Journal: Molecular Medicine Reports

Article Title: Concentrated growth factor exudate enhances the proliferation of human periodontal ligament cells in the presence of TNF-α

doi: 10.3892/mmr.2018.9714

Figure Lengend Snippet: Identification and characterization of primary hPDLCs. (A) Primary cells grew out from the tissue explants and (B) were spindle shaped. Scale bar, 200 µm. (C) Immunocytochemistry staining showed that cells were vimentin positive and (D) cytokeratin negative. Scale bar, 100 µm. hPDLCs, human periodontal ligament cells.

Article Snippet: Next, cells were incubated with anti-vimentin (1:100; cat. no. ab24525; Abcam, Cambridge, MA, USA) and anti-cytokeratin (1:200; cat. no. AM06387SU-N; OriGene Technologies, Inc., Beijing, China) primary antibodies overnight at 4°C.

Techniques: Immunocytochemistry, Staining

Journal: Scientific Reports

Article Title: GATA4 blocks squamous epithelial cell gene expression in human esophageal squamous cells

doi: 10.1038/s41598-021-82557-x

Figure Lengend Snippet: Ectopic expression of GATA4 in human squamous esophageal epithelial NES-B10T cells alters the expression of squamous cell marker genes. Expression of four squamous epithelial cell marker genes (p63, KRT5, KRT13, KRT15 ) were examined by qRT-PCR and/or immunoblot in three pInducer20-GATA4 NES-B10T cell clones (G4_1, G4_2, G4_3) and one pInducer20 NES-B10T control cell clone (C) after 72 h of doxycycline treatment to induce GATA4. ( A – D ) qRT-PCR demonstrated reduced steady-state levels of p63, KRT5, KRT13 , and KRT15 mRNA in NES-B10T cells expressing GATA4 compared with controls cells lacking GATA4 protein (n = 3 experiments; *P ≤ 0.05). ( E – I ) Quantitative infrared immunoblotting (LI-COR) was used to analyze p63, KRT5, and KRT13 protein expression in control and GATA4 expressing NES-B10T cell clones. Revert Total Protein Stain was used for normalization. Immunoblots were performed using protein extracts from two independent doxycycline induction experiments. Representative blots are shown in ( E ) and ( F ). Quantification of blots is shown in panels G-I with the abundance of protein detected in GATA-expressing clones expressed as the percent relative to the non-expressing control cells. Full immunoblots used to generate panels are shown in Supplementary Fig. .

Article Snippet: For quantitative reverse-transcription polymerase chain reaction (qRT-PCR), cDNA synthesized using MMLV and random hexamer primers was amplified using TaqMan Gene Expression Mastermix and TaqMan gene expression assays ( GATA4 , HS00171403_m1; KRT5 , Hs00361185_m1; KRT8 , Hs01595539_m1; KRT13 , Hs00357961_g1; KRT15 , Hs00951967_m1; KRT20 , Hs00300643_m1; p63 , Hs00978340_m1; CDX2 , Hs01078080_m1; VILLIN , Hs01031724_m1; Gata4 , Mm00484689_m1; p63 , Mm00495793_m1; Krt5 , Mm01305291_g1; Krt15 , Mm00492972_m1).

Techniques: Expressing, Marker, Quantitative RT-PCR, Western Blot, Clone Assay, Control, Staining

Journal: Scientific Reports

Article Title: GATA4 blocks squamous epithelial cell gene expression in human esophageal squamous cells

