Journal: Biology Open

Article Title: Generation and characterization of a knockout mouse of an enhancer of EBF3

doi: 10.1242/bio.062070

Figure Lengend Snippet: Ebf3 regulatory landscape and associated hs737/Rr169617 enhancer deletion mouse lines. Genome browser view of the topologically associating domain region containing Rr169617 and its target gene Ebf3 (GRCm38/mm10). The first track shows the two independent founder mouse lines generated in this study: line 299 (C57BL/6J-Rr169617 em1Tnt /J) and line 304 (C57BL/6J-Rr169617 em2Tnt /J). The second track shows the location of the regulatory region, Rr169617. The third track shows the location of human VISTA enhancers lifted over to the mouse genome. Included is hs737 that resides within the Rr169617 region. The fourth track shows enhancer-promoter interactions of Rr169617 and Ebf3 from . The fifth track shows the genes within the region. The sixth track shows human topologically associating domains lifted over to this region and show high conservation. Finally, the chromatin state data available from ENCODE3 is shown across the different timepoints in mouse development. The colors in this track refer to their chromHMM status as described in .

Article Snippet: Taqman mouse Ebf3 (Mm00438642_m1) and GAPDH (Mm99999915_g1) gene expression assays were performed on a QuantStudio 6 Flex quantitative thermocycler using four reactions for each sample.

Techniques: Generated

Journal: Biology Open

Article Title: Generation and characterization of a knockout mouse of an enhancer of EBF3

doi: 10.1242/bio.062070

Figure Lengend Snippet: qRT-PCR analysis for Ebf3 expression in E12.5 forebrain. (A) Results of five independent qRT-PCR for Ebf3 expression. Samples for each genotype were as follows: Rr169617 +/+ ( n =12), Rr169617 +/− ( n =14), and Rr169617 −/− ( n =10). (B) Relative fold expression aggregating data across all five independent qPCR experiments. For both A and B, relative fold expression is in comparison to the Rr169617 +/+ results.

Article Snippet: Taqman mouse Ebf3 (Mm00438642_m1) and GAPDH (Mm99999915_g1) gene expression assays were performed on a QuantStudio 6 Flex quantitative thermocycler using four reactions for each sample.

Techniques: Quantitative RT-PCR, Expressing, Comparison

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Essential roles of Ebf3 in sternum ossification. (A) Whole-mount immunostaining of E10.5 mouse embryo with antibodies against Ebf3 (green), Pax7 (cyan) and Sox9 (red). Ebf3 was expressed in the lateral plate mesoderm, as well as in syndetomes located between Pax7 + dermomyotomes and Sox9 + sclerotomes. Insets 1 and 2 show higher magnifications of the indicated regions. (B,B′) Ebf3 (green) was expressed in Prx1:Tdtomato + cells (red) at the thoracic lateral plate mesoderm and in syndetomes adjacent to Pax7 + dermomyotomes (cyan) at E10.5. B′ shows an enlarged view of the boxed region. (C) WISH for Ebf1 , Ebf2 and Ebf3 in E10.5 and E11.5 mouse embryos. Red arrowheads indicate that Ebf3 , but not other Ebfs, was expressed in the thoracic lateral plate mesoderm tissue at E10.5 and show highest expression of Ebf3 in the sternal primordial bars at E11.5. (D) Schema of sternum development during murine embryogenesis. Red regions represent sternal bars at E12.5 and ossified zones of the sternum at E18.5. (E) Ossification and chondrogenesis analyses of Ebf3 flox/flox CAG-Cre and Ebf3 flox/+ CAG-Cre embryos at E18.5 and E17.5 based on skeletal preparations. Ebf3-KO mice exhibited defective sternum ossification without any defects in chondrogenesis. Scale bars: 100 μm (B); 1 mm (C); 2 mm (E).

