Journal: Frontiers in Neuroscience

Article Title: Modeling Protein Aggregation and the Heat Shock Response in ALS iPSC-Derived Motor Neurons

doi: 10.3389/fnins.2018.00086

Figure Lengend Snippet: Antibodies.

Article Snippet: Mouse α-Islet1 , DSHB , 40.2D6.

Techniques:

Journal: Frontiers in Neuroscience

Article Title: Modeling Protein Aggregation and the Heat Shock Response in ALS iPSC-Derived Motor Neurons

doi: 10.3389/fnins.2018.00086

Figure Lengend Snippet: iPSC-derived motor neurons from multiple ALS genotypes do not exhibit reduced viability in culture. (A) Representative images at 3 weeks of differentiation of control and ALS motor neurons immunostained for Tuj1 (green), ChAT (red), and Islet1 (white). Nuclei are labeled with Hoechst nuclear dye (blue). Scale bar = 50 μm. (B) Quantification shows no difference in the number of motor neurons at 3 or 4 weeks of differentiation. n = 3–4. Not significant by one-way ANOVA with Tukey post-hoc test.

Article Snippet: Mouse α-Islet1 , DSHB , 40.2D6.

Techniques: Derivative Assay, Control, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Islet-1 Regulates Arx Transcription during Pancreatic Islet ?-Cell Development *

doi: 10.1074/jbc.M111.231670

Figure Lengend Snippet: The Isl-1 binding sites in Re1 and Re2 are necessary for αTC1–6 cell activity. Sequence identity within Arx Re1 (A) and Arx Re2 (B) between mouse, human, and rat. Core Isl-1 binding site sequences are shown in bold, and the lines above demarcate the EMSA probe. Nucleotide mutated for luciferase reporter (C) and EMSA (Fig. 6C) assays are labeled in red. Nonconserved nucleotides are labeled in green. C, the activity of wild-type and Isl-1 binding site mutants (red) of pGL4.27-Re1 and pGL4.27-Re2 in αTC1–6 cells. The pRL-SV40 normalized data were presented as the mean ± S.E. *, p <0.05.

Article Snippet: Competition experiments were performed using a 100-fold molar excess of the unlabeled oligonucleotide. α-Isl-1 (Developmental Studies Hybridoma Bank at the University of Iowa, 40.2D6) or α-Myc antibody (SC-40, Santa Cruz Biotechnology) was incubated with the nuclear extract for 10 min on ice prior to probe addition in the supershift assays.

Techniques: Binding Assay, Activity Assay, Sequencing, Luciferase, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Islet-1 Regulates Arx Transcription during Pancreatic Islet ?-Cell Development *

doi: 10.1074/jbc.M111.231670

Figure Lengend Snippet: Isl-1 binding sites in Re1 and Re2. A and B, as a screen of putative Isl-1 binding elements, the radiolabeled MafA-R3 Isl-1 site probe (17) was used in reactions with in vitro translated Isl-1-Myc and MafA-R3, Re1, or Re2 competitor oligonucleotides. Isl-1 and Myc-epitope antibodies were used to localize the Isl-1-Myc:MafA complex. Ab, antibody; IVT, in vitro translation; Neg, without Isl-1-Myc; Pos, with Isl-1-Myc. C, Re2–2, Re2–3, and Re2–6 probes were incubated with αTC1–6 nuclear extract. The specificity of Isl-1:Arx-Re2 binding was determined by Isl-1 antibody addition and competition with excess of unlabeled wild-type (WT) and Isl-1 binding site mutant (MT).

Article Snippet: Competition experiments were performed using a 100-fold molar excess of the unlabeled oligonucleotide. α-Isl-1 (Developmental Studies Hybridoma Bank at the University of Iowa, 40.2D6) or α-Myc antibody (SC-40, Santa Cruz Biotechnology) was incubated with the nuclear extract for 10 min on ice prior to probe addition in the supershift assays.