doi: 10.1038/s41598-021-82557-x

Figure Lengend Snippet: Effects of ectopic expression of GATA4 on the expression of squamous epithelial cell marker genes in human squamous esophageal epithelial NES-B3T cells were less pronounced than those observed in GATA4-expressing NES-B10T cells. Expression of four squamous epithelial cell marker genes (p63, KRT5, KRT13, KRT15 ) were examined by qRT-PCR and/or immunoblot in three pInducer20-GATA4 NES-B3T cell clones (G4_1, G4_2, G4_3) and one pInducer20 NES-B3T control cell clone (C) after 72 h of doxycycline treatment to induce GATA4. ( A – D ) qRT-PCR demonstrated a trend for reduction in the steady-state levels of p63 , KRT5 , and KRT15 , but not KRT13 , in NES-B3T cells expressing GATA4 compared with controls cells lacking GATA4 protein. Statistically significant reductions were observed for only one cell clone, G4_2, for p63 expression and two clones, G4_2 and G4_3, for KRT5 expression (n = 3 experiments; *P ≤ 0.05). ( E – I ) Quantitative infrared immunoblotting (LI-COR) was used to analyze p63, KRT5, and KRT13 protein expression in control and GATA4 expressing NES-B3T cell clones. Revert Total Protein Stain was used for normalization. Immunoblots were performed using extracts from two independent doxycycline induction experiments. Representative blots are shown in panels ( E ) and ( F ). Quantification of blots is shown in panels ( G – I ) with the abundance of protein detected in GATA-expressing clones expressed as the percent relative to the non-expressing control cells. Full immunoblots used to generate panels are shown in Supplementary Fig. .

Article Snippet: For quantitative reverse-transcription polymerase chain reaction (qRT-PCR), cDNA synthesized using MMLV and random hexamer primers was amplified using TaqMan Gene Expression Mastermix and TaqMan gene expression assays ( GATA4 , HS00171403_m1; KRT5 , Hs00361185_m1; KRT8 , Hs01595539_m1; KRT13 , Hs00357961_g1; KRT15 , Hs00951967_m1; KRT20 , Hs00300643_m1; p63 , Hs00978340_m1; CDX2 , Hs01078080_m1; VILLIN , Hs01031724_m1; Gata4 , Mm00484689_m1; p63 , Mm00495793_m1; Krt5 , Mm01305291_g1; Krt15 , Mm00492972_m1).

Techniques: Expressing, Marker, Quantitative RT-PCR, Western Blot, Clone Assay, Control, Staining

Journal: Scientific Reports

Article Title: GATA4 blocks squamous epithelial cell gene expression in human esophageal squamous cells

doi: 10.1038/s41598-021-82557-x

Figure Lengend Snippet: GATA4 occupies the proximal promoters of squamous epithelial cell marker genes suggesting that GATA4 can repress expression of these genes. ( A ) qRT-PCR was used to examine the expression profiles of Gata4 , p63 , Krt5 , and Krt15 genes in mouse columnar, glandular hindstomach and stratified squamous forestomach epithelial cells. As expected, Gata4 transcript was detected only in columnar hindstomach epithelium, whereas p63 , Krt5 , and Krt15 transcripts were detected only in the stratified squamous forestomach. ( B ) Diagrammatic representation of GATA consensus binding sites identified as evolutionarily conserved among human, mouse, and chimpanzee in the p63 , Krt5 , and Krt15 genes. ( C ) GATA4 Bio-ChIP–PCR showed enriched amplification of regions containing predicted evolutionarily conserved GATA binding sites in the p63 , Krt5 , and Krt15 genes in cells expressing biotinylated GATA4 compared with control cells lacking biotinylated GATA4. No amplification enrichment was observed for a gene lacking a GATA4 binding site (Hprt ). Autoradiographic band intensity was measured using a Storm820 Phosphor Imager and ImageQuant software. Representative autoradiographic data for two of four animals per genotype assayed shown. Amplification enrichment per sample was normalized to input (n = 4 GATA4-BIO and 4 GATA4-WT). Error bars show SEM, ** P ≤ 0.01. The full autoradiograph used to generate panels is shown in Supplementary Fig. .

Article Snippet: For quantitative reverse-transcription polymerase chain reaction (qRT-PCR), cDNA synthesized using MMLV and random hexamer primers was amplified using TaqMan Gene Expression Mastermix and TaqMan gene expression assays ( GATA4 , HS00171403_m1; KRT5 , Hs00361185_m1; KRT8 , Hs01595539_m1; KRT13 , Hs00357961_g1; KRT15 , Hs00951967_m1; KRT20 , Hs00300643_m1; p63 , Hs00978340_m1; CDX2 , Hs01078080_m1; VILLIN , Hs01031724_m1; Gata4 , Mm00484689_m1; p63 , Mm00495793_m1; Krt5 , Mm01305291_g1; Krt15 , Mm00492972_m1).

Techniques: Marker, Expressing, Quantitative RT-PCR, Binding Assay, Amplification, Control, Software, Autoradiography