Journal: Frontiers in cell and developmental biology

Article Title: Production of human entorhinal stellate cell-like cells by forward programming shows an important role of Foxp1 in reprogramming.

doi: 10.3389/fcell.2022.976549

Figure Lengend Snippet: FIGURE 4 Unique transcriptional factors upregulated in RELN positive clusters. (A) Expression of the top twenty enriched transcription factors (TFs, x-axis) were identified for each of the RELN-positive and putative stellate cell (SC) populations (RELN6, 7) and are visualized across the identified progenitor and excitatory neuron populations (y-axis) in the developing medial entorhinal cortex (MEC). The selected genes for reprogramming induced pluripotent stem cells (iPSCs) are highlighted in blue within the RELN7 cluster. (B) The laminar expression of the six stellate cell TFs (Runx1t1, Eya2, Foxp1, Sox5, Tcf4, and Mef2c) was investigated by ISH in sagittal sections of the publicly available Allen Mouse Brain Atlas (Lein et al., 2004; 2007). Image credit: Allen Institute. The red dotted lines demarcate the lamina dissecans.

Article Snippet: Plasmids containing EYA2 (Addgene #49264), RUNX1T1 (Addgene #49264), TCF4 (Addgene #109144), Foxp1 (Addgene #16362), Sox5 (Addgene #48707) and MEF2C (Addgene #61538) were all acquired from Addgene.

Techniques: Expressing

Journal: Frontiers in cell and developmental biology

Article Title: Production of human entorhinal stellate cell-like cells by forward programming shows an important role of Foxp1 in reprogramming.

doi: 10.3389/fcell.2022.976549

Figure Lengend Snippet: FIGURE 6 Efficiency and morphology of transduced induced pluripotent stem cells (iPSCs) after dropping out a single transcription factor. (A) Co- expression of Reelin and Nestin in cells 10 days post induction (dpi) was significantly decreased when Foxp1 was dropped out (p = 0.0013 using a one-way ANOVA) in three human iPSC lines (B) Morphology of transduced cells revealed that removal of RUNX1T1 resulted in a higher number of neurons in the SFC180 iPSC line. (C) Co-expression of Reelin and Nestin in cells 10 dpi with Foxp1 but with drop out of two factors, showed no significant difference between transcription factor combinations and high conversion rates in the SFC180 iPSC line. (D) Assessment of morphology 10 dpi with Foxp1 but drop out of two factors showed loss of EYA2 resulted in higher numbers of neurons indicating EYA2 is redundant in the reprogramming process. (E) Immunocytochemistry of forward programmed cells 10 dpi shows that dropping out RUNX1T1 in combination with either TCF4 or MEF2C results in cells co-expressing Nestin and Reelin with neural progenitor morphology, whereas dropping out of EYA2 in combination with RUNX1T1 or MEF2C results in more Nestin+/Reelin + neurons. More neurons can be observed in the -RUNX1T1, -EYA2 and in the -MEF2C, -EYA2 dropout images. Scale bar: 25 μm.

Article Snippet: Plasmids containing EYA2 (Addgene #49264), RUNX1T1 (Addgene #49264), TCF4 (Addgene #109144), Foxp1 (Addgene #16362), Sox5 (Addgene #48707) and MEF2C (Addgene #61538) were all acquired from Addgene.

Techniques: Expressing, Immunocytochemistry

Journal: Frontiers in cell and developmental biology

Article Title: Production of human entorhinal stellate cell-like cells by forward programming shows an important role of Foxp1 in reprogramming.

doi: 10.3389/fcell.2022.976549

Figure Lengend Snippet: FIGURE 4 Unique transcriptional factors upregulated in RELN positive clusters. (A) Expression of the top twenty enriched transcription factors (TFs, x-axis) were identified for each of the RELN-positive and putative stellate cell (SC) populations (RELN6, 7) and are visualized across the identified progenitor and excitatory neuron populations (y-axis) in the developing medial entorhinal cortex (MEC). The selected genes for reprogramming induced pluripotent stem cells (iPSCs) are highlighted in blue within the RELN7 cluster. (B) The laminar expression of the six stellate cell TFs (Runx1t1, Eya2, Foxp1, Sox5, Tcf4, and Mef2c) was investigated by ISH in sagittal sections of the publicly available Allen Mouse Brain Atlas (Lein et al., 2004; 2007). Image credit: Allen Institute. The red dotted lines demarcate the lamina dissecans.

