Journal:

Article Title: Protein Kinase C alpha (PKC?) dependent signaling mediates endometrial cancer cell growth and tumorigenesis

doi: 10.1002/ijc.24633

Figure Lengend Snippet: PKCα knockdown impairs tumorigenesis in vivo. (a) Ishikawa cell clones expressing luciferase (Luc) or PKCα shRNA constructs were isolated as described in materials and methods. Lysate from clonal populations was harvested after 24h of serum deprivation and probed for PKCα PKCβI, PKCβII, and PKCγ. β-actin is included as loading control and arrow (<) indicates the correct molecular weight band for PKCγ. (b) Luc and PKCα shRNA clones were deprived of serum for 24h and the proportion of cells in the indicated phases of the cell cycle was determined by flow cytometry. Data represent an average derived from two independent experiments. (c) Volume of xenograft tumors resulting from the subcutaneous injection of 10 million cells from Luc and PKCα shRNA clonal cell lines into athymic, nude mice. Each PKCα knockdown clone was injected contra-laterally to a Luc control in a total of 5 mice, thus data represent the combined average from n = 15 animals. (d) Average final weight of excised xenograft tumors assessed after the final dimension measurement. Bar graphs represent mean ± SEM; * denotes P < 0.01.

Article Snippet: Antibodies used for immunoblotting included PKCα (sc-208), PKC βI (sc-209), PKCβII (sc-210), PKCγ (sc-211), p53 (sc-126), p57 (sc-8298), Pol II (sc-900) and secondary anti-rabbit (sc-2004) or anti-mouse (sc-2005) IgG-HRP antibodies obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Knockdown, In Vivo, Clone Assay, Expressing, Luciferase, shRNA, Construct, Isolation, Control, Molecular Weight, Flow Cytometry, Derivative Assay, Injection

Journal: Development (Cambridge, England)

Article Title: Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1.

doi: 10.1242/dev.090910

Figure Lengend Snippet: Fig. 1. Mcl1 protein expression is tightly regulated in mouse embryonic NPCs. Mcl1 protein expression 48 hours post-electroporation (h.p.e.) in brains transfected (GFP+) with control (Ctl), Mcl1 or mutated Mcl1 (mt Mcl1) plasmid. In merge: GFP, green; Mcl1, red; both, yellow. Scale bar: 50 μm.

Article Snippet: Western blotting was performed as described (Vanderluit et al., 2004) with antibodies to Mcl1 (Rockland, 600-401-394), p27Kip1 (Santa Cruz Biotechnology, SC-528), p57Kip2 (Santa Cruz Biotechnology, SC-8298) and actin (Santa Cruz Biotechnology, SC-1616), followed by horseradish peroxidase-tagged anti-mouse IgG or anti-rabbit IgG (Bio-Rad), and developed by chemiluminescence using Western Lightning Plus ECL (PerkinElmer, NEL100) according to the manufacturer’s instructions.

Techniques: Expressing, Electroporation, Transfection, Control, Plasmid Preparation

Journal: Development (Cambridge, England)

Article Title: Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1.

doi: 10.1242/dev.090910

Figure Lengend Snippet: Fig. 2. Mcl1 gain-of-function promotes the migration of NPCs into the cortical plate. (A) Transfected (GFP+) NPCs located in the (a) dorsomedial and the (b) dorsolateral cortex. Scale bar: 200 μm. (B-D) Transfected NPCs in the dorsolateral cortex of (B) Ctl-, (C) Mcl1- and (D) mt Mcl1-electroporated brains at 48 h.p.e. Scale bar: 100 μm. (E) Quantification of the percentage of GFP+ cells located in the ventricular zone (VZ), combined subventricular and intermediate zones (SVZ and IZ) and cortical plate (CP). n=5 embryos/treatment. Results are expressed as mean ± s.e.m. One-way analysis of variance (ANOVA) with Tukey’s post-hoc test. *P<0.05.

Article Snippet: Western blotting was performed as described (Vanderluit et al., 2004) with antibodies to Mcl1 (Rockland, 600-401-394), p27Kip1 (Santa Cruz Biotechnology, SC-528), p57Kip2 (Santa Cruz Biotechnology, SC-8298) and actin (Santa Cruz Biotechnology, SC-1616), followed by horseradish peroxidase-tagged anti-mouse IgG or anti-rabbit IgG (Bio-Rad), and developed by chemiluminescence using Western Lightning Plus ECL (PerkinElmer, NEL100) according to the manufacturer’s instructions.

Techniques: Migration, Transfection

Journal: Development (Cambridge, England)

Article Title: Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1.

doi: 10.1242/dev.090910

Figure Lengend Snippet: Fig. 3. Mcl1 promotes premature NPC terminal mitosis. (A) Timeline of the BrdU birth dating experiment. (B,C) Low (B) and high (C) magnification photomicrographs of anti-BrdU immunostained sections of mt Mcl1-transfected versus Ctl-transfected brains. Arrows indicate double-labeled [BrdU+ (red) GFP+

Article Snippet: Western blotting was performed as described (Vanderluit et al., 2004) with antibodies to Mcl1 (Rockland, 600-401-394), p27Kip1 (Santa Cruz Biotechnology, SC-528), p57Kip2 (Santa Cruz Biotechnology, SC-8298) and actin (Santa Cruz Biotechnology, SC-1616), followed by horseradish peroxidase-tagged anti-mouse IgG or anti-rabbit IgG (Bio-Rad), and developed by chemiluminescence using Western Lightning Plus ECL (PerkinElmer, NEL100) according to the manufacturer’s instructions.

