Journal: Journal of the American College of Cardiology

Article Title: Pim1 Kinase Overexpression Enhances Ckit + Cardiac Stem Cell Cardiac Repair Following Myocardial Infarction in Swine

doi: 10.1016/j.jacc.2016.09.925

Figure Lengend Snippet: Localization of human mitochondrial marker is shown: (A) Confocal image depicts human mitochondria (red) costained with myosin light chain (green) and DAPI in Pim1+ hCSC animals 8 weeks after injection. Colocalization of human mitochondria (green), PIM1 (red), and nuclei (blue) is seen in hCSC animals (B) and PIM1+ hCSC-treated animals (C) at 8 weeks post-injection (arrows point to cells costained for both human mitochondria and PIM1). Scale bar = 10 μm. DAPI = 4’,6-diamidino-2- phenylindole; hMito = human mitochondria; other abbreviations as in Figure 1.

Article Snippet: Primary antibodies used: human mitochondria (1:600; Abcam, Cambridge, Massachusetts); phospho-histone H3 (pH3) (1:1500; Abcam); PIM-1 (1:30; Santa Cruz Biotechnology, Dallas, Texas; 1:30; Abcam), ckit (1:30; Sigma-Aldrich Co. LLC, St. Louis, Missouri), myosin light chain (1:100; Novus Biologicals, Littleton, Colorado), smooth muscle actin (sm22a) (1:1000, Abcam), endothelial cell (cd31) (1:1000, Abcam ).

Techniques: Marker, Injection

Journal: Journal of the American College of Cardiology

Article Title: Pim1 Kinase Overexpression Enhances Ckit + Cardiac Stem Cell Cardiac Repair Following Myocardial Infarction in Swine

doi: 10.1016/j.jacc.2016.09.925

Figure Lengend Snippet: (A) Confocal image depicts proliferating cells phospho-histone H3 (pH3) costained with myosin light chain (MLC) and DAPI. (B) Mitotic activity of endogenous cardiomyocytes (pH3) increased in the border zone in Pim1+ hCSC-injected animals as compared to placebo 8 weeks post-injection (p < 0.05). (C) There were no significant differences in mitotic activity within noncardiomyocyte cells. Scale bar = 20 μm. *p < 0.05 Pim1+ hCSCs vs. placebo. Abbreviations as in Figures 1 and ​and66.

Article Snippet: Primary antibodies used: human mitochondria (1:600; Abcam, Cambridge, Massachusetts); phospho-histone H3 (pH3) (1:1500; Abcam); PIM-1 (1:30; Santa Cruz Biotechnology, Dallas, Texas; 1:30; Abcam), ckit (1:30; Sigma-Aldrich Co. LLC, St. Louis, Missouri), myosin light chain (1:100; Novus Biologicals, Littleton, Colorado), smooth muscle actin (sm22a) (1:1000, Abcam), endothelial cell (cd31) (1:1000, Abcam ).

Techniques: Activity Assay, Injection

Journal: bioRxiv

Article Title: Role of Klhl14 in senescence and epithelial-to-mesenchymal transition via TGF-β modulation

doi: 10.1101/2025.09.12.675818

Figure Lengend Snippet: ( a ) Experimental setup: single cells obtained from 7-day-old organoids were transduced with lentiviruses after 24 hours, seeded in BMM domes, and analyzed one week later. ( b ) Western blot analysis to validate KLHL14 silencing, using GAPDH as housekeeping. On the right is the relative normalized quantification of the bands. ( c ) Microscope images of organoids grown from transduced cells for one week, using the GFP channel, 4x objective. Scalebar 100 μm. ( d ) Data obtained from the Incucyte Live-Cell Analysis system exploiting the GFP expression of the organoids: on the left, the green integrated intensity. On the right, the ratio between the green integrated intensity and the total object count indicates the organoids’ dimension. ( e ) 7-day-old organoids obtained from transduced cells were collected after the disruption of the BMM domes, and relative OFE was calculated by counting organoids with a size > 50 mm (left graph). After trypsinization of the collected organoids, single cells were counted to calculate the population doublings (right graph) according to the formula described in the Materials and Methods. Data are shown as mean ± SEM. Group differences were evaluated with two-tailed Student’s t -tests. N=4. ** p < 0.01

Article Snippet: KLHL14 and GAPDH primary antibodies (Proteintech, 16693-1-AP; Fitzgeral, 10R-G109a) were used respectively with a 1:1000 and 1:10000 dilution in BSA 3% PBS-Tween20; secondary antibodies were used with a 1:3000 dilution in BSA 3% PBS-Tween20.