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: Immunostaining, Expressing

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Ebf3 is required in lateral plate mesoderm-derived cells for sternum osteoblast differentiation. (A) Left: The generation of lateral plate mesoderm-specific Ebf3-KO mice embryos by crossing the Ebf3 flox/+ Prx1-Cre line with Ebf3 flox/flox Rosa26 Tdtomato/Tdtomato mice. Right: The expression pattern of Prx1-Cre-activated Tdtomato in the sternum tissue and muscle connective tissues based on longitudinal sections of E15.5 embryos together with immunostaining for tenascin (green). (B) Ossification analysis based on Alizarin Red skeletal preparations of thoracic bones in Ebf3 flox/flox Prx1-Cre embryos at E18.5. (C) Longitudinal sections of the sternum of Ebf3 flox/flox Prx1-Cre and Ebf3 flox/+ Prx1-Cre embryos at E18.5, immunostained for osteoblast (green, osterix) and pre-osteoblast (green, Runx2) markers. Upper panels show the distribution of lateral plate mesoderm-derived cells labeled with Prx1-Cre-activated TdTomato (red) in interstitial cells surrounding the sternum, which was decreased in Ebf3-KO embryos ( n =7) compared with heterozygous mice ( n =7). Lower-enlarged panels show much fewer osterix/Runx2-positive lateral plate mesoderm-derived cells in the periosteum of Ebf3-KO mice compared with heterozygous mice (white arrowheads). Scale bars: 100 μm.

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: Derivative Assay, Expressing, Immunostaining, Labeling

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Decreased number of Runx2 + pre-osteoblasts in Ebf3-deficient thoracic lateral plate mesoderm at E12.5. (A) Transverse section of thoracic lateral plate mesoderm (as indicated in the schematic) at E12.5 immunostained with anti-Ebf3 and anti-Runx2 antibodies. Most but not all Ebf3-expressing cells were Runx2 positive. White and yellow arrowheads show Ebf3/Runx2 double-positive cells. (B) Decrease in the number of Prx1: Tdtomato + Runx2 + cells in Ebf3 flox/flox Prx1-Cre compared with that in heterozygous embryos (white arrowheads). Immunostained transverse sections of the thoracic lateral plate mesoderm at E12.5 are shown. A and upper panels of B show images of Ebf3/Runx2-double-positive cells and those of Prx1: Tdtomato + Runx2 + LPMs, respectively, prepared from a single section of a heterozygous embryo stained with antibodies against Ebf3, Runx2 and DAPI. Yellow arrowheads indicate Prx1: Tdtomato + Ebf3 + Runx2 + LPMs. (C) The ratio of Runx2 + cells in Prx1: Tdtomato + thoracic LPMs was lower in Ebf3 flox/flox Prx1-Cre KO mice than in Ebf3 heterozygous mice at E12.5. Error bars represent s.e.m. ** P <0.01 ( n =4). (D) Ratio of Sox9 + cells in Prx1: Tdtomato + thoracic LPMs at E12.5. Error bars represent s.e.m.; ns, non-significant ( n =4). Scale bars: 100 μm.

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: Expressing, Staining

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Ebf3 plays a crucial role in sternum ossification at approximately E9.5-E10.5. (A) Generation of tamoxifen-induced temporal Ebf3-KO mouse embryos by crossing the Ebf3 flox/+ Ubc-CreER T2 line with Ebf3 flox/flox mice. (B) Strategy to determine Ebf3 functional timing in sternal ossification in Ubc-CreER T2 Ebf3-KO embryos. (C) Evaluation of sternum phenotypes in Ebf3 flox/flox Ubc-CreER T2 embryos upon tamoxifen injection at different developmental stages (E8.5-E12.5) based on Alizarin Red skeletal preparations. Embryonic days when tamoxifen was injected into pregnant dams are indicated.

