Journal:

Article Title: Role of Metalloprotease Disintegrin ADAM12 in Determination of Quiescent Reserve Cells during Myogenic Differentiation In Vitro †

doi: 10.1128/MCB.23.19.6725-6738.2003

Figure Lengend Snippet: Overexpression of ADAM12 leads to induction of p130. Subconfluent C2C12 cells transfected with ADAM12 (A to F) or the L73P ADAM12 mutant (G to L) and incubated in medium containing 1% FBS were fixed and costained with anti-ADAM12 antibody (red; A, D, G, and J) and anti-p130 antibody (green; B, E, H, and K). Two representative microscopic fields are shown for ADAM12 and L73P transfectants. Panels C, F, I, and L are merged images of panels A and B, D and E, G and H, and J and K, respectively. Arrows indicate ADAM12- or L73P-overexpressing cells with induced expression of p130. Arrowheads mark ADAM12- or L73P-overexpressing cells that are p130 negative. (M) The percentage of p130-positive cells among ADAM12-overexpressing (solid bar), L73P-overexpressing (grey bar), and cells that were not stained with ADAM12 antibody (nontransfected cells, open bar) was quantified. Results shown are the averages from three experiments ± standard errors. At least 100 transfected cells were analyzed in each experiment.

Article Snippet: The membrane was then hybridized at 65°C with a 2.7-kbp ADAM12 cDNA probe labeled with [α- 32 P]dCTP (3,000 Ci/mmol) by random priming (Prime a Gene labeling system; Promega, Madison, Wis.), washed at 68°C, and exposed for autoradiography at −70°C with an intensifying screen.

Techniques: Over Expression, Transfection, Mutagenesis, Incubation, Expressing, Staining

Journal:

Article Title: Role of Metalloprotease Disintegrin ADAM12 in Determination of Quiescent Reserve Cells during Myogenic Differentiation In Vitro †

doi: 10.1128/MCB.23.19.6725-6738.2003

Figure Lengend Snippet: ADAM12 protein is more abundant in reserve cells than in myotubes during C2C12 cell differentiation. (A) Western blot analysis of ADAM12 expression in proliferating C2C12 cells grown in 10% FBS and in confluent C2C12 cells incubated for 4 days in differentiation medium containing 2% horse serum (HS). Cells incubated in 2% horse serum were separated by mild trypsinization into two fractions: nondifferentiated reserve cells (R), characterized by a lack of expression of myogenin, downregulation of MyoD, and upregulation of p130 expression; and differentiated myotubes (M) characterized by high expression of myogenin and integrin α7A, sustained expression of MyoD, and low expression of p130. Notice that while the cell cycle inhibitors p21 and p27 are both upregulated in cells switched to differentiation medium, p21 is more abundant in myotubes and p27 is more abundant in reserve cells. To detect endogenous ADAM12, cell extracts were enriched for glycoproteins on concanavalin A columns prior to Western blotting; the levels of all other proteins were analyzed by using total cell lysates. Since the mature 90-kDa cell surface form of ADAM12 (8) is degraded during the trypsinization used to separate reserve cells and myotubes, only the nascent 120-kDa intracellular form of ADAM12 is shown. Tubulin was a loading control. (B) The amount of ADAM12 mRNA in the same three populations of C2C12 cells as in panel A was analyzed by Northern blotting with ADAM12 cDNA as a probe (left). rRNA on the same membrane was stained with methylene blue to control for sample loading (right). The positions of RNA standards (in kilobases) are indicated.

Article Snippet: The membrane was then hybridized at 65°C with a 2.7-kbp ADAM12 cDNA probe labeled with [α- 32 P]dCTP (3,000 Ci/mmol) by random priming (Prime a Gene labeling system; Promega, Madison, Wis.), washed at 68°C, and exposed for autoradiography at −70°C with an intensifying screen.

