Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: (a) – Phase contrast light microscopy of proliferating myoblasts and myotubes formed on day 6 of differentiation. (b) - WB analysis of specific protein marker expression during C2C12 cell differentiation assessed with antibodies against myogenin, SERCA1, Cav3, and Cav1. Actin expression levels are shown as controls for protein load. (c) – Myotubes and reserve cells were separated at days 4 (4d) and 6 (6d) of differentiation, as shown in the schematic representation and described under “Experimental Procedures”, and analyzed by WB with antibodies specific for myogenin, SERCA1, Cav3, and Cav1. The abbreviations used for the cell types are M - proliferating myoblasts; T – myotubes; R - reserve cells.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Light Microscopy, Marker, Expressing, Cell Differentiation

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: (a) - Proliferating myoblasts (day 0) and differentiating C2C12 cells were harvested on days 2 - 6 after the onset of differentiation and analyzed by SDS-PAGE and WB analysis with antibodies against Hsp70, Bcl-2, Bax, Bak, and Bad. (b) – Analysis of these proteins separately in myoblasts (M), myotubes (T) and reserve cells (R) along with cell type-specific protein markers Pax7 and MyoD at days 4 (4d) and day 6 (6d) of differentiation. (c) Densitometry analysis of data from (b).

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: SDS Page

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: (a) – Sequence specificity of SERCA2A and SERCA2B in the C-terminal part. (b) – WB analysis of protein expression during C2C12 cell differentiation assessed with antibodies against SERCA2. (c) - Densitometry analysis of WB data of SERCA2 level in GM and in DM after 6 days of differentiation from three independent experiments.(d) – Analysis of SERCA2 isoforms separately in myoblasts (M), myotubes (T) and reserve cells (R): myotubes and reserve cells were separated as described under “Experimental Procedures” at day 6 of differentiation and analyzed by WB with antibodies specific for SERCA2 and SERCA2B (polyclonal custom antibody raised against the C-terminal peptide underlined in panel A). (e) - Capillary LC-LTQ-FT-MS/MS spectrum of the C-terminal tryptic peptide of SERCA2A, identified in the tryptic digests of C2C12 cell lysates.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Sequencing, Expressing, Cell Differentiation, Tandem Mass Spectroscopy

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: Phase contrast light microscopy of differentiated C2C12 cells prior to (a) and after exposure to 0.5 (b), 1 (c), 2 (d), and 4 mM H2O2 (e) for 4 h or 2 h (f) as described under “Experimental Procedures”.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Light Microscopy

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: Differentiated C2C12 cells were incubated without (control) or with 4 mM H2O2 for different times as indicated (a) or incubated overnight with smaller concentrations of H2O2 (b), and adherent cells were analyzed by WB with anti-SERCA1, anti-Cav3 and anti-Bcl-2 antibodies. Actin expression levels are presented for control of protein load. (c) Differentiated C2C12 cells were incubated in the absence or in the presence of 4 mM H2O2 for 4 h, and expression levels of SERCA1 and Bcl-2 were analyzed by WB separately for adherent and detached cells.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Incubation, Control, Expressing

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: (a) – Detached cells obtained after different treatment were harvested as depicted in the inserted scheme and analyzed by WB for Cav3, Bcl-2, and actin. (b) – Differentiated C2C12 cells treated as above were lysed, digested with trypsin, and submitted to a “shotgun” capLC-LTQ-FT-MS/MS analysis coupled to a protein database search, followed by a quantification of peptide TIC values for selected proteins as described under “Experimental Procedures”. Protein IDs in the IPI (mouse) database: SERCA1, 00311654; SERCA2, 00468900; Myosin light chain, skeletal muscle, 00224549; Myosin 3, 00380895; Myosin 9, 00123181; Troponin c, slow-twitch skeletal muscle, 00113712; Tubulin beta, 00117352. (Note: error bars on logarithmic scale).

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Tandem Mass Spectroscopy

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: Differentiated C2C12 cells were incubated without (control) or in the presence of 4 mM of H2O2 for 2 h as shown in the schematic representation, and myotubes and reserve cells were separated by mild trypsinization as described under “Experimental Procedure”. (a) – Scheme of experiment. (b) – Expression profiles of Bcl-2, SERCA1, Cav3, and Pax7 in myotubes and reserve cells analyzed by WB after incubation without (control) or with 4 mM H2O2 for 2 h. (c) - Bcl-2 levels in non-treated myotubes (control) and myotubes remaining attached to the dish after incubation with 4 mM H2O2 for 2 h, collected by mild trypsinization, and analyzed by a densitometry analysis of the respective blots presented in panel B.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Incubation, Control, Expressing

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

doi: 10.1007/s10495-013-0922-7

Figure Lengend Snippet: Proliferating C2C12 myoblasts were transfected with either empty vector or Bcl-2 expression vector for 24 h before the onset of differentiation. (a) - Cells detached from the dishes at days 2, 4, and 6 after the initiation of differentiation were harvested and analyzed by WB for Bcl-2, SERCA1, Cav3 and caspase 9. (b) - Myotubes and reserve cells remaining attached to the dishes at day 6 after initiation of differentiation were separated via differential trypsinization as described under “Experimental Procedures”, and analyzed by WB with anti-Bcl-2 and anti-Bax antibodies. (c) – Control and Bcl-2 transfected C2C12 cells at day 6 after initiation of differentiation were incubated without or with 4 mM H2O2 for 2 hours, followed by WB analysis for Cav3, Bcl-2, and caspase 9 of both adherent and detached cells. (d) - Densitometry quantification of Cav-3 on the blots (marker of myotubes) shown in panel C, normalized to the level of Cav-3 in Bcl-2 transfected cells, remaining adherent to the dish after exposure to 4 mM H2O2 for 2 hours.

Article Snippet: Cell Culture and incubations C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Incubation, Marker

Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control