bcl xl shrna gfp lentiviral particles  (Santa Cruz Biotechnology)

Journal: Biology

Article Title: Bcl-xL Is Required by Primary Hippocampal Neurons during Development to Support Local Energy Metabolism at Neurites

doi: 10.3390/biology10080772

Figure Lengend Snippet: Bcl-xL depletion alters ATP/ADP ratio in primary hippocampal neurons. Primary rat hippocampal neurons were transduced with control shRNA or Bcl-xL shRNA. Immunoblotting ( A ) was performed to show depletion of the Bcl-xL protein levels in Bcl-xL shRNA transduced neurons ( n = 4, * p = 0.0286, Mann–Whitney test). ATP/ADP ratio ( B , C ) was measured using the GW1-PercevalHR sensor (F 488nm /F 405nm ). Bcl-xL depleted neurons showed lower ATP/ADP ratios in both the soma ( n = 20) and neurites ( n = 20). * p < 0.05, and *** p < 0.001, two-tailed Student’s t -test. Scale bar = 50 μm. Pseudocolour ratiometric micrographs ( D ) of neurons indicating the ATP/ADP ratio using the PercevalHR sensor. Red, higher ATP/ADP; Blue, lower ATP/ADP. Primary hippocampal neurons lacking Bcl-xL showed a significantly decreased TMRM signal ( E – G ), indicating a loss of mitochondrial inner membrane potential (Δψ) ( n = 12). ** p < 0.01, and **** p < 0.0001, two-tailed Student’s t -test. Scale bar = 20 μm.

Article Snippet: Briefly, neurons (0.3 × 10 6 cells/35 mm plate) were seeded on poly-l-lysin coated plates and grown in a neurobasal medium supplemented with B-27, glutamine, and antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) for 21 days in vitro (DIV). shRNA lentiviral particle transduction: Primary hippocampal neurons were transduced with control shRNA lentiviral particles or Bcl-xL shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA); copGFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) at DIV 7.

Techniques: Transduction, shRNA, Western Blot, MANN-WHITNEY, Two Tailed Test

Journal: Biology

Article Title: Bcl-xL Is Required by Primary Hippocampal Neurons during Development to Support Local Energy Metabolism at Neurites

doi: 10.3390/biology10080772

Figure Lengend Snippet: Bcl-xL depletion disrupts mitochondrial motility in primary hippocampal neurons. Primary rat hippocampal neurons were transduced with control shRNA or Bcl-xL shRNA. Kymographs ( A ) were produced and analysis using the KymoAnalyzer was completed to give the percentage of mitochondria that showed mitochondria moving in the following directions: only anterograde ( B ), n = 40, net anterograde ( C ), n = 40, ** p = 0.0022, Mann-Whitney test, only retrograde ( D ), n = 40, net retrograde ( E ), n = 40, ** p = 0.0075, Mann–Whitney test, stationary ( F ), n = 40, *** p = 0.0001, Mann–Whitney test, and net stationary ( G ), n = 40, *** p = 0.0002, Mann–Whitney test. The analysis also provided the mitochondrial density (number of mitochondria/µm) for mitochondria exhibiting anterograde ( H ), n = 40 or retrograde ( I ), n = 40 motion, as well as mitochondria that reversed directions ( J ), n = 40, ** p = 0.0029, Mann–Whitney test during the image sequence and stationary mitochondria ( K ), n = 40, * p = 0.0177, Mann–Whitney test. The COX IV protein was labeled to visualize mitochondria ( L , M ), and mitochondria length was measured ( n = 63). * p < 0.05, two-tailed Student’s t -test. Scale bar = 20 μm.

Article Snippet: Briefly, neurons (0.3 × 10 6 cells/35 mm plate) were seeded on poly-l-lysin coated plates and grown in a neurobasal medium supplemented with B-27, glutamine, and antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) for 21 days in vitro (DIV). shRNA lentiviral particle transduction: Primary hippocampal neurons were transduced with control shRNA lentiviral particles or Bcl-xL shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA); copGFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) at DIV 7.

Techniques: Transduction, shRNA, Produced, MANN-WHITNEY, Sequencing, Labeling, Two Tailed Test

Journal: Biology

Article Title: Bcl-xL Is Required by Primary Hippocampal Neurons during Development to Support Local Energy Metabolism at Neurites

doi: 10.3390/biology10080772

Figure Lengend Snippet: Bcl-xL depletion impairs neurite arborization and synapse formation in primary hippocampal neurons. Primary hippocampal neurons were transduced with conGFP control or Bcl-xL shRNA-GFP. The Sholl analysis ( A ) determined the number of neurite intersections ( n = 12). Pseudocolour images ( B ) show a qualitative difference in neurite intersections resulting from Bcl-xL depletion. Red, higher intersections; blue, fewer intersections. Scale bar = 50 µm. Immunocytochemistry ( C ) was performed, and Bassoon-positive puncta were counted from 50–100 µm from the soma ( n = 63). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, two-tailed Student’s t -test. ( D ) Red, Bassoon; Green, MAP2; Blue, DAPI. Scale bar = 20 μm.

Article Snippet: Briefly, neurons (0.3 × 10 6 cells/35 mm plate) were seeded on poly-l-lysin coated plates and grown in a neurobasal medium supplemented with B-27, glutamine, and antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) for 21 days in vitro (DIV). shRNA lentiviral particle transduction: Primary hippocampal neurons were transduced with control shRNA lentiviral particles or Bcl-xL shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA); copGFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) at DIV 7.

Techniques: Transduction, shRNA, Immunocytochemistry, Two Tailed Test

Journal: Biology

Article Title: Bcl-xL Is Required by Primary Hippocampal Neurons during Development to Support Local Energy Metabolism at Neurites

doi: 10.3390/biology10080772

Figure Lengend Snippet: Bcl-xL depletion makes primary hippocampal neurons more susceptible to excitotoxicity. Primary hippocampal neurons transduced with control shRNA or Bcl-xL shRNA were treated with or without glutamate to induce excitotoxicity. Neurons were treated with Fluo-4 ( A , B ) to measure intracellular calcium concentration ( n = 121). ** p < 0.01 and **** p < 0.0001, Kruskal–Wallis test with Dunn’s multiple comparisons test. Calcein-AM and Hoechst staining ( C , D ) shows the proportion of healthy cells via fluorescent image analysis ( n = 100). ** p < 0.01, and *** p < 0.001, one-way ANOVA with a Tukey’s post-hoc-analysis. Scale bar = 20 μm. PI and Hoechst staining ( E , F ) shows the proportion of dead cells via fluorescent image analysis ( n = 20). **** p < 0.0001, one-way ANOVA with a Tukey’s post-hoc analysis. Scale bar = 50 μm.

Article Snippet: Briefly, neurons (0.3 × 10 6 cells/35 mm plate) were seeded on poly-l-lysin coated plates and grown in a neurobasal medium supplemented with B-27, glutamine, and antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) for 21 days in vitro (DIV). shRNA lentiviral particle transduction: Primary hippocampal neurons were transduced with control shRNA lentiviral particles or Bcl-xL shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA); copGFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) at DIV 7.

Techniques: Transduction, shRNA, Concentration Assay, Staining

Journal: Biology

Article Title: Bcl-xL Is Required by Primary Hippocampal Neurons during Development to Support Local Energy Metabolism at Neurites

doi: 10.3390/biology10080772

Figure Lengend Snippet: Bcl-xL depletion alters ATP/ADP ratio in primary hippocampal neurons. Primary rat hippocampal neurons were transduced with control shRNA or Bcl-xL shRNA. Immunoblotting ( A ) was performed to show depletion of the Bcl-xL protein levels in Bcl-xL shRNA transduced neurons ( n = 4, * p = 0.0286, Mann–Whitney test). ATP/ADP ratio ( B , C ) was measured using the GW1-PercevalHR sensor (F 488nm /F 405nm ). Bcl-xL depleted neurons showed lower ATP/ADP ratios in both the soma ( n = 20) and neurites ( n = 20). * p < 0.05, and *** p < 0.001, two-tailed Student’s t -test. Scale bar = 50 μm. Pseudocolour ratiometric micrographs ( D ) of neurons indicating the ATP/ADP ratio using the PercevalHR sensor. Red, higher ATP/ADP; Blue, lower ATP/ADP. Primary hippocampal neurons lacking Bcl-xL showed a significantly decreased TMRM signal ( E – G ), indicating a loss of mitochondrial inner membrane potential (Δψ) ( n = 12). ** p < 0.01, and **** p < 0.0001, two-tailed Student’s t -test. Scale bar = 20 μm.

Article Snippet: Briefly, neurons (0.3 × 10 6 cells/35 mm plate) were seeded on poly-l-lysin coated plates and grown in a neurobasal medium supplemented with B-27, glutamine, and antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) for 21 days in vitro (DIV). shRNA lentiviral particle transduction: Primary hippocampal neurons were transduced with control shRNA lentiviral particles or Bcl-xL shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA); copGFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) at DIV 7.

Techniques: Transduction, shRNA, Western Blot, MANN-WHITNEY, Two Tailed Test

Journal: Biology

Article Title: Bcl-xL Is Required by Primary Hippocampal Neurons during Development to Support Local Energy Metabolism at Neurites

doi: 10.3390/biology10080772

Figure Lengend Snippet: Bcl-xL depletion disrupts mitochondrial motility in primary hippocampal neurons. Primary rat hippocampal neurons were transduced with control shRNA or Bcl-xL shRNA. Kymographs ( A ) were produced and analysis using the KymoAnalyzer was completed to give the percentage of mitochondria that showed mitochondria moving in the following directions: only anterograde ( B ), n = 40, net anterograde ( C ), n = 40, ** p = 0.0022, Mann-Whitney test, only retrograde ( D ), n = 40, net retrograde ( E ), n = 40, ** p = 0.0075, Mann–Whitney test, stationary ( F ), n = 40, *** p = 0.0001, Mann–Whitney test, and net stationary ( G ), n = 40, *** p = 0.0002, Mann–Whitney test. The analysis also provided the mitochondrial density (number of mitochondria/µm) for mitochondria exhibiting anterograde ( H ), n = 40 or retrograde ( I ), n = 40 motion, as well as mitochondria that reversed directions ( J ), n = 40, ** p = 0.0029, Mann–Whitney test during the image sequence and stationary mitochondria ( K ), n = 40, * p = 0.0177, Mann–Whitney test. The COX IV protein was labeled to visualize mitochondria ( L , M ), and mitochondria length was measured ( n = 63). * p < 0.05, two-tailed Student’s t -test. Scale bar = 20 μm.

Article Snippet: Briefly, neurons (0.3 × 10 6 cells/35 mm plate) were seeded on poly-l-lysin coated plates and grown in a neurobasal medium supplemented with B-27, glutamine, and antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) for 21 days in vitro (DIV). shRNA lentiviral particle transduction: Primary hippocampal neurons were transduced with control shRNA lentiviral particles or Bcl-xL shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA); copGFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) at DIV 7.

Techniques: Transduction, shRNA, Produced, MANN-WHITNEY, Sequencing, Labeling, Two Tailed Test

Journal: Biology

Article Title: Bcl-xL Is Required by Primary Hippocampal Neurons during Development to Support Local Energy Metabolism at Neurites

doi: 10.3390/biology10080772

Figure Lengend Snippet: Bcl-xL depletion impairs neurite arborization and synapse formation in primary hippocampal neurons. Primary hippocampal neurons were transduced with conGFP control or Bcl-xL shRNA-GFP. The Sholl analysis ( A ) determined the number of neurite intersections ( n = 12). Pseudocolour images ( B ) show a qualitative difference in neurite intersections resulting from Bcl-xL depletion. Red, higher intersections; blue, fewer intersections. Scale bar = 50 µm. Immunocytochemistry ( C ) was performed, and Bassoon-positive puncta were counted from 50–100 µm from the soma ( n = 63). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, two-tailed Student’s t -test. ( D ) Red, Bassoon; Green, MAP2; Blue, DAPI. Scale bar = 20 μm.

Article Snippet: Briefly, neurons (0.3 × 10 6 cells/35 mm plate) were seeded on poly-l-lysin coated plates and grown in a neurobasal medium supplemented with B-27, glutamine, and antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) for 21 days in vitro (DIV). shRNA lentiviral particle transduction: Primary hippocampal neurons were transduced with control shRNA lentiviral particles or Bcl-xL shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA); copGFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) at DIV 7.

Techniques: Transduction, shRNA, Immunocytochemistry, Two Tailed Test

Journal: Biology

Article Title: Bcl-xL Is Required by Primary Hippocampal Neurons during Development to Support Local Energy Metabolism at Neurites

doi: 10.3390/biology10080772

Figure Lengend Snippet: Bcl-xL depletion makes primary hippocampal neurons more susceptible to excitotoxicity. Primary hippocampal neurons transduced with control shRNA or Bcl-xL shRNA were treated with or without glutamate to induce excitotoxicity. Neurons were treated with Fluo-4 ( A , B ) to measure intracellular calcium concentration ( n = 121). ** p < 0.01 and **** p < 0.0001, Kruskal–Wallis test with Dunn’s multiple comparisons test. Calcein-AM and Hoechst staining ( C , D ) shows the proportion of healthy cells via fluorescent image analysis ( n = 100). ** p < 0.01, and *** p < 0.001, one-way ANOVA with a Tukey’s post-hoc-analysis. Scale bar = 20 μm. PI and Hoechst staining ( E , F ) shows the proportion of dead cells via fluorescent image analysis ( n = 20). **** p < 0.0001, one-way ANOVA with a Tukey’s post-hoc analysis. Scale bar = 50 μm.

Article Snippet: Briefly, neurons (0.3 × 10 6 cells/35 mm plate) were seeded on poly-l-lysin coated plates and grown in a neurobasal medium supplemented with B-27, glutamine, and antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) for 21 days in vitro (DIV). shRNA lentiviral particle transduction: Primary hippocampal neurons were transduced with control shRNA lentiviral particles or Bcl-xL shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA); copGFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) at DIV 7.