Techniques: Binding Assay, In Vitro, Incubation, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Islet-1 Regulates Arx Transcription during Pancreatic Islet ?-Cell Development *

doi: 10.1074/jbc.M111.231670

Figure Lengend Snippet: Arx expression is elevated Pdx1PB-Isl-1-Myc mice. A, diagram representing the Pdx1PB-Isl-1-Myc transgenic construct, which contains the pdx1PB fragment of the pdx1 gene and the hsp68 minimal promoter. B, Western blot by α-Isl-1 and α-Myc analysis of total cell lysate from islets of 8-week-old control and Pdx1PB-Isl-1-Myc mice. The asterisk denotes a nonspecific band. C, Isl-1 mRNA levels in Pdx1PB-Isl-1-Myc and littermate control mice. Results are presented as mean ± S.E. *, p <0.05. D–G, immunohistochemical analysis for glucagon or insulin and Myc expression in 8-week-old Pdx1PB-Isl-1-Myc and control mice. H, Arx mRNA levels in Pdx1PB-Isl-1-Myc and control mice. Results are presented as mean ± S.E. *, p <0.05. I–L, costaining of Arx with glucagon or insulin in 8-week-old Pdx1PB-Isl-1-Myc and control mice. Insets show the magnified views of the outlined areas. The asterisk denotes autofluorescence from red blood cells.

Article Snippet: Competition experiments were performed using a 100-fold molar excess of the unlabeled oligonucleotide. α-Isl-1 (Developmental Studies Hybridoma Bank at the University of Iowa, 40.2D6) or α-Myc antibody (SC-40, Santa Cruz Biotechnology) was incubated with the nuclear extract for 10 min on ice prior to probe addition in the supershift assays.

Techniques: Expressing, Transgenic Assay, Construct, Western Blot, Immunohistochemical staining

Journal: The Journal of Biological Chemistry

Article Title: Islet-1 Regulates Arx Transcription during Pancreatic Islet ?-Cell Development *

doi: 10.1074/jbc.M111.231670

Figure Lengend Snippet: Isl-1 binds to Re1 and Re2 of the mouse Arx gene. A, schematic diagram of the Arx locus illustrating the locations of Re1 (orange box, +5.6 to +6.1) and Re2 (green box, +23.6 to +24 kb). TSS, transcription start site; Ex, exon. B, the ChIP-Seq image depicts Isl-1 occupancy within Arx Re1 (chrX:89544959–89545500) and Re2 (chrX:89563002–89563440) in αTC1–6 cells. The conservation between mouse and other species within this region was shown using the University of California Santa Cruz genome browser. C and D, Re1 and Re2 binding to Isl-1 was readily detected in adult mouse islets and αTC1–6 cells by standard ChIP but to a lesser extent in βTC3 cells. Results were normalized to Isl-1 binding to the PEPCK promoter. Data are presented as the mean ± S.E. n = 3; p value <0.05.

Article Snippet: Competition experiments were performed using a 100-fold molar excess of the unlabeled oligonucleotide. α-Isl-1 (Developmental Studies Hybridoma Bank at the University of Iowa, 40.2D6) or α-Myc antibody (SC-40, Santa Cruz Biotechnology) was incubated with the nuclear extract for 10 min on ice prior to probe addition in the supershift assays.

Techniques: ChIP-sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Islet-1 Regulates Arx Transcription during Pancreatic Islet ?-Cell Development *

doi: 10.1074/jbc.M111.231670

Figure Lengend Snippet: Re1 and Re2 mediate Arx transcription in αTC1–6 cells. A, pGL4.27-Re1, pGL4.27-Re2, and pGL4.27-Re1/2 were transfected into αTC1–6 cells in the presence or absence of a Isl-1-Myc expression plasmid. B, Western blot analysis using Isl-1 and control α-tubulin antibodies demonstrates a ∼70% reduction of Isl-1 protein levels in Isl-1 shRNA lentivirus-treated αTC1–6 cells. C, endogenous Arx mRNA expression was down-regulated relative to HPRT in Isl-1 shRNA treated αTC1–6 cells. D, Isl-1 shRNA knockdown reduced pGL4.27-Re1 and pGL4.27-Re2 reporter activity in αTC1–6 cells. Results are presented as the mean ± S.E. *, p <0.05. pGL4.27-Arx activity was normalized to that of the pRL-SV40 Renilla luciferase in A and D.

