Journal: Cryobiology

Article Title: Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I—Comparative Fundamental Cryobiology of Multiple Mouse Embryonic Stem Cell Lines and the Implications for Embryonic Stem Cell Cryopreservation Protocols

doi: 10.1016/j.cryobiol.2013.12.007

Figure Lengend Snippet: Percent post-thaw recoveries of membrane-intact BALB/c, CBA, FVB, and 129R1 (R1) mouse embryonic stem cells frozen in cryovials in standard freezing conditions (106 cells/mL, 1 °C/minute cooling rate, 1.0 M Me2SO, -80 °C plunge temperature). Percent post-thaw recovery is expressed as mean ± SEM. Different superscripts indicate significantly different means (p<0.05). Previously published results for C57BL/6 mouse embryonic stem cells cryopreserved under the same conditions (indicated with the asterisk) are included for reference [35], but are not included in the statistical analysis.

Article Snippet: Anecdotal reports (personal communication, D. Nielsen, Stem Cell Technologies technical support, 2004; personal communication, Xin Yu, University of California-Davis, 2004) and a recent publication from Kashuba Benson et al . [ 35 ] concerning the C57BL/6 cell line show PTR of mESCs to be highly variable across cell lines.

Techniques: Membrane

Journal: Cryobiology

Article Title: Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I—Comparative Fundamental Cryobiology of Multiple Mouse Embryonic Stem Cell Lines and the Implications for Embryonic Stem Cell Cryopreservation Protocols

doi: 10.1016/j.cryobiol.2013.12.007

Figure Lengend Snippet: Osmotic tolerance limits (OTL) of five mouse embryonic stem cell (mESC) lines as determined by plasma membrane integrity. Previously published data for the C57BL/6 mESC line [35] are included for comparative purposes but were not included in statistical analyses. Equal quantities of mESCs were exposed to solutions of 38, 75, 150, 600, 1200, 2400, and 4800 mOsm for 10 minutes at room temperature, and compared with controls exposed to isosmotic solution (285 mOsm) in the same manner. Plasma membrane integrity was assessed by flow cytometry analysis of propidium iodide exclusion. A. Inverted U-shaped osmotic tolerance curves of BALB/c, C57BL/6 (B6), CBA, FVB, and 129R1 (R1) mESC lines. B. OTL, defined as the range of osmolalities in which 80% of cells maintained plasma membrane integrity, for each of 5 different mESC lines (n=3). “0” represents isosmotic (285 mOsm). Distance from isosmotic is in terms of mOsm. Inset numbers indicate the extrapolated mean osmolalities (through linear regression) ± SEM at which 80% of mESCs retained membrane integrity. SEM for all lower OTL was ± 5 mOsm. Different lower case superscripts indicate statistically significant differences in lower OTL (p<0.05).

Article Snippet: Anecdotal reports (personal communication, D. Nielsen, Stem Cell Technologies technical support, 2004; personal communication, Xin Yu, University of California-Davis, 2004) and a recent publication from Kashuba Benson et al . [ 35 ] concerning the C57BL/6 cell line show PTR of mESCs to be highly variable across cell lines.

Techniques: Clinical Proteomics, Membrane, Flow Cytometry

Journal: Cell Death and Differentiation

Article Title: FOXQ1 controls the induced differentiation of melanocytic cells

doi: 10.1038/s41418-018-0066-y

Figure Lengend Snippet: FOXQ1 induces MITF-dependent differentiation. a, b NHM transduced with empty vector (VECTOR) or FOXQ1-expressing vector (FOXQ1) were probed in immunoblotting with indicated antibodies (left) or in Q-RT-PCR (right). FOXQ1/ACTB, MITF/ACTB, and TYR/ACTB signal ratios are shown. c Cells described in a were imaged as adherent cells or as cell pellets followed by quantification of total melanin content (white boxes). d, e Cells transduced with control (CL) or FOXQ1 (F1, F2) shRNAs were probed in immunoblotting with the indicated antibodies (left panel) or in Q-RT-PCR (right panel). FOXQ1/ACTB, MITF/ACTB, and TYR/ACTB signal ratios are shown. f NHM described in d, e) were imaged as adherent cells or as cell pellets followed by quantification of total melanin content (white boxes). g NHM expressing empty vector (V), FOXQ1 cDNA f, or control (CL) or FOXQ1 shRNAs (F1, F2) were counted for 72 h starting 48 h post infection. The cell numbers at 72 h were normalized by those before plating and by the ratio of these numbers in vector or control cells. h Human MITF promoter. Shown are FOXQ1-binding sites (diamonds) and PCR primers (arrows). i Q-PCR signals in reactions with DNA precipitated with FOXQ1-specific antibodies from untreated and FSK-treated NHM cells were normalized by corresponding signals in DNA precipitated with IgG antibodies and by signals obtained with MITF nonspecific control primers (NS). All data represent mean ± SEM. Statistical significance was assessed using two-tailed Student’s t-tests. A p < 0.05 (*) was considered significant

Article Snippet: Generation of Foxq1 knockout mice Two Foxq1 tm1 (KOMP)Vlcg mouse embryonic stem cell clones were obtained from the Velocigene (Regeneron) branch of the Knockout Mouse Project (KOMP).