Article Snippet: Plasmids containing EYA2 (Addgene #49264), RUNX1T1 (Addgene #49264), TCF4 (Addgene #109144), Foxp1 (Addgene #16362), Sox5 (Addgene #48707) and MEF2C (Addgene #61538) were all acquired from Addgene.

Techniques: Expressing

Journal: Frontiers in cell and developmental biology

Article Title: Production of human entorhinal stellate cell-like cells by forward programming shows an important role of Foxp1 in reprogramming.

doi: 10.3389/fcell.2022.976549

Figure Lengend Snippet: FIGURE 6 Efficiency and morphology of transduced induced pluripotent stem cells (iPSCs) after dropping out a single transcription factor. (A) Co- expression of Reelin and Nestin in cells 10 days post induction (dpi) was significantly decreased when Foxp1 was dropped out (p = 0.0013 using a one-way ANOVA) in three human iPSC lines (B) Morphology of transduced cells revealed that removal of RUNX1T1 resulted in a higher number of neurons in the SFC180 iPSC line. (C) Co-expression of Reelin and Nestin in cells 10 dpi with Foxp1 but with drop out of two factors, showed no significant difference between transcription factor combinations and high conversion rates in the SFC180 iPSC line. (D) Assessment of morphology 10 dpi with Foxp1 but drop out of two factors showed loss of EYA2 resulted in higher numbers of neurons indicating EYA2 is redundant in the reprogramming process. (E) Immunocytochemistry of forward programmed cells 10 dpi shows that dropping out RUNX1T1 in combination with either TCF4 or MEF2C results in cells co-expressing Nestin and Reelin with neural progenitor morphology, whereas dropping out of EYA2 in combination with RUNX1T1 or MEF2C results in more Nestin+/Reelin + neurons. More neurons can be observed in the -RUNX1T1, -EYA2 and in the -MEF2C, -EYA2 dropout images. Scale bar: 25 μm.

Article Snippet: Plasmids containing EYA2 (Addgene #49264), RUNX1T1 (Addgene #49264), TCF4 (Addgene #109144), Foxp1 (Addgene #16362), Sox5 (Addgene #48707) and MEF2C (Addgene #61538) were all acquired from Addgene.

Techniques: Expressing, Immunocytochemistry

Journal: Blood Advances

Article Title: Non-del(5q) myelodysplastic syndromes–associated loci detected by SNP-array genome-wide association meta-analysis

doi: 10.1182/bloodadvances.2019000922

Figure Lengend Snippet: Summary of MDS associated poymorphisms

Article Snippet: THP-1 cells were stably transfected with pcDNA3.1 Flag-Eya2 vector purchased from Addgene (plasmid #49264; Cambridge, MA) using an Amaxa Nucleofector (Lonza, Walkersville, MD) with the manufacturer’s THP-1–optimized transfection protocol.

Techniques:

Journal: Blood Advances

Article Title: Non-del(5q) myelodysplastic syndromes–associated loci detected by SNP-array genome-wide association meta-analysis

doi: 10.1182/bloodadvances.2019000922

Figure Lengend Snippet: EYA2 expression and impact on survival and hematopoiesis. (A) Relative gene expression of EYA2 using the NTUH data set showing significantly higher EYA2 expression in MDS case subjects compared with control subjects. (B) EYA2 gene expression is higher in lower risk disease subtypes, including RARS and RCMD. (C) Kaplan-Meier plot showing that higher EYA2 expression is associated with improved OS. (D) Western blot showing activation of innate immune signaling after EYA2 transfection. (E) Representative micrographs (40×) of colony-forming capacity assays of control (i) or EYA2 inhibitor–treated (ii, 1 µM; iii, 10 µM) MDS bone marrow mononuclear cells showing significantly greater erythroid colonies in treated samples with accompanying quantitation for each of 3 independent samples (mean ± standard error). *P < .005, **P < .0001.