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: Functional Assay, Injection

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Ebf3 downstream analysis in mesenchymes of the thoracic lateral plate mesoderm by RNA-seq. (A) Thoracic LPMs were excised from Ebf3 flox/flox Prx1-Cre and Ebf3 flox/+ Prx1-Cre embryos at E10.5, and Prx1: Tdtomato + cells were isolated by FACS ( n =3). (B) Differentially expressed genes based on RNA-seq data between Ebf3 flox/flox Prx1-Cre and Ebf3 flox/+ Prx1-Cre embryos (cut off: 10 read, P ≤0.1) were categorized by their biological functions using IPA software into three groups: (1) genes required for the differentiation of connective-tissue (CT) cells, (2) genes required for the differentiation of bone cells and osteoblasts, and (3) genes required for the differentiation of mesenchymal cells. (C) Venn diagram of these three groups. Genes in red were significantly upregulated and genes in blue were significantly downregulated in Ebf3 flox/flox Prx1-Cre compared with expression in Ebf3 flox/+ Prx1-Cre embryos ( P <0.05) (from RNA-seq data). (D) Relative expression levels of MCT cell/tenocyte-associated genes in Ebf3 flox/flox Prx1-Cre embryos compared with those in Ebf3 flox/+ Prx1-Cre embryos (from RNA-seq data). ** P <0.01, * P <0.05; ns, not significant ( n =3). (E) Relative expression levels of osteoblast-associated genes in Ebf3 flox/flox Prx1-Cre embryos compared with those in Ebf3 flox/+ Prx1-Cre embryos. ** P <0.01, * P <0.05 (from RNA-seq data) ( n =3). (F) Relative expression level of Islet1 in Ebf3 flox/flox Prx1-Cre embryos was increased significantly compared with that in Ebf3 flox/+ Prx1-Cre embryos. ** P <0.01 (from RNA-seq data) ( n =3). (G) Islet1 was expressed in Prx1:Tdtomato + Ebf3 + cells at the lateral somitic frontier of the thoracic lateral plate mesoderm (as indicated in the schematic) at E10.5 (white arrowheads). (H) Increase in the number of Prx1:Tdtomato + Islet1 + cells in Ebf3 flox/flox Prx1-Cre ( n =3) compared with that in heterozygous embryos ( n =3) (white arrowheads). Immunostained transverse sections of the lateral somitic frontier at E10.5 are shown. (I) The ratio of Islet1 + cells in Prx1:Tdtomato + LPMs was higher in Ebf3 flox/flox Prx1-Cre compared with that in heterozygous mice at E10.5. ** P <0.01 ( n =3). Error bars represent s.e.m. Scale bars: 100 μm.

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: RNA Sequencing, Isolation, Software, Expressing

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Ebf3 regulates mesenchymal progenitors in Scx-Cre lineage cells of the thoracic lateral plate mesoderm. (A) Ebf3 was expressed in Prx1:Tdtomato + Scx:GFP + cells at the lateral somitic frontier of the thoracic lateral plate mesoderm at E10.5 (white arrowheads). Scale bar: 100 μm. Schematic shows Ebf3, Scx:GFP and Islet1 expression in the lateral somitic frontier. (B) Ossification analysis based on Alizarin Red skeletal preparations of thoracic bones in Ebf3 flox/flox Scx-Cre embryos at E18.5. Red rectangle highlights ossification defects of the sternum. (C) Schematic summarizing the working hypothesis on the roles of Ebf3 in sternum ossification. Osteo-chondrogenic progenitors in the lateral plate mesoderm give rise to osteo-fibro progenitors marked with Islet1 + cells in the lateral somitic frontier (green area). Ebf3 regulates differentiation of these cells towards Runx2 + pre-osteoblasts (light blue), which differentiate into osteoblasts and osteocytes to form the sternum bone. In the absence of Ebf3, osteo-fibro progenitors differentiate towards fibroblastic cells (yellow) or self-renew.

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Identification of genes expressed in a mesenchymal subset regulating prostate organogenesis using tissue and single cell transcriptomics

doi: 10.1038/s41598-017-16685-8

Figure Lengend Snippet: Validation of VMP- and SU-specific transcript expression in female and male P0 rat tissues. Quantitative real-time PCR (qPCR) showed significantly elevated levels of both control (Fgf10, Ptn and Scube1) and candidate (Ebf3, Gfra3, Nmur2, Rspo2, Scara5, Slc26a7, Robo1 and Meis2) VMP-specific transcripts versus SU. Fgf10, Ptn, Scube1, Ebf3, Gfra3, Scara5 and Meis2 were expressed in VP and DP, while Rspo2, Nmur2 and Slc26a7 showed low expression in VP and DP. SU candidate transcripts Anxa1, Enpp2 and Unc5b were enriched versus VMP. Data is represented as mean fold difference to VMP ± SD of duplicate biological replicates and duplicate technical replicates (n = 4). Significance was detected using One-way ANOVA with TUKEY multiple comparison *p < 0.05. Figures in red indicate fold difference compared to VMP.