Techniques: Transduction, Plasmid Preparation, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Infection, Binding Assay, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: FOXQ1 controls the induced differentiation of melanocytic cells

doi: 10.1038/s41418-018-0066-y

Figure Lengend Snippet: FOXQ1 regulates cAMP/CREB1-dependent pigmentation. a NHM were treated with the indicated doses of Forskolin (FSK) for 5 h (left panel) or with 50 μM of FSK for indicated duration (right panel) followed by immunoblotting with the indicated antibodies. b NHM were transduced with control (CL) or FOXQ1 (F1, F2) shRNAs and treated with vehicle (DMSO) or the indicated doses of Forskolin (FSK) for 5 h followed by immunoblotting with the indicated antibodies. c Melanin content in cells described in b. d Representative images of ears from Foxq1+/+ or Foxq1–/– mice (n = 3) treated with either vehicle (DMSO, left ear) or vehicle containing 100μM Forskolin (FSK, right ear). e Pigmentation was quantified using a reflectance spectrometer and represented as fold change in the coefficient of absorption “mua”. f Representative images of H&E staining of mouse ear tissues shown in d. All data represent mean ± SEM. Statistical significance was assessed using two-tailed Student’s t-tests. A p < 0.05 (*) was considered significant

Article Snippet: Generation of Foxq1 knockout mice Two Foxq1 tm1 (KOMP)Vlcg mouse embryonic stem cell clones were obtained from the Velocigene (Regeneron) branch of the Knockout Mouse Project (KOMP).

Techniques: Western Blot, Transduction, Control, Staining, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: FOXQ1 controls the induced differentiation of melanocytic cells

doi: 10.1038/s41418-018-0066-y

Figure Lengend Snippet: FOXQ1 is important for cAMP/CREB1-dependent pigmentation. a, b Cells transduced with control shRNA (CL) or CREB1 shRNA (C1) were probed in immunoblotting with the indicated antibodies or in Q-RT-PCR. CREB1/Actb, Foxq1/Actb, and Mitf/Actb signal ratios are shown. c Melanin content and representative cell pellet images of cells described in a, b. d Schematic representation of human and mouse FOXQ1 promoter. Diamonds represent potential CREB1 consensus binding sites. e NHM, Melan-a, and B16 cells treated with vehicle (DMSO) or 10 μM Forskolin were probed in ChIP assay. Shown are ratios of Q-PCR signals in reactions with DNA precipitated with CREB1 or IgG antibodies using primers corresponding to potential CREB1-binding sites in FOXQ1 promoter. NS corresponds to CREB1-nonspecific primer targeting a distal genomic region. All data represent mean ± SEM. Statistical significance was assessed using two-tailed Student’s t-tests. A p < 0.05 (*) was considered significant

Article Snippet: Generation of Foxq1 knockout mice Two Foxq1 tm1 (KOMP)Vlcg mouse embryonic stem cell clones were obtained from the Velocigene (Regeneron) branch of the Knockout Mouse Project (KOMP).

Techniques: Transduction, Control, shRNA, Western Blot, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: FOXQ1 controls the induced differentiation of melanocytic cells

doi: 10.1038/s41418-018-0066-y

Figure Lengend Snippet: BRAF-β-catenin-FOXQ1 axis controls MITF-dependent phenotypes. a NHM and Melan-a cells were transduced with indicated constructs (V = empty vector, B = BRAFV600E, F = FOXQ1) followed by immunoblotting with the indicated antibodies. b Cells were transduced with the indicated constructs and probed in Q-RT-PCR. (MITF/ACTB (Mitf/Actb) and FOXQ1/ACTB (Foxq1/Actb) signal ratios are shown. c Total melanin content in cells described in a. d NHMs transduced with the indicated constructs were probed in immunoblotting with the indicated antibodies (left panel) and in Q-RT-PCR (right panel). FOXQ1/ACTB signal ratios are shown. e Melan-a cells were transduced with the indicated constructs and assayed for the ability to form colonies in soft agar. Shown are average numbers of colonies per view field (n = 5). All data represent mean ± SEM. Statistical significance was assessed using two-tailed Student’s t-tests. A p < 0.05 (*) was considered significant

Article Snippet: Generation of Foxq1 knockout mice Two Foxq1 tm1 (KOMP)Vlcg mouse embryonic stem cell clones were obtained from the Velocigene (Regeneron) branch of the Knockout Mouse Project (KOMP).

Techniques: Transduction, Construct, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Cancer Science

Article Title: NeuroD1 promotes neuroblastoma cell growth by inducing the expression of ALK

doi: 10.1111/cas.12628

Figure Lengend Snippet: Relationship of NeuroD1 and ALK in neurospheres. (a) NeuroD1 in neurosphere cells cultured from the hippocampus region of wild-type E13.5 embryos was knocked down by two independent shRNA. 4 days later, the phase contrast pictures were taken. Scale bar, 200 μm. (b) The sphere numbers in (a) were counted. (c) The mRNA levels of NeuroD1 were examined by real-time PCR. (d) The mRNA levels of ALK were examined by real-time PCR. * P < 0.05. ** P < 0.01.

Article Snippet: Embryonic neural stem cells were prepared from an E13.5 ICR mouse (Japan SLC, Hamamatsu, Japan).

Techniques: Cell Culture, shRNA, Real-time Polymerase Chain Reaction