Techniques: Cell Differentiation, Western Blot, Expressing, Incubation, Control, Northern Blot, Membrane, Staining

Journal:

Article Title: Role of Metalloprotease Disintegrin ADAM12 in Determination of Quiescent Reserve Cells during Myogenic Differentiation In Vitro †

doi: 10.1128/MCB.23.19.6725-6738.2003

Figure Lengend Snippet: Downregulation of ADAM12 by siRNA is correlated with decreased expression of p130, p27, myogenin, and integrin α7A, but not p21. C2C12 cells were transfected with small interfering RNA (siRNA) designed to suppress ADAM12 expression or with control siRNA against GFP. Two days after transfection, cells reached confluence and were transferred to differentiation medium containing 2% horse serum. After incubation for 2 additional days in differentiation medium, glycoprotein-enriched fractions obtained from C2C12 cells transfected with ADAM12 or GFP siRNA were subjected to Western blotting with anti-ADAM12 antibody (A). To control for equal protein loading, the same glycoprotein-enriched fractions were stained with anti-integrin β1 antibody. In cells transfected with GFP siRNA, the nascent (≈120 kDa) and the mature form (≈90 kDa) of ADAM12 are detected (8); in cells transfected with ADAM12 siRNA, the mature form is below the antibody detection limit. (B) The levels of expression of p130, p27, myogenin, integrin α7A, and p21 in cells transfected with ADAM12 or GFP siRNA were analyzed by Western blotting. Tubulin was a loading control. (C) The intensities of the bands in A and B were quantified by densitometry with ScionImage software. Solid and open bars represent cells transfected with ADAM12 and GFP siRNA, respectively. The experiment was repeated three times with similar results. Panels A to C show the results of a representative experiment. (D) Cells transfected with ADAM12 or GFP siRNA and incubated for 2 days in differentiation medium containing 2% horse serum were separated by mild trypsinization into myotube (M) and reserve cell (R) fractions, as described for Fig. ​Fig.1.1. One fifth of the total myotube and reserve cell samples was then analyzed by Western blotting with the indicated antibodies. Notice that the amount of myotubes is lower in ADAM12 siRNA-transfected cells than in GFP siRNA-transfected cells, as documented by antitubulin blot.

Article Snippet: The membrane was then hybridized at 65°C with a 2.7-kbp ADAM12 cDNA probe labeled with [α- 32 P]dCTP (3,000 Ci/mmol) by random priming (Prime a Gene labeling system; Promega, Madison, Wis.), washed at 68°C, and exposed for autoradiography at −70°C with an intensifying screen.

Techniques: Expressing, Transfection, Small Interfering RNA, Control, Incubation, Western Blot, Staining, Software

Journal:

Article Title: Role of Metalloprotease Disintegrin ADAM12 in Determination of Quiescent Reserve Cells during Myogenic Differentiation In Vitro †

doi: 10.1128/MCB.23.19.6725-6738.2003

Figure Lengend Snippet: Enhanced expression of the wild type but not the L73P mutant of ADAM12 inhibits S-phase entry in C2C12 cells. (A and B) C2C12 cells were transfected with a vector encoding the wild-type ADAM12. Twenty-four hours after transfection, cells were transferred to medium containing 1% FBS. After an additional 24 h, cells were supplemented with 10 μM BrdU, and incubation was continued for 16 h. Cells were then fixed and coimmunostained with anti-ADAM12 (red) and anti-BrdU antibody (green); two representative microscopic fields are shown. Bar, 25 μm. (C) The percentage of BrdU-positive nuclei in the experiment shown in panels A and B was determined for cells that were not stained with ADAM12 antibody (control), for cells overexpressing ADAM12, and for cells overexpressing the L73P mutant form of ADAM12, which is not transported to the cell surface (8). (D) Transfected cells were incubated in the presence of 10% FBS, and BrdU labeling was determined as in panel C. (E) Following transfection, cells were incubated for 24 h with 1% FBS. BrdU labeling was then performed for 16 h in the presence of 10% FBS. In panels C, D, and E, at least 150 to 200 ADAM12-overexpressing cells in multiple fields were scored for BrdU staining. Results shown are the averages from five experiments ± standard errors. (F) Analysis of the phosphorylation status of the retinoblastoma protein in C2C12 cells incubated for 24 h in the presence of 10% FBS, 1% FBS, 1% FBS in the absence of methionine, or 2% horse serum. ppRb and pRb designate the hyperphosphorylated and hypophosphorylated forms of the retinoblastoma protein, respectively. Tubulin was a loading control.

Article Snippet: The membrane was then hybridized at 65°C with a 2.7-kbp ADAM12 cDNA probe labeled with [α- 32 P]dCTP (3,000 Ci/mmol) by random priming (Prime a Gene labeling system; Promega, Madison, Wis.), washed at 68°C, and exposed for autoradiography at −70°C with an intensifying screen.

Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Incubation, Staining, Control, Labeling, BrdU Staining, Phospho-proteomics

Journal:

Article Title: Role of Metalloprotease Disintegrin ADAM12 in Determination of Quiescent Reserve Cells during Myogenic Differentiation In Vitro †

doi: 10.1128/MCB.23.19.6725-6738.2003

Figure Lengend Snippet: Metalloprotease domain of ADAM12 is not essential for inhibition of cell cycle progression. (A) Expression constructs used to transfect C2C12 cells included mouse ADAM12, ADAM12(ΔPM) (a truncated form of ADAM12 lacking the prodomain and metalloprotease domain), ADAM9(ΔPM) (a truncated form of mouse ADAM9 lacking the prodomain and metalloprotease domain), a chimeric construct, ADAM12(ΔPM)/9, composed of the extracellular and transmembrane domains of ADAM12(ΔPM) and the cytoplasmic domain of ADAM9, and ADAM9(ΔPM)/12, containing the extracellular and transmembrane domains of ADAM9(ΔPM) and the cytoplasmic domain of ADAM12. (B) C2C12 cells were transfected with the expression constructs shown in panel A and incubated for 24 h in the presence of 1% FBS and then for 16 h with BrdU (as in Fig. 4A and B), fixed, and stained with mouse anti-BrdU antibody and either anti-ADAM12 antibody [ADAM12, ADAM12(ΔPM), and ADAM9(ΔPM)/12 transfectants] or anti-ADAM9 antibody [ADAM9(ΔPM) and ADAM12(ΔPM)/9 transfectants], as described in Materials and Methods. Cells were then viewed by confocal microscopy and scored for the expression of recombinant proteins and nuclear BrdU labeling. At least 150 to 200 positively transfected cells in multiple fields were analyzed in each experiment, and transfection with each construct was repeated at least three times.

Article Snippet: The membrane was then hybridized at 65°C with a 2.7-kbp ADAM12 cDNA probe labeled with [α- 32 P]dCTP (3,000 Ci/mmol) by random priming (Prime a Gene labeling system; Promega, Madison, Wis.), washed at 68°C, and exposed for autoradiography at −70°C with an intensifying screen.

Techniques: Inhibition, Expressing, Construct, Transfection, Incubation, Staining, Confocal Microscopy, Recombinant, Labeling

Journal:

Article Title: Role of Metalloprotease Disintegrin ADAM12 in Determination of Quiescent Reserve Cells during Myogenic Differentiation In Vitro †

doi: 10.1128/MCB.23.19.6725-6738.2003

Figure Lengend Snippet: Overexpression of ADAM12 arrests C2C12 cells between M and S phases of the cycle. (A) Cells were transfected with the full-length ADAM12 or the L73P ADAM12 mutant and incubated for 24 h in the presence of 1% FBS and then for 16 h with BrdU (as in Fig. 4A and B) (unsynchronized cells [unsync]). Alternatively, transfected cells were incubated for 24 h in the presence of 10% FBS, mitotic cells were dislodged by shake-off, plated on a coverslip, and incubated for 8 h in the presence of 1% FBS (M-synchronized cells [sync]). Cells were then fixed and stained with anti-ADAM12 and anti-BrdU antibodies. The percentage of BrdU labeling among cells that were not stained with anti-ADAM12 antibody (open bars), among ADAM12-overexpressing cells (solid bars), and among L73P-overexpressing cells (grey bars) was determined. At least 150 to 200 transfected cells in multiple fields were analyzed in the experiment with unsynchronized cells, and 50 to 100 transfected cells were scored in the experiments with M-synchronized cells. Results shown are the averages from four experiments ± standard errors. (B) Total cells before the shake-off procedure (T), dislodged cells (D), and cells that remained attached after shake-off (A) were analyzed by Western blotting with anti-cyclin B to verify the effective synchronization of the dislodged cells in M phase. Tubulin was a loading control.