Article Snippet: THP-1 cells were stably transfected with pcDNA3.1 Flag-Eya2 vector purchased from Addgene (plasmid #49264; Cambridge, MA) using an Amaxa Nucleofector (Lonza, Walkersville, MD) with the manufacturer’s THP-1–optimized transfection protocol.

Techniques: Expressing, Western Blot, Activation Assay, Transfection, Quantitation Assay

Journal: BioMed Research International

Article Title: Eya2 Is Overexpressed in Human Prostate Cancer and Regulates Docetaxel Sensitivity and Mitochondrial Membrane Potential through AKT/Bcl-2 Signaling

doi: 10.1155/2019/3808432

Figure Lengend Snippet: Eya2 expression in prostate cancers. (a) Negative or weak immunostaining of Eya2 in normal prostate tissue. (b) Negative Eya2 staining in prostate cancer. (c) Moderate nuclear Eya2 staining in a prostate cancer. (d) Strong nuclear Eya2 immunostaining in a case of prostate cancer. (e) Analysis of the Singh Prostate dataset of Oncomine. Eya2 mRNA was significantly elevated in prostate cancers compared with normal prostate tissues. (f) Eya2 mRNA in prostate cancers (TCGA). Eya2 mRNA positively correlated with Gleason score. (g) Eya2 mRNA was positively correlated with T stage (TCGA dataset). (h) Eya2 mRNA expression was significantly higher in prostate cancers with nodal metastasis (TCGA dataset). Statistical methods and p values are indicated in the figure.

Article Snippet: Cells were cultured in PRMI-1640 with 10% fetal bovine serum (FBS). pcDNA 3.1 Eya2 plasmid was obtained from Addgene.

Techniques: Expressing, Immunostaining, Staining

Journal: BioMed Research International

Article Title: Eya2 Is Overexpressed in Human Prostate Cancer and Regulates Docetaxel Sensitivity and Mitochondrial Membrane Potential through AKT/Bcl-2 Signaling

doi: 10.1155/2019/3808432

Figure Lengend Snippet: Distribution of Eya2 status in prostate cancer according to clinicopathological characteristics.

Article Snippet: Cells were cultured in PRMI-1640 with 10% fetal bovine serum (FBS). pcDNA 3.1 Eya2 plasmid was obtained from Addgene.

Techniques: Expressing

Journal: BioMed Research International

Article Title: Eya2 Is Overexpressed in Human Prostate Cancer and Regulates Docetaxel Sensitivity and Mitochondrial Membrane Potential through AKT/Bcl-2 Signaling

doi: 10.1155/2019/3808432

Figure Lengend Snippet: Eya2 regulates the proliferation and invasion of prostate cancer. (a) Western blot analysis of Eya2 protein in prostate cancer cell lines (LNCaP, PC-3, and DU145) and RWPE-1 (normal prostate cell line): there were higher levels of Eya2 protein in cancer cell lines than the normal cell line. Eya2 transfection significantly increased the levels of Eya2 protein and mRNA levels in PC-3 and LNCaP cells. The application of Eya2 shRNA significantly reduced the levels of Eya2 protein and mRNA levels in PC-3 and LNCaP cells. (b) Eya2 transfection led to an increase in proliferation rate in both PC-3 and LNCaP cell lines, while Eya2 shRNA knockdown reduced the proliferation rate. (c) Colony formation demonstrated that the overexpression of Eya2 upregulated colony number in both PC-3 and LNCaP cell lines, while Eya2 shRNA knockdown downregulated colony number. (d) Matrigel invasion assays demonstrated that the overexpression of Eya2 in both PC-3 and LNCaP cell lines increased the number of invading cells, while Eya2 shRNA knockdown decreased invading cell numbers. ∗ p<0.05.

Article Snippet: Cells were cultured in PRMI-1640 with 10% fetal bovine serum (FBS). pcDNA 3.1 Eya2 plasmid was obtained from Addgene.

