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, Control, shRNA, Western Blot, MANN-WHITNEY, Two Tailed Test, Membrane

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, Control, 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, Control, 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, Control, shRNA, Concentration Assay, Staining

Journal: Antioxidants & Redox Signaling

Article Title: Bcl-xL Is Necessary for Neurite Outgrowth in Hippocampal Neurons

doi: 10.1089/ars.2013.5570

Figure Lengend Snippet: Depletion of Bcl-xL by siRNA in rAAV-transduced neurons. (A) Immunoblot of Bcl-xL protein amount from cell lysates isolated at 1, 2.5, or 4 weeks after rAAV transduction (n=3 samples in each group from three independent cultures). *p<0.05 compared with corresponding scrambled siRNA-expressing group. ANOVA, Tukey's test. GAPDH serves as a protein loading control. (B) Immunocytochemistry of cultured hippocampal neurons at 2.5 weeks after rAAV transduction showing level of Bcl-xL expression. Green: Bcl-xL; Red: dsRED; Blue: DAPI-stained nuclei. The high power image of a single neuron is shown in the box. (C) Immunocytochemistry of cultured hippocampal neurons at 2.5 weeks after rAAV transduction showing colocalization of rAAV with neuronal markers. Green: NeuN; Red: dsRED; Blue: DAPI-stained nuclei. The high power image of a single neuron is shown in the box. Scale bar=100 μm for (B) and (C). Bcl-xL, B-cell lymphoma-extra large; rAAV, recombinant adeno-associated virus. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: The scrambled siRNA sequences were as follows: scrambled up: 5′-tttgcaagctgaccctgaagttcttttt-3′ scrambled down: 5′-cgaaaaagaacttcagggtcagcttg-3′ The Bcl-xL shRNA sequences were: Bcl-xL up: 5′-TTTggagtcagtttagcgatgtcgGTGAAGCCACAGATGcgacatcgctaaactgactccTTTTT-3′ Bcl-xL down: 5′-CGAAAAAggagtcagtttagcgatgtcgCATCTGTGGCTTCACcgacatcgctaaactgactc-3′ Neuronal growth analysis Primary hippocampal neurons were transduced with rAAVs or a combination of lenti-DR6 shRNA or lenti-copGFP control (Santa Cruz, Dallas, TX) at DIV 7.

Techniques: Western Blot, Isolation, Transduction, Expressing, Control, Immunocytochemistry, Cell Culture, Staining, Recombinant, Virus

Journal: Antioxidants & Redox Signaling

Article Title: Bcl-xL Is Necessary for Neurite Outgrowth in Hippocampal Neurons

doi: 10.1089/ars.2013.5570

Figure Lengend Snippet: Depletion of Bcl-xL inhibits growth of neurites. Primary hippocampal neurons were transduced with rAAV for 2.5 weeks. Morphometric analysis was performed on the fluorescent images. (A) Number of neurites extending directly from the soma (n=number of cells; three independently isolated cultures). (B, D) Length of neurites (n=number of cells; three independently isolated cultures). The full extent of neurites is clearly seen in fluorescent images. (C, E) Number of bifurcations per cell (n=number of cells; three independently isolated cultures, blue dots show bifurcations). ****p<0.0001, compared with scrambled siRNA-expressing neurons, two-tailed Student's t-test. Scale bar for (D)=100 μm and (E)=50 μm. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: The scrambled siRNA sequences were as follows: scrambled up: 5′-tttgcaagctgaccctgaagttcttttt-3′ scrambled down: 5′-cgaaaaagaacttcagggtcagcttg-3′ The Bcl-xL shRNA sequences were: Bcl-xL up: 5′-TTTggagtcagtttagcgatgtcgGTGAAGCCACAGATGcgacatcgctaaactgactccTTTTT-3′ Bcl-xL down: 5′-CGAAAAAggagtcagtttagcgatgtcgCATCTGTGGCTTCACcgacatcgctaaactgactc-3′ Neuronal growth analysis Primary hippocampal neurons were transduced with rAAVs or a combination of lenti-DR6 shRNA or lenti-copGFP control (Santa Cruz, Dallas, TX) at DIV 7.