Techniques: Transfection, Labeling

Journal: Development (Cambridge, England)

Article Title: Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1.

doi: 10.1242/dev.090910

Figure Lengend Snippet: Fig. 4. Mcl1 alters the laminar fate of NPCs in the mouse embryonic brain. (A) The location of GFP+ cells in Ctl- and mt Mcl1-electroporated brains from 2-week-old mice. CC, corpus callosum. Scale bar: 200 μm. (B) Layer II-IV-specific Cux1 immunohistochemistry. Scale bar: 100 μm. (C) Quantification of the percentage of GFP+ cells in layers II-IV and layers V-VI. n=10 embryos/treatment. Means were analysed by two-way ANOVA and Bonferroni post hoc. ***P<0.001. Error bars indicate s.e.m. (D,E) mt Mcl1-transfected brain sections immunostained with antibodies to (D) Tbr1 and (E) Ctip2. Arrows indicate double-labeled cells. Scale bars: 50 μm.

Article Snippet: Western blotting was performed as described (Vanderluit et al., 2004) with antibodies to Mcl1 (Rockland, 600-401-394), p27Kip1 (Santa Cruz Biotechnology, SC-528), p57Kip2 (Santa Cruz Biotechnology, SC-8298) and actin (Santa Cruz Biotechnology, SC-1616), followed by horseradish peroxidase-tagged anti-mouse IgG or anti-rabbit IgG (Bio-Rad), and developed by chemiluminescence using Western Lightning Plus ECL (PerkinElmer, NEL100) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Transfection, Labeling

Journal: Development (Cambridge, England)

Article Title: Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1.

doi: 10.1242/dev.090910

Figure Lengend Snippet: Fig. 5. Mcl1 upregulates p27Kip1 in NPCs. (A) Western analysis of NPCs transfected with Ctl or mt Mcl1 plasmids revealed no significant difference in p57Kip2 protein levels. (B) FACS analysis of p57Kip2 protein from Ctl-, Mcl1- and mt Mcl1-transfected NPCs. n=3/treatment. (C) Western analysis revealed a greater than threefold increase in p27Kip1

Article Snippet: Western blotting was performed as described (Vanderluit et al., 2004) with antibodies to Mcl1 (Rockland, 600-401-394), p27Kip1 (Santa Cruz Biotechnology, SC-528), p57Kip2 (Santa Cruz Biotechnology, SC-8298) and actin (Santa Cruz Biotechnology, SC-1616), followed by horseradish peroxidase-tagged anti-mouse IgG or anti-rabbit IgG (Bio-Rad), and developed by chemiluminescence using Western Lightning Plus ECL (PerkinElmer, NEL100) according to the manufacturer’s instructions.

Techniques: Western Blot, Transfection

Journal: Development (Cambridge, England)

Article Title: Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1.

doi: 10.1242/dev.090910

Figure Lengend Snippet: Fig. 6. p27Kip1 reduces NPC proliferation in vitro similar to Mcl1. E12.5 NPCs were transfected with Ctl, mt Mcl1 or p27Kip1 plasmids and cultured in proliferating conditions. Quantification of the percentage of proliferating (PCNA+ GFP+) cells at 24, 48 and 72 hours post-transfection (h.p.t.). n=3 embryos/treatment. Means were analysed by two-way ANOVA and Bonferroni post-hoc test. *P<0.05. Error bars indicate s.e.m.

Article Snippet: Western blotting was performed as described (Vanderluit et al., 2004) with antibodies to Mcl1 (Rockland, 600-401-394), p27Kip1 (Santa Cruz Biotechnology, SC-528), p57Kip2 (Santa Cruz Biotechnology, SC-8298) and actin (Santa Cruz Biotechnology, SC-1616), followed by horseradish peroxidase-tagged anti-mouse IgG or anti-rabbit IgG (Bio-Rad), and developed by chemiluminescence using Western Lightning Plus ECL (PerkinElmer, NEL100) according to the manufacturer’s instructions.

Techniques: In Vitro, Transfection, Cell Culture

Journal: Development (Cambridge, England)

Article Title: Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1.

doi: 10.1242/dev.090910

Figure Lengend Snippet: Fig. 7. Mcl1 promotes NPC cell cycle exit through p27Kip1 activity. (A) GFP+ and BrdU+ cells at 48 h.p.t. of Ctl and mt Mcl1 plasmids into E13 NPCs from wild-type, p27Kip1+/−and p27Kip1−/−mouse embryos. Arrowheads indicate double-labeled cells. In merge: GFP, green; BrdU, red; Hoechst, blue. Scale bar: 20 μm. (B) Quantification of the percentage of double-labeled cells at 24 and 48 h.p.t. n=5 wild type (wt), n=3 p27Kip1+/−, n=4 p27Kip1−/−. Means were analysed by two-way ANOVA and Bonferroni post-hoc test. *P<0.05, **P<0.01. Error bars indicate s.e.m.

Article Snippet: Western blotting was performed as described (Vanderluit et al., 2004) with antibodies to Mcl1 (Rockland, 600-401-394), p27Kip1 (Santa Cruz Biotechnology, SC-528), p57Kip2 (Santa Cruz Biotechnology, SC-8298) and actin (Santa Cruz Biotechnology, SC-1616), followed by horseradish peroxidase-tagged anti-mouse IgG or anti-rabbit IgG (Bio-Rad), and developed by chemiluminescence using Western Lightning Plus ECL (PerkinElmer, NEL100) according to the manufacturer’s instructions.

Techniques: Activity Assay, Labeling