Article Snippet: Immunostaining of Ebf3 and Meis2 on serial sections of female and male rat P0 urogenital sinus tissue (isolated as per ) was performed as per using Ebf3 IgG (Clone 8D6, mouse monoclonal, Novus Biologicals, Littleton, Colorado, USA; dilution 1:1000) and Meis2 IgG (Clone 63-T, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, USA; dilution 1:750).

Techniques: Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Control, Comparison

Journal: Scientific Reports

Article Title: Identification of genes expressed in a mesenchymal subset regulating prostate organogenesis using tissue and single cell transcriptomics

doi: 10.1038/s41598-017-16685-8

Figure Lengend Snippet: Immunohistochemistry of Ebf3 and Meis2 in female VMP and male prostate. To examine the distribution of Ebf3 and Meis2 in mesenchymal subsets in VMP and ventral prostate (VP) we performed immunohistochemistry using P0 rat female and male reproductive tracts. Panels ( a–d ) show Ebf3 expression; in female ( a and c ) and male ( b and d ). Ebf3 showed a highly selective distribution within VMP and VP mesenchyme, and was absent from smooth muscle (SM) and the urethra ( c , a and b ). Panels ( e–h ) show Meis2 distribution; in female ( e and g ) and male ( f and h ) mesenchyme. Urethral epithelia (URE) and prostatic epithelia (E) were negative for both Ebf3 and Meis2. Scale bars ( a , b , e and f ) = 600 µm. Scale bars ( c , d , g and h ) = 300 µm.

Article Snippet: Immunostaining of Ebf3 and Meis2 on serial sections of female and male rat P0 urogenital sinus tissue (isolated as per ) was performed as per using Ebf3 IgG (Clone 8D6, mouse monoclonal, Novus Biologicals, Littleton, Colorado, USA; dilution 1:1000) and Meis2 IgG (Clone 63-T, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, USA; dilution 1:750).

Techniques: Immunohistochemistry, Expressing

Journal: Scientific Reports

Article Title: Identification of genes expressed in a mesenchymal subset regulating prostate organogenesis using tissue and single cell transcriptomics

doi: 10.1038/s41598-017-16685-8

Figure Lengend Snippet: Summary of primers used for qPCR.

Article Snippet: Immunostaining of Ebf3 and Meis2 on serial sections of female and male rat P0 urogenital sinus tissue (isolated as per ) was performed as per using Ebf3 IgG (Clone 8D6, mouse monoclonal, Novus Biologicals, Littleton, Colorado, USA; dilution 1:1000) and Meis2 IgG (Clone 63-T, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, USA; dilution 1:750).

Techniques:

Journal: Cancer Research

Article Title: Disruption of Transforming Growth Factor-β Signaling by Five Frequently Methylated Genes Leads to Head and Neck Squamous Cell Carcinoma Pathogenesis

doi: 10.1158/0008-5472.can-09-3073

Figure Lengend Snippet: Figure 1. Interaction between frequently methylated genes and TGF-β pathway components in HNSCC. A, immunoprecipitation was done with SMAD2 antibody using lysates from WT SCC22B and SCC22B + IRX1 overexpression. This revealed no IRX1 association with SMAD2. B, chromatin immunoprecipitation analysis using EBF3 and SMAD2 antibodies in SCC22B cells. p21 and IRX1 promoter pulldown was assessed through quantitative PCR, which revealed increased pulldown of both promoters with ther respective antibody compared with the negative IgG control. C, immunoprecipitation analysis of SLC5A8 pulldown by survivin antibody. Arrow, SLC5A8, which is only pulled down by survivin and not by the IgG control. D, overexpression of SEPT9 in SCC22B and immunoprecipitation analysis of SEPT9 pulldown by HIF-1α antibody. HIF-1α associates with SEPT9, and this binding is increased in SEPT9-overexpressing cells (+SEPT9). Furthermore, SEPT9 expression correlates with decreased HIF-1α expression.