Techniques: Transduction, Isolation, Expressing, Two Tailed Test

Journal: Antioxidants & Redox Signaling

Article Title: Bcl-xL Is Necessary for Neurite Outgrowth in Hippocampal Neurons

doi: 10.1089/ars.2013.5570

Figure Lengend Snippet: Prolonged depletion of Bcl-xL further inhibits neurite outgrowth and is associated with delayed neuronal death. Primary hippocampal neurons were transduced with rAAV for 4 weeks. Morphometric analysis was performed on the fluorescent images. (A) LDH release (n=number of plates). (B) Number of neurites extending directly from the soma (n=number of cells; three independently isolated cultures). (C, H) Length of neurites (n=number of cells; three independently isolated cultures). (D, I) Number of bifurcations per cell (n=number of cells; three independently isolated cultures, blue dots show bifurcations). ***p<0.001 and ****p<0.0001 compared with scrambled siRNA-expressing neurons. Two-tailed Student's t-test. (E) Number of neurites at three different time points (Figs. 3A, ​,4A4A and ​and5B5B as combined data). (F) Length of neurites at three different time points (Figs. 3B, ​,4B4B and ​and5C5C as combined data). (G) Number of bifurcations at three different time points (Figs. 3C, ​,4C4C and ​and5D5D as combined data). *p<0.05 compared with corresponding 1 week group, §p<0.05 compared with corresponding 2.5 weeks group. ANOVA, Tukey's test. Scale bar for (H)=100 μm and (I)=50 μm. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: The scrambled siRNA sequences were as follows: scrambled up: 5′-tttgcaagctgaccctgaagttcttttt-3′ scrambled down: 5′-cgaaaaagaacttcagggtcagcttg-3′ The Bcl-xL shRNA sequences were: Bcl-xL up: 5′-TTTggagtcagtttagcgatgtcgGTGAAGCCACAGATGcgacatcgctaaactgactccTTTTT-3′ Bcl-xL down: 5′-CGAAAAAggagtcagtttagcgatgtcgCATCTGTGGCTTCACcgacatcgctaaactgactc-3′ Neuronal growth analysis Primary hippocampal neurons were transduced with rAAVs or a combination of lenti-DR6 shRNA or lenti-copGFP control (Santa Cruz, Dallas, TX) at DIV 7.

Techniques: Transduction, Isolation, Expressing, Two Tailed Test

Journal: Antioxidants & Redox Signaling

Article Title: Bcl-xL Is Necessary for Neurite Outgrowth in Hippocampal Neurons

doi: 10.1089/ars.2013.5570

Figure Lengend Snippet: Depletion of DR6 partially rescues neuronal growth. Primary hippocampal neurons were coexpressed with lenti-copGFP or lenti-DR6 siRNA along with rAAV. After 2.5 weeks of transduction, images of cells were analyzed. (A) Immunoblot of DR6 protein level from cell lysate isolated at 2.5 weeks after viral transduction. ****p<0.0001, compared with copGFP-expressing neurons, two-tailed Student's t-test. (B, D) Length of neurites (n=number of cells; three independently isolated cultures) (C, D) Number of bifurcations per cell (n=number of cells; three independently isolated cultures, blue dots show bifurcations). *p<0.05 compared with corresponding copGFP-expressing group, §p<0.05 compared with corresponding scrambled siRNA-expressing group. ANOVA, Tukey's test. Scale bar=100 μm (50 μm for the last column). DR6, death receptor 6. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: The scrambled siRNA sequences were as follows: scrambled up: 5′-tttgcaagctgaccctgaagttcttttt-3′ scrambled down: 5′-cgaaaaagaacttcagggtcagcttg-3′ The Bcl-xL shRNA sequences were: Bcl-xL up: 5′-TTTggagtcagtttagcgatgtcgGTGAAGCCACAGATGcgacatcgctaaactgactccTTTTT-3′ Bcl-xL down: 5′-CGAAAAAggagtcagtttagcgatgtcgCATCTGTGGCTTCACcgacatcgctaaactgactc-3′ Neuronal growth analysis Primary hippocampal neurons were transduced with rAAVs or a combination of lenti-DR6 shRNA or lenti-copGFP control (Santa Cruz, Dallas, TX) at DIV 7.

Techniques: Transduction, Western Blot, Isolation, Expressing, Two Tailed Test

Journal: Antioxidants & Redox Signaling

Article Title: Bcl-xL Is Necessary for Neurite Outgrowth in Hippocampal Neurons

doi: 10.1089/ars.2013.5570

Figure Lengend Snippet: Depletion of Bcl-xL aggravates hypoxia-induced neuronal death. Cultures were transduced for 2.5 weeks before 6 h of hypoxic exposure. Activity of caspase 6 (A), caspase 3 and 7 (B) was measured (n=number of wells). (C–F) Primary hippocampal neurons were transduced with rAAV for 2.5 weeks, and cells were exposed to hypoxia for 24 h. First two bars in each group represent the same data as in Figure 2. (C) LDH release (n=number of plates). (D) Number of TUNEL-positive cells (number listed on each histogram refers to the number of plates for at least three independently isolated cultures, data represent 48–56 micrographs). (E, F) Calcein retention (number listed on each histogram refers to the number of plates for at least three independently isolated cultures, data represent 48–60 micrographs). (G) TUNEL staining in hypoxic neurons. Green: TUNEL; Red: dsRED; Blue: DAPI-stained nuclei. (H) Calcein staining in hypoxic neurons. Green: Calcein; Red: dsRED; Blue: DAPI-stained nuclei. *p<0.05 compared with corresponding normoxic group, §p<0.05 compared with corresponding scrambled siRNA-expressing group. ANOVA, Tukey's test. Scale bar for (G) and (H)=50 μm. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: The scrambled siRNA sequences were as follows: scrambled up: 5′-tttgcaagctgaccctgaagttcttttt-3′ scrambled down: 5′-cgaaaaagaacttcagggtcagcttg-3′ The Bcl-xL shRNA sequences were: Bcl-xL up: 5′-TTTggagtcagtttagcgatgtcgGTGAAGCCACAGATGcgacatcgctaaactgactccTTTTT-3′ Bcl-xL down: 5′-CGAAAAAggagtcagtttagcgatgtcgCATCTGTGGCTTCACcgacatcgctaaactgactc-3′ Neuronal growth analysis Primary hippocampal neurons were transduced with rAAVs or a combination of lenti-DR6 shRNA or lenti-copGFP control (Santa Cruz, Dallas, TX) at DIV 7.

Techniques: Activity Assay, Transduction, TUNEL Assay, Isolation, Staining, Expressing

Journal: Antioxidants & Redox Signaling

Article Title: Bcl-xL Is Necessary for Neurite Outgrowth in Hippocampal Neurons

doi: 10.1089/ars.2013.5570

Figure Lengend Snippet: Depletion of Bcl-xL increases loss of neurites after hypoxic exposure. Primary hippocampal neurons were transduced with rAAV for 2.5 weeks, and cells were exposed to hypoxia for 24 h. (A) Number of neurites arising from the soma (n=number of cells; three independently isolated cultures). (B, D) Neurite length (n=number of cells; three independently isolated cultures). (C, E) Number of bifurcations per cell (n=number of cells; three independently isolated cultures, blue dots show bifurcations). ****p<0.0001, compared with scrambled siRNA-expressing neurons exposed to hypoxia, two-tailed Student's t-test. Scale bar for (D)=100 μm and (E)=50 μm. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: The scrambled siRNA sequences were as follows: scrambled up: 5′-tttgcaagctgaccctgaagttcttttt-3′ scrambled down: 5′-cgaaaaagaacttcagggtcagcttg-3′ The Bcl-xL shRNA sequences were: Bcl-xL up: 5′-TTTggagtcagtttagcgatgtcgGTGAAGCCACAGATGcgacatcgctaaactgactccTTTTT-3′ Bcl-xL down: 5′-CGAAAAAggagtcagtttagcgatgtcgCATCTGTGGCTTCACcgacatcgctaaactgactc-3′ Neuronal growth analysis Primary hippocampal neurons were transduced with rAAVs or a combination of lenti-DR6 shRNA or lenti-copGFP control (Santa Cruz, Dallas, TX) at DIV 7.

Techniques: Transduction, Isolation, Expressing, Two Tailed Test

Journal: Antioxidants & Redox Signaling

Article Title: Bcl-xL Is Necessary for Neurite Outgrowth in Hippocampal Neurons

doi: 10.1089/ars.2013.5570

Figure Lengend Snippet: Depletion of DR6 rescues hypoxia-induced neurite loss. Primary hippocampal neurons were coexpressed with lenti-copGFP or lenti-DR6 siRNA along with rAAV. After 2.5 weeks of transduction, cells were exposed to hypoxia for 24 h. (A, C) Neurite length (n=number of cells; three independently isolated cultures). (B, C) Number of bifurcations per cell (n=number of cells; three independently isolated cultures, blue dots show bifurcations). *p<0.05 compared with corresponding copGFP-expressing group, §p<0.05 compared with corresponding scrambled siRNA-expressing group. ANOVA, Tukey's test. Scale bar=100 μm (50 μm for the last column). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: The scrambled siRNA sequences were as follows: scrambled up: 5′-tttgcaagctgaccctgaagttcttttt-3′ scrambled down: 5′-cgaaaaagaacttcagggtcagcttg-3′ The Bcl-xL shRNA sequences were: Bcl-xL up: 5′-TTTggagtcagtttagcgatgtcgGTGAAGCCACAGATGcgacatcgctaaactgactccTTTTT-3′ Bcl-xL down: 5′-CGAAAAAggagtcagtttagcgatgtcgCATCTGTGGCTTCACcgacatcgctaaactgactc-3′ Neuronal growth analysis Primary hippocampal neurons were transduced with rAAVs or a combination of lenti-DR6 shRNA or lenti-copGFP control (Santa Cruz, Dallas, TX) at DIV 7.

Techniques: Transduction, Isolation, Expressing

Journal: Antioxidants & Redox Signaling

Article Title: Bcl-xL Is Necessary for Neurite Outgrowth in Hippocampal Neurons

doi: 10.1089/ars.2013.5570

Figure Lengend Snippet: Depletion of Bcl-xL increases DR6 and activates Bax in hippocampal neurons in the absence and presence of hypoxic insult. (A) Immunoblots with the indicated antibodies. (B) Group data indicating band intensity normalized to GAPDH (n=3 independent cultures). (C) The same group data as in (B) now normalized to Bcl-xL. (D) Immunoblots with the indicated antibodies. (E) Group data indicating band intensity normalized to GAPDH (n=3–5 independent cultures). (F) The same group data as in (E) now normalized to Bcl-xL. *p<0.05 compared with corresponding normoxic group, §p<0.05 compared with corresponding scrambled siRNA-expressing group, ‡p<0.05 compared with Bcl-xL-depleted normoxic group. ANOVA, Tukey's test.

Article Snippet: The scrambled siRNA sequences were as follows: scrambled up: 5′-tttgcaagctgaccctgaagttcttttt-3′ scrambled down: 5′-cgaaaaagaacttcagggtcagcttg-3′ The Bcl-xL shRNA sequences were: Bcl-xL up: 5′-TTTggagtcagtttagcgatgtcgGTGAAGCCACAGATGcgacatcgctaaactgactccTTTTT-3′ Bcl-xL down: 5′-CGAAAAAggagtcagtttagcgatgtcgCATCTGTGGCTTCACcgacatcgctaaactgactc-3′ Neuronal growth analysis Primary hippocampal neurons were transduced with rAAVs or a combination of lenti-DR6 shRNA or lenti-copGFP control (Santa Cruz, Dallas, TX) at DIV 7.

Techniques: Western Blot, Expressing