Journal: The International journal of developmental biology

Article Title: Regeneration of neuronal cell types in Schmidtea mediterranea: an immunohistochemical and expression study.

doi: 10.1387/ijdb.113428sf

Figure Lengend Snippet: Fig. 3. Regeneration of the anterior Central Nervous System. (A-H) Anti-5-HT immunos- taining. All samples correspond to trunk fragments regenerating a new head. (A) Arrow- heads point to 5-HT-positive cells at the level of the truncated nervous system at day 1 of regeneration. (B) At day 3, a thin fibre (arrowhead) connects the developing cephalic ganglia (asterisks). (C) Five days of regeneration. (D) Seven-day regenerated cephalic ganglia with a central neuropil (asterisks) and peripheral cell bodies (arrowheads). (E-H) Anti-GYRFamide immunostaining. (E) Arrowheads point to GYRFamide-positive cells at the level of the trun- cated nervous system at day 1 of regeneration. (F) Developing cephalic ganglia (asterisks) connected by a thin commissure (arrowhead in F) at day 3 of regeneration. (G) Double immunostaining with anti-5-HT (in red). Arrowheads point to 5-HT-positive cells. Asterisks point to the cephalic ganglia. (H) Double immunostaining with anti-5-HT (in red) at day 5 of regeneration. Arrowheads indicate 5-HT-positive cells surrounding the central neuropil (asterisks). (I-L) Anti-5F10 (allatostatin) immunostaining. (J,L) Double immunostaining with anti-5-HT (in red). Arrowheads in (I,J) point to 5F10-positive cells. Asterisks in (J, L) mark the developing cephalic ganglia. In (A,B,F,G,I,J) anterior is to the top left; in (C,D,E,H,K,L) anterior is to the top. Scale bar, 100 +m.

Article Snippet: Primary antibodies were used at the following dilutions: 5-HT (Sigma, ref. S5545), 1:1,000; GYRFamide (Johnston et al., 1996), 1:1000; 5F10 (Developmental Studies Hybridoma Bank), 1:2; neuropeptide F (Maule et the regeneration process.

Techniques: Immunostaining, Double Immunostaining

Journal: Cell Death & Disease

Article Title: Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation

doi: 10.1038/cddis.2015.110

Figure Lengend Snippet: Q p site substitutions lead to reduced cell death induction and ROS generation in response to MitoVES, and the induced cell death is dependent on CII-derived ROS. ( a ) Variant cell lines were exposed to 20 μ M MitoVES for 24 h and the percentage of annexin V-positive cells was determined by flow cytometry. n ≥5, mean±S.E.M., * values significantly different from WT. ( b ) Variant cell lines were exposed to 2 μ M MitoVES for 30 min and the level of ROS was determined by dihydroethidium (DHE) staining and flow cytometry. n ≥5, mean±S.E.M., * values significantly different from WT. ( c ) Cells were transfected with catalase-coding or control vector, exposed to 20 μ M MitoVES for 20 h and the percentage of annexin V-positive cells was determined by flow cytometry. n ≥4, mean±S.E.M., * values significantly different between catalase and mock-transfected cells. Inset, catalase overexpression verified by western blot. ( d ) Cells were exposed to 30 μ M MitoVES for 12 h in the presence or absence of 20 mM malonate (30 min pretreatment). The percentage of annexin V-positive cells was determined by flow cytometry. n ≥3, mean±S.E.M., * values significantly different in the presence and absence of malonate. ( e ) Cells were exposed to 5 μ M MitoVES for 30 min in the presence or absence of 50 mM malonate (30 min pretreatment) and the level of ROS was determined by DHE staining and flow cytometry. n =5, mean±S.E.M., * values significantly different in the presence and absence of malonate

Article Snippet: All these fractions were combined, washed by PBS and incubated with PE- or Dy647-labeled annexin V (Becton Dickinson and Apronex, Vestec, Czech Republic) in the supplied binding buffer for at least 10 min. Hoechst 33258 was added to mark the cells with ruptured cell membrane.

Techniques: Derivative Assay, Variant Assay, Flow Cytometry, Staining, Transfection, Plasmid Preparation, Over Expression, Western Blot

Journal: Cell Death & Disease

Article Title: Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation

doi: 10.1038/cddis.2015.110

Figure Lengend Snippet: Suppression of CII-driven respiration correlates with cell death for Q p site inhibitors that do not rapidly increase the succinate level. ( a ) Digitonin-permeabilized respiration-competent variant cell lines respiring on 10 mM succinate in the presence of 0.5 μ M rotenone and FCCP were exposed to increasing concentrations of MitoVES. The variants show reduced inhibition compared with WT. ( b ) Inhibition by the dicarboxylate-binding site inhibitor malonate in the same experimental set-up shows similar efficiency for all Q p site substitutions. ( c ) Similar to a and b , but the Q p site inhibitor TTFA was used. ( a – c ) The data represent the mean±S.E.M. of 3–4 independent experiments. ( d ) Variant cell lines were exposed to 0.5 mM TTFA for 24 h and the percentage of annexin V-positive cells was determined by flow cytometry. n =5, mean±S.E.M., * values significantly different from WT. ( e ) Cells were exposed to 250 μ M TTFA for 30 min and the level of ROS was determined by dihydroethidium (DHE) staining and flow cytometry. n =5, mean±S.E.M., * values significantly different from WT. ( f ) Cells were transfected with catalase-coding or control vector, exposed to 2 mM TTFA for 20 h, and the percentage of annexin V-positive cells was determined by flow cytometry. n =4, mean±S.E.M., * values significantly different between catalase and mock-transfected cells. Inset, catalase overexpression verified by western blot. ( g ) Cells were exposed to 250 μ M TTFA for 30 min in the presence or absence of 50 mM malonate (30 min pretreatment), and the level of ROS was determined by DHE staining and flow cytometry. n =4, mean±S.E.M., * values significantly different in the presence and absence of malonate. ( h ) Cells were exposed to 1.5 mM TTFA for 12 h in the presence or absence of 20 mM malonate (30 min pretreatment). The percentage of annexin V-positive cells was determined by flow cytometry. n =4, mean±S.E.M., * values significantly different in the presence and absence of malonate. ( i ) Atpenin A5-induced inhibition of respiration of permeabilized cells as described in a . n ≥3, mean±S.E.M. ( j ) Cells were exposed to 1 μ M Atpenin A5 for 24 h and the percentage of annexin V-positive cells was determined by flow cytometry. n =3, mean±S.E.M. ( k ) Cells were exposed to 0.5 μ M Atpenin A5 for 30 min and the level of ROS was determined by DHE staining and flow cytometry. n =5, mean±S.E.M. ( l ) Succinate levels were determined in WT cells exposed to 20 μ M MitoVES, 1 mM TTFA or 1 μ M Atpenin A5 for 30 min. n =3, mean±S.E.M., * values significantly different from control

Article Snippet: All these fractions were combined, washed by PBS and incubated with PE- or Dy647-labeled annexin V (Becton Dickinson and Apronex, Vestec, Czech Republic) in the supplied binding buffer for at least 10 min. Hoechst 33258 was added to mark the cells with ruptured cell membrane.

Techniques: Variant Assay, Inhibition, Binding Assay, Flow Cytometry, Staining, Transfection, Plasmid Preparation, Over Expression, Western Blot