Journal: The Journal of Cell Biology

Article Title: Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

doi: 10.1083/jcb.200904144

Figure Lengend Snippet: MyoD occupies the Cdc6 promoter in myoblasts. (A) Schematic of the Cdc6 promoter. Closed ovals (E1 and E2) and squares identify the putative MyoD (E-boxes) and E2F sites within the promoter, respectively. The inverted arrows represent the primers used in the PCR amplification reactions. The transcription start site is depicted with an arrow. (B) Cross-linked chromatin from C2C12 myoblasts or primary mouse myoblasts cultured in GM was immunoprecipitated in parallel with anti-MyoD antibody (Ab) and then analyzed by semiquantitative PCR using primers surrounding the E1 and E2 sites in the Cdc6 promoter. A control reaction with the use of normal rabbit IgG (NR IgG) is shown along with input DNA (0.05%), which was amplified by the same set of primers. Black lines indicate that intervening lanes have been spliced out. (C) ChIP experiments were performed in parallel on chromatin from C2C12 myoblasts before and after differentiation using normal rabbit IgG or an antibody specific for MyoD. Precipitated DNA was then analyzed by PCR using the E1 primers, and afterward, the bands were quantified by ImageJ (version 1.36b; National Institutes of Health). Controls for PCR included the use of normal rabbit IgG and the titration of input DNA to ensure all amplifications were within the linear range ( Fig. S1 D ). (D) Values in the histogram represent the ratio of chromatin-bound MyoD to input genomic DNA and are the mean of three independent experiments with standard deviation.

Article Snippet: Antibodies recognizing MyoD (C-20 and M-318), Myf5 (C-20), E2F3a (C-20), Cdc6 (180.2 and H-304), E2F4 (C-20), E2F1 (KH95), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 6C5), and normal rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. Hybridoma cells producing the anti-Pax7 mouse monoclonal antibody were provided by M.A.

Techniques: Amplification, Cell Culture, Immunoprecipitation, Control, Titration, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

doi: 10.1083/jcb.200904144

Figure Lengend Snippet: The E1 and E2F sites are functionally important to the activity of the Cdc6 promoter. (A) A schematic representation of three luciferase (Luc) reporter constructs, empty vector (pGL3-basic), and a construct driven by a wild-type (wt) Cdc6 promoter (Cdc6-wt) or a Cdc6 promoter harboring mutations within the E1 site (Cdc6-mut1). (B) C3H10T1/2 cells were separately transfected with empty vector and with Cdc6-wt or Cdc6-mut1, either alone or with increasing amounts of a plasmid encoding MyoD (pCMV-MyoD). Activity is expressed as fold increase in comparison with activity observed with empty vector after correcting for transfection efficiency (see Materials and methods). (C) Transcriptional-activity of the empty vector or the Cdc6-luciferase constructs in C2C12 myoblasts with or without mutations within E1, E2F, or both sites. (B and C) Error bars represent SEM.

Article Snippet: Antibodies recognizing MyoD (C-20 and M-318), Myf5 (C-20), E2F3a (C-20), Cdc6 (180.2 and H-304), E2F4 (C-20), E2F1 (KH95), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 6C5), and normal rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. Hybridoma cells producing the anti-Pax7 mouse monoclonal antibody were provided by M.A.

Techniques: Activity Assay, Luciferase, Construct, Plasmid Preparation, Transfection, Comparison

Journal: The Journal of Cell Biology

Article Title: Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

doi: 10.1083/jcb.200904144

Figure Lengend Snippet: MyoD is expressed before Cdc6 after C2C12 myoblasts or primary satellite cells transition from quiescence. (A) C2C12 myoblasts were cultured either in GM or in methionine-free medium (−Met) with 1% serum for 36 h and then restimulated with GM for the indicated times. For Western blot analysis, whole cell extracts were prepared, resolved on SDS-PAGE, and then probed with antibodies specific to MyoD, Cdc6, and GAPDH. MyoD and Cdc6 mRNAs were measured by RT-PCR. GAPDH was used as the internal control. White lines indicate that intervening lanes have been spliced out. (B) Isolated extensor digitorum longus myofibers were cultured in basal medium (0 h) before switching to mitogen-rich medium for the activation of satellite cells. The myofibers were fixed at the indicated times and then coimmunostained for Pax7 (green) and MyoD (red) or Cdc6 (red). Immunostaining for Pax7 identifies the satellite cells . Arrows point to the individual satellite cells. Approximately 200 myofibers were analyzed in this experiment. Bar, 20 µm.