Article Snippet: Competition experiments were performed using a 100-fold molar excess of the unlabeled oligonucleotide. α-Isl-1 (Developmental Studies Hybridoma Bank at the University of Iowa, 40.2D6) or α-Myc antibody (SC-40, Santa Cruz Biotechnology) was incubated with the nuclear extract for 10 min on ice prior to probe addition in the supershift assays.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, shRNA, Activity Assay, Luciferase

Journal: EMBO Molecular Medicine

Article Title: Stimulation of endogenous cardioblasts by exogenous cell therapy after myocardial infarction

doi: 10.1002/emmm.201303626

Figure Lengend Snippet: Still images of GFP + cardioblasts exhibiting spontaneous contractile activity in vitro after 1 day in culture (see Supplementary Movies S1 and S2 for the corresponding Movies) (scale bars, 10 μm) [green: GFP, bright field (BF)]. Fluorescent immunocytochemistry of FACS-sorted GFP + cardioblasts for nuclear expression of cardiac transcription factors NKX2-5, GATA4, and MEF2C. Note that GFP − cells (middle & right panels, resulting from imperfect FACS for GFP) are negative for cardiac transcription factors (blue: DAPI, green: GFP, red: NKX2-5, GATA4, MEF2C). Fluorescent immunocytochemistry of FACS-sorted GFP + cardioblasts for cytoplasmic expression of α-sarcomeric actinin (αSA) and α-myosin heavy chain (αMHC). Image on the right shows a GFP + cardioblast expressing αMHC in the cytoplasm and NKX2-5 in the nucleus. Note that GFP − cells (left panel) do not express sarcomeric proteins (blue: DAPI, green: GFP, red: αSA/αMHC). Quantification of expression of cardiac transcription factors and sarcomeric proteins in GFP + cardioblasts by fluorescent immunocytochemistry ( n = 4 mice). No expression of TBX5 or ISL1 could be detected in GFP + cells. Fluorescent immunocytochemistry of FACS-sorted GFP + cardioblasts for Ki67 and phosphorylated histone H3 (H3P). Note that the GFP − cell is Ki67 − (left panel) and the GFP + dividing cardioblast is expressing H3P in both nuclei (right panel) (blue: DAPI, green: GFP, red: Ki67/αSA, white: H3P). Confocal microscopy revealed the presence of GFP + cardioblasts appearing to undergo mitosis in the border zone. Image on the right is a magnified image of highlighted area on left. Image of confocal scanning across the XZ plane is also provided (blue: DAPI, green: GFP, red: αSA). Quantification of expression of cycling proteins in GFP + cardioblasts by fluorescent immunocytochemistry ( n = 4 mice).

Article Snippet: Fluorescent immunocytochemistry was performed with antibodies against GFP (ab13970, Abcam), α-sarcomeric actin (A7811, Sigma), α-myosin heavy chain (ab15, Abcam), NKX2-5 (ab35842, Abcam), GATA4 (ab84593, Abcam), MEF2C (ab64644, Abcam), TBX5 (ab101227, Abcam), Isl1 (ab20670, Abcam), Ki67 (RM-9106, Thermo Scientific), H3P (ab5176, Abcam), lacZ (ab9361, Abcam), Sca-1 (ab25195, Abcam), Nestin (ab6142, Abcam), c-kit (ab5506, Abcam), SSEA1 (ab16285, Abcam), CD34 (ab8158, Abcam), PDGFRα (ab61219, Abcam), CD31 (ab28364, Abcam), CD 45 (ab10558, Abcam), CXCR4 (ab2074, Abcam), VEGFR1 (ab32152, Abcam), VEGFR2 (ab2349, Abcam), and VEGFR3 (ab27278, Abcam).

Techniques: Activity Assay, In Vitro, Immunocytochemistry, Expressing, Confocal Microscopy