

Journal: PLoS ONE

Article Title: Sex Hormones in Autism: Androgens and Estrogens Differentially and Reciprocally Regulate RORA, a Novel Candidate Gene for Autism

doi: 10.1371/journal.pone.0017116

Figure Lengend Snippet: (A) Schematic diagram showing the upstream region of the aromatase gene (edited from the UCSC Genome Browser). Potential RORA binding sites are labeled (RORAbs = RORA potential binding sites). Because sites II and III are so close to each other, a single pair of primers was designed to cover both sites. (B) ChIP-qPCR analyses of potential binding sites for RORA on the aromatase gene promoter region. Sonicated chromatin from SH-SY5Y cells was immunoprecipitated using anti-RORA or control IgG antibody. The enrichment of each RORA binding site in anti-RORA-IP DNA was determined by qPCR analyses (n = 3). Error bars indicate SEM (* P <0.05, ** P <0.001, versus the enrichment in IgG-IP DNA). Aromatase expression is enhanced by RORA overexpression . SH-SY5Y cells were transfected with 1.25 µg or 2.50 µg pSG5.HA-RORA plasmid or empty plasmid for 24 hrs, and qRT-PCR analyses (n = 3) were conducted to measure RORA and aromatase expression (Mock = mock-treatment control, Neg = empty plasmid, RORA = pSG5.HA-RORA plasmid). (C) Relative quantity of RORA transcript levels in RORA-transfected cells which is increased by more than a factor of 10 5 relative to the controls. (** P <0.001 versus the empty plasmid control) (D) Relative quantity of aromatase transcript levels in RORA-transfected cells. Error bars indicate SEM (* P <0.05 versus the empty plasmid control).

Article Snippet: Rabbit polyclonal antibody (1∶50 final dilution) against RORA (4 μg/ml, Santa Cruz Biotechnology: sc-28612), goat polyclonal antibody (1∶50 dilution) against aromatase (4 μg/ml, Santa Cruz Biotechnology: sc-14245), and mouse monoclonal antibody (1∶100 dilution) against MAP2 (5 μg/ml, Invitrogen) were pooled and added to the tissue array in a humidified chamber.

Techniques: Binding Assay, Labeling, ChIP-qPCR, Sonication, Immunoprecipitation, Control, Expressing, Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Sex Hormones in Autism: Androgens and Estrogens Differentially and Reciprocally Regulate RORA, a Novel Candidate Gene for Autism

doi: 10.1371/journal.pone.0017116

Figure Lengend Snippet: A tissue array containing postmortem frontal cortex specimen from 12 autistic individuals and 22 age- and sex-matched controls was co-immunolabeled for aromatase (red), RORA (blue), and MAP2, and counterstained with DAPI to reveal nuclei (see for example of quadruple staining). (A) Representative images from confocal immunofluorescence analyses of the frontal cortex tissue arrays. Arrows mark neurons in the frontal cortex. Scale bars, 20 µm. F: female; M: male; #'s indicate the respective ages of the subjects from whom the brain tissues were obtained. (B) Quantitative analyses of confocal fluorescence images of RORA, aromatase, and DAPI. Frontal cortex neurons expressing MAP2 protein were selected according to its green immunofluorescence (shown in ), and fluorescence signal intensity for RORA, aromatase (ARO), and DAPI, in the neurons were extracted using image analysis software and normalized by background subtraction. For each sample, ∼40–50 neurons were selected for quantification of RORA and aromatase fluorescence in a “sample blind” fashion such that the identities of the samples were unknown to the person performing the analyses. Error bars indicate SEM (* P <0.05 versus the control samples). (C) Correlation analysis of RORA and aromatase protein expression in the frontal cortex neurons.

Article Snippet: Rabbit polyclonal antibody (1∶50 final dilution) against RORA (4 μg/ml, Santa Cruz Biotechnology: sc-28612), goat polyclonal antibody (1∶50 dilution) against aromatase (4 μg/ml, Santa Cruz Biotechnology: sc-14245), and mouse monoclonal antibody (1∶100 dilution) against MAP2 (5 μg/ml, Invitrogen) were pooled and added to the tissue array in a humidified chamber.

Techniques: Immunolabeling, Staining, Immunofluorescence, Fluorescence, Expressing, Software, Control

Journal: PLoS ONE

Article Title: Sex Hormones in Autism: Androgens and Estrogens Differentially and Reciprocally Regulate RORA, a Novel Candidate Gene for Autism

doi: 10.1371/journal.pone.0017116

Figure Lengend Snippet: The schematic illustrates a mechanism through which the observed reduction in RORA in autistic brain may lead to increased testosterone levels through downregulation of aromatase. Through AR, testosterone negatively modulates RORA, whereas estrogen upregulates RORA through ER.

Article Snippet: Rabbit polyclonal antibody (1∶50 final dilution) against RORA (4 μg/ml, Santa Cruz Biotechnology: sc-28612), goat polyclonal antibody (1∶50 dilution) against aromatase (4 μg/ml, Santa Cruz Biotechnology: sc-14245), and mouse monoclonal antibody (1∶100 dilution) against MAP2 (5 μg/ml, Invitrogen) were pooled and added to the tissue array in a humidified chamber.

Techniques: