Journal: Development (Cambridge, England)

Article Title: Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development

doi: 10.1242/dev.200250

Figure Lengend Snippet: Dynamics of the H3K27ac mark following NeuroD1 expression. (A) Schematic of analysing H3K27ac dynamics upon NeuroD1 expression. (B) Global dynamics of H3K27ac enrichment before and after NeuroD1 induction (12 h and 48 h). R1 and R2 are replicates. (C) Boxplots show average H3K27ac enrichment in each cluster (C1 to C7). Middle bars show median values, boxes show first to third interquartile ranges, whiskers show the minimum and maximum of data range and beyond that are outliers. (D) NeuroD1-bound and transcriptionally induced sites. (E) Motif enrichment profiles at transcriptionally active and inactive sites.

Article Snippet: The eluted fractions were immunoblotted with Tcf12 antibody (SCBT sc-28364 X) and NeuroD1 antibody (CST 4373S) (1:1000).

Techniques: Expressing

Journal: Development (Cambridge, England)

Article Title: Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development

doi: 10.1242/dev.200250

Figure Lengend Snippet: Identification of potential NeuroD1 co-factors. (A) Schematic of ISMARA analysis for co-enriched TF selection. (B) Overlap of upregulated motifs predicted from ISMARA with bHLH transcription factors. (C) Activity profiles for selected motifs from the overlap of ISMARA motifs and bHLH factors. R1 and R2 are the replicates for each time point (0 h, 12 h and 48 h). Data are mean±s.d. (D) Expression of selected factors in different cortical layers. (E) In situ hybridisation images (Allen Brain Atlas) showing the expression of NeuroD1 and Tcf12 in developing cortex at E11.5, E13.5 and E15.5. (F) Bar chart showing the expression level of NeuroD1 in VZ, SVZ and CP regions. (G) Bar chart showing the expression level of Tcf12 in VZ, SVZ and CP regions. (H) Bar chart showing the expression level of Tcf12 in proliferating progenitors (PP), differentiating progenitors (DPs) and neurons (NNs). Data are mean±s.d. Statistical significance was calculated using a paired two-tailed Student's t -test. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The eluted fractions were immunoblotted with Tcf12 antibody (SCBT sc-28364 X) and NeuroD1 antibody (CST 4373S) (1:1000).

Techniques: Selection, Activity Assay, Expressing, In Situ, Hybridization, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development

doi: 10.1242/dev.200250

Figure Lengend Snippet: NeuroD1 and Tcf12 are co-expressed in distinct subpopulations during cortical development. (A) Expression of NeuroD1 and Tcf12 in different brain cell types in E14.5 cortex shows the highest NeuroD1 expression in the migrating neuronal population and the Tcf12 expression levels. (B) Pseudo-timecourse trajectory of SVZ migrating cells. Numbers show the clusters arranged in developmental pseudotime. (C) Expression of progenitor, neurogenic and neuronal markers in a SVZ migrating subpopulation arranged by pseudotime, where we see increasing NeuroD1 expression levels.

Article Snippet: The eluted fractions were immunoblotted with Tcf12 antibody (SCBT sc-28364 X) and NeuroD1 antibody (CST 4373S) (1:1000).

Techniques: Expressing

Journal: Development (Cambridge, England)

Article Title: Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development

doi: 10.1242/dev.200250

Figure Lengend Snippet: Tcf12-NeuroD1 complex induces active chromatin and expression of neuronal migration genes. (A) Co-IP experiment showing GFP-tagged NeuroD1 interacting with HA-tagged Tcf12 in vitro . This specific interaction was absent in control immunoprecipitates where we co-expressed HA-tagged Tcf12 with only GFP protein. (B) IP experiment showing the specific enrichment of endogenous Tcf12 by NeuroD1 only in the negative control siRNA (left panel). It was absent in Tcf12 knockdown conditions (right panel) during in vitro neurogenesis. (C) IP experiment from the E14.5 mouse cortex showing specific interaction of Tcf12 with NeuroD1 during cortical development in vivo . (D) ChIP qPCR results showing the enrichment of Tcf12 at NeuroD1 target sites after 12 h and 48 h of NeuroD1 induction. (E) ChIP Re-ChIP qPCR experiment showing the NeuroD1 and Tcf12 co-binding at the same target genes. (F) qPCR results showing a change in H3K27ac levels at NeuroD1 target sites upon Tcf12 knockdown. (G) qPCR results showing the expression of NeuroD1 target genes upon Tcf12 knockdown. All the ChIP experiments were performed as three independent biological replicates. The ChIP-Re-ChIP experiment was repeated twice. Data are mean±s.d. Statistical significance was calculated using a paired two-tailed Student's t -test. * P <0.05, ** P <0.01.

Article Snippet: The eluted fractions were immunoblotted with Tcf12 antibody (SCBT sc-28364 X) and NeuroD1 antibody (CST 4373S) (1:1000).

Techniques: Expressing, Migration, Co-Immunoprecipitation Assay, In Vitro, Control, Negative Control, Knockdown, In Vivo, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development

doi: 10.1242/dev.200250

Figure Lengend Snippet: A schematic showing how the functional cooperativity of Tcf12 and NeuroD1 in specific subpopulations of the developing cortex creates the gene regulatory program essential for neuronal migration. Both Tcf12 and NeuroD1 are highly co-expressed during the transition of cortical progenitors from proliferative to neurogenic divisions. During this phase, Tcf12 forms a complex with NeuroD1 and co-occupies a subset of NeuroD1 target loci. This Tcf12-NeuroD1 cooperativity is essential for a gain in active chromatin and expression of neuronal migration genes, and therefore for the correct migration of newborn neurons.