Article Snippet: The membrane was then hybridized at 65°C with a 2.7-kbp ADAM12 cDNA probe labeled with [α- 32 P]dCTP (3,000 Ci/mmol) by random priming (Prime a Gene labeling system; Promega, Madison, Wis.), washed at 68°C, and exposed for autoradiography at −70°C with an intensifying screen.

Techniques: Over Expression, Transfection, Mutagenesis, Incubation, Staining, Labeling, Western Blot, Control

Journal:

Article Title: Role of Metalloprotease Disintegrin ADAM12 in Determination of Quiescent Reserve Cells during Myogenic Differentiation In Vitro †

doi: 10.1128/MCB.23.19.6725-6738.2003

Figure Lengend Snippet: Overexpression of ADAM12 reduces MyoD expression. C2C12 cells transfected with ADAM12 (A) or the L73P ADAM12 mutant (B) were synchronized by mitotic shake-off, plated on coverslips, incubated for 4 h in the presence of 1% FBS, fixed, and stained with anti-ADAM12 (red) or anti-myoD antibody (green). Arrows indicate L73P-transfected cells with nuclear MyoD staining. Arrowheads mark ADAM12- or L73P-overexpressing cells, which are MyoD negative. (C) The percentage of MyoD-positive cells among nontransfected, ADAM12-overexpressing, and L73P-overexpressing cells was quantified 4 h after shake-off. The results shown are the averages from three experiments ± standard errors; 50 to 100 transfected cells were analyzed in each experiment.

Article Snippet: The membrane was then hybridized at 65°C with a 2.7-kbp ADAM12 cDNA probe labeled with [α- 32 P]dCTP (3,000 Ci/mmol) by random priming (Prime a Gene labeling system; Promega, Madison, Wis.), washed at 68°C, and exposed for autoradiography at −70°C with an intensifying screen.

Techniques: Over Expression, Expressing, Transfection, Mutagenesis, Incubation, Staining

Journal:

Article Title: Role of Metalloprotease Disintegrin ADAM12 in Determination of Quiescent Reserve Cells during Myogenic Differentiation In Vitro †

doi: 10.1128/MCB.23.19.6725-6738.2003

Figure Lengend Snippet: Overexpression of ADAM12 induces p27 expression. Subconfluent C2C12 cells transfected with ADAM12 (A and B) or the L73P ADAM12 mutant (C and D) and incubated in medium containing 1% FBS were fixed and costained with anti-ADAM12 antibody (red) and anti-p27 antibody (green). Two representative microscopic fields are shown for ADAM12 and L73P transfections. Arrows indicate ADAM12- and L73P-overexpressing cells with induced expression of p27. Arrowheads mark ADAM12- and L73P-overexpressing cells that are p27 negative. (E) The percentage of p27-positive cells among ADAM12-overexpressing (solid bar), L73P-overexpressing (grey bar), and cells that were not stained with ADAM12 antibody (nontransfected cells, open bar) was quantified. Results shown are the averages from three experiments ± standard errors. At least 100 transfected cells were analyzed in each experiment.

Article Snippet: The membrane was then hybridized at 65°C with a 2.7-kbp ADAM12 cDNA probe labeled with [α- 32 P]dCTP (3,000 Ci/mmol) by random priming (Prime a Gene labeling system; Promega, Madison, Wis.), washed at 68°C, and exposed for autoradiography at −70°C with an intensifying screen.

Techniques: Over Expression, Expressing, Transfection, Mutagenesis, Incubation, Staining