Article Snippet: Antibodies recognizing MyoD (C-20 and M-318), Myf5 (C-20), E2F3a (C-20), Cdc6 (180.2 and H-304), E2F4 (C-20), E2F1 (KH95), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 6C5), and normal rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. Hybridoma cells producing the anti-Pax7 mouse monoclonal antibody were provided by M.A.

Techniques: Cell Culture, Western Blot, SDS Page, Reverse Transcription Polymerase Chain Reaction, Control, Isolation, Activation Assay, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

doi: 10.1083/jcb.200904144

Figure Lengend Snippet: Knockdown of MyoD dramatically reduces the expression of Cdc6 in growth-stimulated quiescent myoblasts. (A) C2C12 myoblasts were separately transduced with lentiviruses expressing two distinct shRNAs specific for MyoD (shMyoD-64 and -66) and a scramble shRNA (shCntrl). After 42 h, the myoblasts were brought to quiescence and then stimulated to grow for the indicated times in GM. (B and C) After stimulation, the myoblasts were harvested, and the expression of MyoD or Cdc6 was then monitored either by RT-PCR (B) or by Western blotting (C). NS denotes a nonspecific band. EV, empty vector. (D and E) Cross-linked chromatin was isolated from growth-stimulated quiescent myoblasts expressing a scramble shRNA (shCntrl) or an shRNA specific for MyoD (shMyoD-66). Afterward, the protein–DNA fragments were immunoprecipitated in parallel with anti-E2F3a or anti-MyoD antibody (Ab) and then analyzed by semiquantitative PCR using primers surrounding the E1 site within the Cdc6 promoter. A control reaction with the use of normal rabbit IgG (NR IgG) is shown along with input DNA, which was amplified by the same set of primers. Black lines indicate that intervening lanes have been spliced out.

Article Snippet: Antibodies recognizing MyoD (C-20 and M-318), Myf5 (C-20), E2F3a (C-20), Cdc6 (180.2 and H-304), E2F4 (C-20), E2F1 (KH95), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 6C5), and normal rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. Hybridoma cells producing the anti-Pax7 mouse monoclonal antibody were provided by M.A.

Techniques: Knockdown, Expressing, Transduction, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Isolation, Immunoprecipitation, Control, Amplification

Journal: The Journal of Cell Biology

Article Title: Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

doi: 10.1083/jcb.200904144

Figure Lengend Snippet: Myf5 can occupy the promoter of Cdc6 in MyoD −/− primary myoblasts and in MyoD-depleted quiescent myoblasts after serum stimulation. (A and B) Cross-linked chromatin prepared from quiescent C2C12 myoblasts expressing shMyoD-66 after stimulation by serum for periods of 6 or 12 h was immunoprecipitated in parallel with anti-Myf5 and normal rabbit IgG (NR IgG). Semiquantitative PCR was then performed with the use of primers surrounding the E-box site E1 within the Cdc6 promoter. Black lines indicate that intervening lanes have been spliced out. (C) Crossed-linked chromatin from cultured MyoD −/− primary myoblasts was immunoprecipitated in parallel with anti-Myf5 or anti-MyoD antibody (Ab) and then analyzed by semiquantitative PCR using primers surrounding the E-box sites (E1 and E2) within the Cdc6 promoter. A control reaction with the use of normal rabbit IgG is shown along with input DNA, which was amplified by the same set of primers.

Article Snippet: Antibodies recognizing MyoD (C-20 and M-318), Myf5 (C-20), E2F3a (C-20), Cdc6 (180.2 and H-304), E2F4 (C-20), E2F1 (KH95), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 6C5), and normal rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. Hybridoma cells producing the anti-Pax7 mouse monoclonal antibody were provided by M.A.

Techniques: Expressing, Immunoprecipitation, Cell Culture, Control, Amplification

Journal: Current Chemical Genomics

Article Title: Galectin-1 and Galectin-3 Mediate Protocadherin-24-Dependent Membrane Localization of β -catenin in Colon Cancer Cell Line HCT116

doi: 10.2174/1875397301206010018

Figure Lengend Snippet: ( a ) Schematic representation of the PCDH24-HaloTag expression clone. The expression of PCDH24-HaloTag is regulated by the CMV-TetO2 promoter, which is induced by DOX through the tetracycline repressor-operator system. A red asterisks indicates a transmembrane region of the PCDH24. ( b ) Inducible expression of PCDH24-HaloTag in the HCT116 PCDH24-HaloTag stable cell line. Lysates from HCT116 PCDH24-HaloTag cells with or without DOX (1 μg/mL; hereafter DOX induction is under identical conditions) were separated on SDS-PAGE and the expression patterns of PCDH24 were analyzed by Western blot using an anti-PCDH24 polyclonal antibody. As a control, α-tubulin was detected with an anti-α-tubulin antibody. PCDH24-HaloTag in the cell lysate was labeled with TMR HaloTag® ligand and detected by a FluoroImager FLA-3000 (Fujifilm). ( c ) Typical images of the subcellular localization of PCDH24-HaloTag. HCT116 PCDH24-HaloTag cells were cultured with or without DOX. The cells were observed by phase contrast and fluorescent staining with the HaloTag® R110Direct™ ligand using an Axiovert S100 microscopy system. The scale bar indicates 100 µm. ( d ) Saturation density of HCT116 and HCT116-PCDH24 stable cell lines. 1.0 × 106 HCT116 cells, HCT116-PCDH24-EGFP cells, or HCT116-PCDH24-HaloTag cells with or without DOX were cultured in 35 mm dishes. Triplicate dishes were counted at the indicated time point of the growth curves, and each number represents the average value. ( e ) Effect of PCDH24 on cell motility. Photographs were taken 0, 8, 16 and 24 h following wound injury using a Biozero microscopy time-lapse system.

Article Snippet: Rabbit anti-PC-LKC (PCDH24) (209-318) polyclonal (Abnova, Taipei, Taiwan), anti-alpha-tubulin (DM1A; Calbiochem, Darmstadt, Germany), anti- β -catenin monoclonal, anti-galectin-1 monoclonal (M01) (Abnova), anti-galectin-3 (H-160) polyclonal (sc-20157), anti-Akt (40D4) (Cell Signaling Technology, Danvers, MA) and anti-phospho Akt (Ser473) (D9E) (Cell Signaling Technology) were used for the primary antibodies.

Techniques: Expressing, Stable Transfection, SDS Page, Western Blot, Control, Labeling, Cell Culture, Staining, Microscopy

Journal: Current Chemical Genomics

Article Title: Galectin-1 and Galectin-3 Mediate Protocadherin-24-Dependent Membrane Localization of β -catenin in Colon Cancer Cell Line HCT116

doi: 10.2174/1875397301206010018

Figure Lengend Snippet: ( a ) SDS-PAGE of pull-down samples by HaloLink™ Resin from HCT116-PCDH24-HaloTag cells cultured with or without DOX and HCT116 cells ectopically expressing HaloTag. The samples were concentrated, then resolved by 5–20% SDS-PAGE and subjected to imida-zole-zinc reverse staining. The excised bands subjected to LC-MS/MS analysis are highlighted by black arrowheads. The numerals of the proteins analyzed in this study are indicated with asterisks (i.e., #11 and #12: PCDH24; #25: galectin-3; #27: galectin-1). The proteins identi-fied by the pull-down assay and MS analysis are shown in Supplemental Table 1. ( b ) Western blot analysis of the pull-down samples. West-ern blot experiments were performed for whole cell lysate (WCL) and pull-down assay samples using anti-galectin-1, anti-galectin-3 and anti- β -catenin antibodies. (c) Delineation of the galectin-interaction domain in the intracellular region of PCDH24. Schematic representa-tion of the luciferase-based pull-down assay is shown. Expression clones for luciferase-fused full-length PCDH24 (PCDH24-luc2, amino acids 1–1310 residues) and the C-terminal deletion mutants (PCDH24d1280-luc2, amino acids 1–1280; PCDH24d1186-luc2, amino acids 1–1186) were co-transfected with HaloTag, HaloTag-fused galectin-1 or HaloTag-fused galectine-3 expression clones into HCT116 cells. The amounts of luciferase-fusion proteins in whole cell lysates (WCL) and the pull-down samples were measured as the luciferase activity with the Dual-Glo™ substrate using a GloMAX™ luminometer (upper panel). Results of the experiments are indicated as an arbitrary unit of the luciferase activities. Transfection of the HaloTag-containing clone without the PCDH24 clones (none) was performed as a negative control (lower panel).

Article Snippet: Rabbit anti-PC-LKC (PCDH24) (209-318) polyclonal (Abnova, Taipei, Taiwan), anti-alpha-tubulin (DM1A; Calbiochem, Darmstadt, Germany), anti- β -catenin monoclonal, anti-galectin-1 monoclonal (M01) (Abnova), anti-galectin-3 (H-160) polyclonal (sc-20157), anti-Akt (40D4) (Cell Signaling Technology, Danvers, MA) and anti-phospho Akt (Ser473) (D9E) (Cell Signaling Technology) were used for the primary antibodies.

Techniques: SDS Page, Cell Culture, Expressing, Staining, Liquid Chromatography with Mass Spectroscopy, Pull Down Assay, Western Blot, Luciferase, Clone Assay, Transfection, Activity Assay, Negative Control

Journal: Current Chemical Genomics

Article Title: Galectin-1 and Galectin-3 Mediate Protocadherin-24-Dependent Membrane Localization of β -catenin in Colon Cancer Cell Line HCT116

doi: 10.2174/1875397301206010018

Figure Lengend Snippet: ( a ) Subcellular localization of endogenous galectin-1 and -3 in HCT116 PCDH24-HaloTag cells. HCT116 PCDH24-HaloTag cells were cultured with or without DOX for 36 h before formalin fixation. For immunofluorescent analysis, galectins were labeled using specific anti-bodies against galectin-1 or -3 (red). PCDH24-HaloTag was labeled with the HaloTag® TMR ligand (green). The scale bar represents 20 µm. ( b ) Translocation of β -catenin to the nucleus in PCDH24-HaloTag-expressing HCT116 cells (DOX+) caused by the over-expression of the galectins. Immunofluorescent analysis of β -catenin in the cells in the presence or absence of ectopically-expressed HaloTag-fused galectins was performed using an antibody against β -catenin (blue). PCDH24-HaloTag was fluorescently-labeled using HaloTag® R110Direct™ ligand prior to the transfection of HaloTag-fused galectins expression clones. HaloTag-fused galectins were fluorescently-double stained using HaloTag® R110Direct™ ligand and coumarin ligand. The HaloTag-fused galectin expression clones used here were obtained from the Kazusa Collection of Flexi HaloTag Clones [26]. The cells were fixed at 24 h after the transfection of HaloTag-galectin-1 or HaloTag-galectin-3 expression clones. The scale bar represents 200 µm. ( c ) Effects of siRNA against the galectins on the subcellular localization of β -catenin. The subcellular localization of the galectins and β -catenin in HCT116 cells was observed in the presence or absence of the ga-lectin siRNAs. Endogenous galectins and β -catenin were labeled with specific antibodies. The scale bar represents 20 µm. (d) Reduction of endogenous galectins and increase in membrane-localized β -catenin by siRNA against galectins. Western blot experiments were performed for whole cell lysate using anti- β -catenin, anti-galectin-1 and -3 and for the membrane fraction using an anti- β -catenin antibody.

Article Snippet: Rabbit anti-PC-LKC (PCDH24) (209-318) polyclonal (Abnova, Taipei, Taiwan), anti-alpha-tubulin (DM1A; Calbiochem, Darmstadt, Germany), anti- β -catenin monoclonal, anti-galectin-1 monoclonal (M01) (Abnova), anti-galectin-3 (H-160) polyclonal (sc-20157), anti-Akt (40D4) (Cell Signaling Technology, Danvers, MA) and anti-phospho Akt (Ser473) (D9E) (Cell Signaling Technology) were used for the primary antibodies.

Techniques: Expressing, Cell Culture, Labeling, Translocation Assay, Over Expression, Transfection, Clone Assay, Staining, Membrane, Western Blot

Journal: Current Chemical Genomics

Article Title: Galectin-1 and Galectin-3 Mediate Protocadherin-24-Dependent Membrane Localization of β -catenin in Colon Cancer Cell Line HCT116

doi: 10.2174/1875397301206010018

Figure Lengend Snippet: ( a ) PI3K activity was assessed using quenched fluorescence signals. Whole cell lysate was extracted and the fluorescence signal was measured. As a control, the PI3K inhibitor LY294002 was used. HCT116-PCDH24-HaloTag cells were exposed to LY294002 (100 µM) for 48 h before the assay. HCT116-PCDH24-HaloTag cells were cultured with or without DOX. Galectin1 and galectin3 indicate cells that were transiently transfected with the HaloTag-fused galectin-1 or galectin-3 expression clone, respectively. %Activity was calculated from the signal intensity of DOX- cells, which was divided by the signal intensity observed for each indicated condition. Triplicate wells were assayed and the data represent the mean ± SD. ( b ) AKT/PKB activity downstream of PI3K signaling modified by the PCDH24 and/or the galectins. HCT116-PCDH24-HaloTag and HCT116 cells were cultured with or without DOX, transiently-expressed HaloTag-fused galectin-1 and galectin3. Western blot analysis of AKT/PKB in the cells was performed using antibodies against total AKT or phosphorylated AKT. Halo-Tag-fusion proteins were fluorescently-labeled using TMR HaloTag® ligand and detected by a FluoroImager FLA-3000 (Fujifilm). As a control, the PI3K inhibitor LY294002 was used. HCT116 cells were exposed to LY294002 (10 µM and 20 µM) for 24 h before the assay. ( c ) Subcellular localization of β -catenin in HCT116 cells treated with LY294002. Immunofluorescent images were obtained using an anti β -catenin antibody. ( d ) Proposed model for the mechanism by which PCDH24 regulates PI3K activation via galectin-1 and galectin-3 in colon cancer cells. In parental HCT116 cells (left), galectin-1 and -3 activate PI3K. The activation of PI3K by galectins activates AKT/PKB and also leads to the nuclear localization of β -catenin. Conversely, in HCT116 cells expressing PCDH24 (right), galectin-1 and -3 are trapped by PCDH24 at the cell membrane, and thus PI3K and AKT/PKB are not activated by the galectins. Subsequently, β -catenin is localized to the cell membrane and is prevented from activating the transcription of its target genes in the nucleus.

Article Snippet: Rabbit anti-PC-LKC (PCDH24) (209-318) polyclonal (Abnova, Taipei, Taiwan), anti-alpha-tubulin (DM1A; Calbiochem, Darmstadt, Germany), anti- β -catenin monoclonal, anti-galectin-1 monoclonal (M01) (Abnova), anti-galectin-3 (H-160) polyclonal (sc-20157), anti-Akt (40D4) (Cell Signaling Technology, Danvers, MA) and anti-phospho Akt (Ser473) (D9E) (Cell Signaling Technology) were used for the primary antibodies.

Techniques: Expressing, Activity Assay, Over Expression, Inhibition, Fluorescence, Control, Cell Culture, Transfection, Modification, Western Blot, Labeling, Activation Assay, Membrane