Techniques: Transduction, Western Blot, Microscopy, Cell Analysis, Expressing, Disruption, Two Tailed Test

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibition results in altered dendritic spine development. (A) Representative dendritic segments of cortical neurons expressing empty vector (EV), Wif1, Sfrp1, Fzd8CRD or Dvl1ΔPDZ. (B) Quantification of dendritic protrusion density. (C) Percent of all dendritic protrusions classified as either spines or filopodia. Quantification of average protrusion length (D) and average spine head width (E) for each treatment. *** p < 0.001, ** p < 0.01, * p < 0.05. n = number of neurons: EV n = 31, Wif1 n = 34 Sfrp1 n = 23, Fzd8CRD n = 25, Dvl1ΔPDZ n = 25. Scale bar: 5 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Inhibition, Expressing, Plasmid Preparation

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Expression of Wnt inhibitors or Wnt2 shifts the distribution of dendritic protrusion lengths. (A) Relative frequency distributions of dendritic protrusion length comparing Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ expressing neurons to EV control. (B) Relative frequency distribution of protrusion length comparing Wnt2 expressing neurons to EV control. n = number of neurons: EV(A) n = 31, Wif1 n = 34, Sfrp1 n = 23, Fzd8CRD n = 25, Dvl1ΔPDZ n = 25; EV(B) n = 29, Wnt2 n = 25. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Expressing, Control

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibition results in decreased dendrite elaboration. (A) Representative cortical neurons expressing EV, Wif1, Sfrp1, Fzd8CRD or Dvl1ΔPDZ. (B) Quantification of the total dendrite length per neuron for each treatment. (C) Quantification of the number of dendritic endpoints per neuron for each treatment. (D–G) Sholl analysis of dendritic complexity comparing neurons treated with each Wnt inhibitor to control neurons. *** p < 0.001, ** p < 0.01, * p < 0.05. n = number of neurons: EV n = 56, Wif1 n = 49, Sfrp1 n = 39, Fzd8CRD n = 39, Dvl1ΔPDZ n = 39. Scale bar: 50 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Inhibition, Expressing, Control

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibition does not affect primary dendrite number on cortical neurons. (A) Representative cell bodies of cortical neurons expressing EV, Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ either alone, or in combination with BDNF. (B) Quantification of the number of primary dendrites per neuron for each treatment. *** p < 0.001. n = number of neurons: EV n = 31, BDNF n = 31, Wif1 n = 34, BDNF + Wif1 n = 29, Sfrp1 n = 22, BDNF + Sfrp1 n = 21, Fzd8CRD n = 25, BDNF + Fzd8CRD n = 25, Dvl1ΔPDZ n = 25, BDNF + Dvl1ΔPDZ n = 30. Scale bar: 5 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Inhibition, Expressing

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibitors interfere with BDNF-induced dendritic spine formation. (A) Representative dendritic segments of cortical neurons expressing EV, BDNF with EV, BDNF with Wif1, BDNF with Sfrp1, BDNF with Fzd8CRD and BDNF with Dvl1ΔPDZ. (B) Quantification of dendritic protrusion density. (C) Fraction of all spines classified as either spines or filopodia. Quantification of average protrusion length (D) and average spine head width (E) for each treatment. *** p < 0.001, ** p < 0.01, * p < 0.5. (* = relative to EV, # = relative to EV + BDNF.) n = number of neurons: EV n = 31, BDNF + EV n = 30, BDNF + Wif1 n = 29, BDNF + Sfrp1 n = 25, BDNF + Fzd8CRD n = 25, BDNF + Dvl1ΔPDZ n = 30. Scale bar: 5 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Expressing

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Transfection with Wnt inhibitors does not prevent BDNF-induced c-Fos expression in cortical neurons. (A) GFP fluorescence, c-Fos immunoreactivity and DAPI fluorescence of representative cell bodies of cortical neurons expressing EV, Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ either alone, or in combination with BDNF. (B) Quantification of normalized nuclear c-Fos immunoreactivity per neuron for each treatment. *** p < 0.001. n = 10 neurons for each treatment.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Transfection, Expressing, Fluorescence