Article Snippet: The antibodies used in the study were as follows: IL-6 (Abcam, ab6672), ARTS (Sigma-Aldrich, A4471), SLC5A8 (Santa Cruz, sc-34189), SEPT9 (aka MSF; Abgent, AP6215a), EBF3 (Aviva Systems Biology, ARP31837_T100), survivin (Santa Cruz, sc-17779), α-tubulin (Cell Signaling Technologies), TGF-β1 (Santa Cruz, sc-146), caspase-3 (Cell Signaling, 9662), actin (Santa Cruz, sc-8432), HIF-1α (Cell Signaling, 3716), and involucrin (Aviva Systems Biology, ARP48675_P050).

Techniques: Methylation, Immunoprecipitation, Over Expression, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Control, Binding Assay, Expressing

Journal: Cancer Research

Article Title: Disruption of Transforming Growth Factor-β Signaling by Five Frequently Methylated Genes Leads to Head and Neck Squamous Cell Carcinoma Pathogenesis

doi: 10.1158/0008-5472.can-09-3073

Figure Lengend Snippet: Figure 3. EBF3 regulates p21 gene expression. A, luciferase analysis of p21 promoter activity in SCC22B cells with and without EBF3 overexpression. Overexpression of EBF3 significantly increases p21 promoter activity. *, P < 0.0006. B, quantitative RT-PCR analysis of p21 mRNA expression in SCC22B cells with and without EBF3 overexpression. EBF3 overexpression results in a significant increase in p21 transcription. *, P < 0.05.

Article Snippet: The antibodies used in the study were as follows: IL-6 (Abcam, ab6672), ARTS (Sigma-Aldrich, A4471), SLC5A8 (Santa Cruz, sc-34189), SEPT9 (aka MSF; Abgent, AP6215a), EBF3 (Aviva Systems Biology, ARP31837_T100), survivin (Santa Cruz, sc-17779), α-tubulin (Cell Signaling Technologies), TGF-β1 (Santa Cruz, sc-146), caspase-3 (Cell Signaling, 9662), actin (Santa Cruz, sc-8432), HIF-1α (Cell Signaling, 3716), and involucrin (Aviva Systems Biology, ARP48675_P050).

Techniques: Gene Expression, Luciferase, Activity Assay, Over Expression, Quantitative RT-PCR, Expressing

Journal: Cancer Research

Article Title: Disruption of Transforming Growth Factor-β Signaling by Five Frequently Methylated Genes Leads to Head and Neck Squamous Cell Carcinoma Pathogenesis

doi: 10.1158/0008-5472.can-09-3073

Figure Lengend Snippet: Figure 4. Functional relevance of the interactions between the methylated candidates and components of the TGF-β pathway. A, Western blot analysis of ARTS, caspase-3, TGF-β, and involucrin expression in the five overexpressing cell lines (tubulin serves as a loading control). B, cell cycle analysis of the five overexpressing cell lines revealed a significant decrease in mitosis in IRX1-, EBF3-, and SLC5A8-overexpressing cells. *, P < 0.05; **, P < 0.01.

Article Snippet: The antibodies used in the study were as follows: IL-6 (Abcam, ab6672), ARTS (Sigma-Aldrich, A4471), SLC5A8 (Santa Cruz, sc-34189), SEPT9 (aka MSF; Abgent, AP6215a), EBF3 (Aviva Systems Biology, ARP31837_T100), survivin (Santa Cruz, sc-17779), α-tubulin (Cell Signaling Technologies), TGF-β1 (Santa Cruz, sc-146), caspase-3 (Cell Signaling, 9662), actin (Santa Cruz, sc-8432), HIF-1α (Cell Signaling, 3716), and involucrin (Aviva Systems Biology, ARP48675_P050).

Techniques: Functional Assay, Methylation, Western Blot, Expressing, Control, Cell Cycle Assay