Techniques: Transduction, shRNA, Concentration Assay, Staining

Journal: Experimental & Molecular Medicine

Article Title: Mitochondrial superoxide dismutase 2 mediates γ-irradiation-induced cancer cell invasion

doi: 10.1038/s12276-019-0207-5

Figure Lengend Snippet: a H460 cells were transfected with a control or a Bcl-X L -expression vector. After 24 and 48 h of incubation, SOD2 mRNA levels were compared by quantitative real-time PCR (top), and the levels of the indicated proteins were compared by western blotting (bottom). b The indicated cells were irradiated with the specified doses of γ-rays. Western blotting for SOD2 and Bcl-X L was performed at 24 and 48 h after irradiation. c Irradiated (2 Gy, 48 h) and untreated control H460 cells were incubated in the presence or absence of NAC (5 mM) and metformin (5 mM) for 1 h, and mitochondrial ROS levels were compared by staining cells with MitoSOX Red probes and analyzing them by flow cytometry. d Irradiated and untreated control cells were analyzed for their invasiveness in the presence or absence of NAC or metformin. The levels of Src and phosphorylated Src in the cells were compared by western blotting

Article Snippet: Small interfering RNA (siRNAs) targeting IL-6 (S7312) and Bcl-X L (120717) were purchased from Ambion (Austin, TX, USA). siRNAs targeting SOD2 (sc-41655), β-catenin (sc-44275), and STAT3 (sc-29209) as well as lentiviruses expressing small hairpin RNAs (shRNAs) targeting SOD2 (sc-41655-V), Bcl-X L (sc-43630-V), and SULF2 (sc-63088-V) were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Irradiation, Staining, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: Mitochondrial superoxide dismutase 2 mediates γ-irradiation-induced cancer cell invasion

doi: 10.1038/s12276-019-0207-5

Figure Lengend Snippet: a H460 cells treated with a control, Bcl-X L -targeting, or SOD2-targeting siRNA were irradiated, and the levels of the indicated proteins were compared 24 h after irradiation. b H460 cells infected with lentiviruses expressing the specified shRNAs were transfected with a Bcl-X L or SOD2 expression vector in the indicated combinations. Cellular invasiveness and the levels of Bcl-X L and SOD2 were compared after 48 h of incubation. c H460 cells were transfected with a Bcl-X L or SOD2 expression vector in the indicated combinations. Cellular invasiveness and the levels of the indicated proteins were compared after 48 h of incubation. d H460 cells treated with a control or SOD2-targeting siRNA were irradiated (2 Gy). After 48 h of incubation, irradiated and untreated control cells were analyzed for their levels of O 2 •− and H 2 O 2 using MitoSox Red and Peroxy Orange-1, respectively. e H460 cells were transfected with a SULF2 expression vector and SOD2-targeting siRNA in the indicated combinations (left). Alternatively, H460 cells infected with lentiviruses expressing a control or SOD2-targeting shRNA were incubated in the presence or absence of 100 ng/mL IL-6 (right). After treatment for 24 h, cellular H 2 O 2 levels were compared using Peroxy Orange-1

Article Snippet: Small interfering RNA (siRNAs) targeting IL-6 (S7312) and Bcl-X L (120717) were purchased from Ambion (Austin, TX, USA). siRNAs targeting SOD2 (sc-41655), β-catenin (sc-44275), and STAT3 (sc-29209) as well as lentiviruses expressing small hairpin RNAs (shRNAs) targeting SOD2 (sc-41655-V), Bcl-X L (sc-43630-V), and SULF2 (sc-63088-V) were obtained from Santa Cruz Biotechnology.

Techniques: Irradiation, Infection, Expressing, Transfection, Plasmid Preparation, Incubation, shRNA

Journal: Experimental & Molecular Medicine

Article Title: Mitochondrial superoxide dismutase 2 mediates γ-irradiation-induced cancer cell invasion

doi: 10.1038/s12276-019-0207-5

Figure Lengend Snippet: STAT3 stimulated by IR via the p53/SULF2/β-catenin/IL-6 pathway induces Bcl-X L and SOD2 expression. Bcl-X L increases the ability of complex I to produce O 2 •− , and SOD2 converts mitochondrial O 2 •− to H 2 O 2 , which, in turn, acts as a signaling molecule to promote cell invasion

Article Snippet: Small interfering RNA (siRNAs) targeting IL-6 (S7312) and Bcl-X L (120717) were purchased from Ambion (Austin, TX, USA). siRNAs targeting SOD2 (sc-41655), β-catenin (sc-44275), and STAT3 (sc-29209) as well as lentiviruses expressing small hairpin RNAs (shRNAs) targeting SOD2 (sc-41655-V), Bcl-X L (sc-43630-V), and SULF2 (sc-63088-V) were obtained from Santa Cruz Biotechnology.

Techniques: Expressing