Techniques: Western Blot, Transfection, shRNA, Knockdown, Over Expression

Journal: BioMed Research International

Article Title: Eya2 Is Overexpressed in Human Prostate Cancer and Regulates Docetaxel Sensitivity and Mitochondrial Membrane Potential through AKT/Bcl-2 Signaling

doi: 10.1155/2019/3808432

Figure Lengend Snippet: Eya2 regulates docetaxel sensitivity and apoptosis. (a) PC-3 and LNCaP cells experiencing Eya2 transfection/shRNA knockdown were treated with different concentration of docetaxel (2.5, 5, 10, and 15 nM). CCK-8 assays were used to determine cell viability. The overexpression of Eya2 led to an increase in cell viability in both cell lines while the depletion of Eya2 led to downregulated cell viability. (b) PC-3 and LNCaP cells experiencing Eya2 transfection/shRNA knockdown were treated with 5nM docetaxel for 24 h. Annexin V/PI staining showed that Eya2 overexpression inhibited the docetaxel-induced rate of apoptosis. Eya2 shRNA increased the rate of docetaxel-induced apoptosis. ∗ p < 0.05.

Article Snippet: Cells were cultured in PRMI-1640 with 10% fetal bovine serum (FBS). pcDNA 3.1 Eya2 plasmid was obtained from Addgene.

Techniques: Transfection, shRNA, Knockdown, Concentration Assay, CCK-8 Assay, Over Expression, Staining

Journal: BioMed Research International

Article Title: Eya2 Is Overexpressed in Human Prostate Cancer and Regulates Docetaxel Sensitivity and Mitochondrial Membrane Potential through AKT/Bcl-2 Signaling

doi: 10.1155/2019/3808432

Figure Lengend Snippet: Eya2 regulates mitochondrial membrane potential and related proteins. (a) The overexpression of Eya2 inhibited the expression of cleaved-caspase3, cleaved-PARP and upregulated caspase3, PARP in docetaxel treated cells. The knockdown of Eya2 using shRNA led to an upregulation in the levels of cleaved-caspase3, cleaved-PARP and downregulated caspase3, PARP in docetaxel treated cells. (b) JC-1 staining was used to determine Δ ψ m. JC-1 is normally visualized as green when Δ ψ m is reduced. Our JC-1 staining results showed that the overexpression of Eya2 upregulated the Δ ψ m (as shown by a reduction in the proportion of green staining) while Eya2 shRNA reduced Δ ψ m (as shown by an increased proportion of green staining) after CDDP treatment. (c) Protein levels of Bcl-2, MMP7, p-AKT, and AKT in prostate cancer cells transfected with the Eya2 plasmid and shRNA; the overexpression of Eya2 increased the expression of Bcl-2, MMP7, and p-AKT proteins while the application of shRNA caused levels of these proteins to decrease. (d) Realtime PCR showed that the overexpression of Eya2 o increased the mRNA levels of Bcl-2 and MMP7 in prostate cancer cells while the application of shRNA caused a reduction in the expression of these proteins. ∗ p<0.05.

Article Snippet: Cells were cultured in PRMI-1640 with 10% fetal bovine serum (FBS). pcDNA 3.1 Eya2 plasmid was obtained from Addgene.

Techniques: Membrane, Over Expression, Expressing, Knockdown, shRNA, Staining, Transfection, Plasmid Preparation

Journal: BioMed Research International

Article Title: Eya2 Is Overexpressed in Human Prostate Cancer and Regulates Docetaxel Sensitivity and Mitochondrial Membrane Potential through AKT/Bcl-2 Signaling

doi: 10.1155/2019/3808432

Figure Lengend Snippet: Eya2 upregulates Bcl-2 via the AKT signaling pathway. (a) We identified positive associations between Eya2 mRNA and Bcl-2/MMP7 mRNA in 497 cases of prostate cancer (Pearson's correlation analysis, RNA-seq data was obtained from the TCGA database). (b) Protein levels of Bcl-2, p-AKT, and AKT in LNCaP cells treated with Eya2 and the AKT inhibitor, perifosine. Realtime PCR analysis of Bcl-2 mRNA in LNCaP cells treated with Eya2 plasmid and the AKT inhibitor, perifosine. Eya2 overexpression failed to upregulate levels of Bcl-2 mRNA and protein in cells treated with the AKT inhibitor.

Article Snippet: Cells were cultured in PRMI-1640 with 10% fetal bovine serum (FBS). pcDNA 3.1 Eya2 plasmid was obtained from Addgene.

Techniques: RNA Sequencing, Plasmid Preparation, Over Expression