Article Snippet: The eluted fractions were immunoblotted with Tcf12 antibody (SCBT sc-28364 X) and NeuroD1 antibody (CST 4373S) (1:1000).

Techniques: Functional Assay, Migration, Expressing

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: LXY30 specifically binds to integrin α3β1-expressing NSCLC cells and their derived exosomes. Chemical structures of LXY30-FITC ( a ) and scrambled-LXY30 (S-LXY30)-FITC ( b ) are illustrated. A549 cells growing in DMEM medium were collected and incubated with LXY30-FITC or S-LXY30-FITC for 2 h, and the binding specificity of LXY30 was evaluated by flow cytometry ( c ) and fluorescence microscopy ( d ). Exosomes were isolated from the supernatant of two integrin α3β1-expressing NSCLC cells (A549 and H1975) and a patient’s malignant pericardial effusion ( e ). Exosomes were incubated with TentaGel bead coated with LXY30 (left panel) or S-LXY30 (right panel) for 2 h, followed by incubation with Alexa 647 mouse anti-CD63 antibody (red) for 1 h before being visualized by fluorescence microscopy ( e ). The expression of integrin α3, integrin β1, and exosomal marker CD63 in 4 NSCLC cell lines and one patient pericardial effusion were determined by Western blots ( f ). Beta-actin was used as loading control for cellular protein expression. The protein loading for cell lysate is around 5 times lower than that of exosomes. h, hour(s); FITC, fluorescein isothiocyanate; NSCLC, non-small cell lung carcinoma

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Expressing, Derivative Assay, Incubation, Binding Assay, Flow Cytometry, Fluorescence, Microscopy, Isolation, Marker, Western Blot, Control

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: Erlotinib-resistant R#2 cells have increased integrin ligands binding compared to their parental erlotinib-sensitive, EGFR-mutant H3255 cells. H3255 (EGFR L585R) and its derived erlotinib-resistant R#2 cells were incubated with different integrin ligands (LXY30, LXW64, and LLP2A) for 2 h, 5 h, or overnight. The binding of tumor cells to integrin ligand-coated beads was visualized by light microscopy, and the intensity of binding was scored from negative (−), weakly, to strongly positive (1+ to 4+) ( a ). The binding of integrin ligands against tumor cells was confirmed by flow cytometry ( b ) and summarized in the table ( c ). h, hour(s); O/N, overnight; EGFR, epidermal growth factor receptor

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Binding Assay, Mutagenesis, Derivative Assay, Incubation, Light Microscopy, Flow Cytometry

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: LXY30 activates EGFR and its downstream signaling molecules independently from another integrin ligand LXW64. Schematic representation for the cross talk between integrin and EGFR is illustrated ( a ). The effect of LXY30 and/or LXW64 on the expression of integrin subtypes, EGFR, and its key downstream signaling molecules in H1975 ( EGFR L858R/T790 M) cells was analyzed after incubating with LXY30-biotin, LXW64-biotin, or LXY30-biotin/LXW64-biotin for 72 h by Western blot ( b ) and subsequently quantified ( c ). * p < 0.01

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Expressing, Western Blot

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: Integrin expression in tumor cells and tumor-derived exosomes isolated from NSCLC patients

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Expressing, Isolation, Binding Assay

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: Optical images showing preferential uptakes of LXY30-biotin-streptavidin-Cy5.5 conjugate (i.e., drug surrogate) in EGFR-resistant lung cancer xenografts in nude mice. After conjugated with streptavidin-Cy5.5, LXY30 shows the capability to deliver imaging dye to subcutaneous xenografts of H1975 ( a , c ) and A549 ( b , d ). Representative images of tumor sections were stained by H&E staining ( e , f ), fluorescent (Alexa 594)-labeled anti-α3 integrin antibody ( g , h ), and integrin α3 IHC ( i , j ). * p < 0.05

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Imaging, Staining, Labeling

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: High α3 integrin expression level is associated with poor prognosis in NSCLC patients. The transcriptome expression data from lung cancer patients were analyzed in The Cancer Genome Atlas (TCGA) database ( http://www.cancergenome.nih.gov ) including 517 lung adenocarcinoma (LUAD) and 502 lung squamous cell carcinoma (LUSC) samples. a The RNA expression levels of α3 integrin (INTA3) gene in LUAD and LUSC, respectively. Kaplan-Meier curves of overall survival were stratified by ITGA3 expression in LUAD ( b ) and LUSC ( c )

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Expressing, RNA Expression

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: LXY30 specifically targets subcutaneous PDX xenograft tumors. A lung squamous cell (LUSC) PDX model was generated by engrafting biopsy specimen into NSG mice as described in the “ ” section. Optical images by white light (WL) and in vivo fluorescence images ( a ) were taken 6 h after the injection of streptavidin-Cy5.5 (SA-Cy5.5) either alone or together with biotinylated LXY30 (LXY30-biotin) into NSG mice bearing subcutaneous LUSC PDX xenografts. Major organs from the NSG mice were subjected to ex vivo imaging immediately after the mice were sacrificed ( b ). The fluorescence signals of SA-Cy5.5 and LXY30-Biotin-SA-Cy5.5 were further quantitated for subcutaneous LUSC PDX tumors ( c ). Representative images of LUSC PDX tumor sections were stained by fluorescent (Alexa594)-labeled anti-α3 integrin antibody, H&E, integrin α3, and integrin β1 immunohistochemistry stains ( d ). PDX, patient-derived xenograft; LUSC, lung squamous carcinoma; WL, white light

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Generated, In Vivo, Fluorescence, Injection, Ex Vivo, Imaging, Staining, Labeling, Immunohistochemistry, Derivative Assay