Journal: PLoS Pathogens

Article Title: STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle

doi: 10.1371/journal.ppat.1006975

Figure Lengend Snippet: A) Representative western blot of C33a cells transfected with GFP tagged HPV18 oncoproteins E5, E6 or E7 and analysed for STAT3 activation using specific antibodies detecting phosphorylated and total STAT3. Expression of HPV oncoproteins was confirmed using a GFP antibody. GAPDH served as a loading control. B) Representative luciferase reporter assays from C33a cells co-transfected with GFP tagged HPV18 oncoproteins E5, E6 or E7 and either a β-casein promoter reporter plasmid or a Pom C reporter plasmid, which contain STAT3 binding sites, and promoter activity measured using a dual-luciferase system. Data are presented as relative to the GFP transfected control cells. Bars represent the means ± standard deviation from at least three independent biological repeats. **P<0.01, N . S . = not significant (Student’s t-test). C) Representative luciferase reporter assays from C33a cells co-transfected with GFP tagged E6 and a β-casein promoter reporter plasmid treated with the STAT3 inhibitors cryptotanshinone and S3I-201, and the STAT5 inhibitor pimozide. Promoter activity was measured using a dual-luciferase system. Data are presented as relative to the GFP transfected control. Bars represent the means ± standard deviation from at least three independent biological repeats. **P<0.01 (Student’s t-test). D) Representative western blot of C33a cells transfected with GFP or GFP tagged HPV18 E6, untreated or treated with the STAT3 inhibitors as above and analysed for total and phosphorylated STAT3 (Y705 and S727), the expression of cyclin D1 and Bcl-xL and GFP to demonstrate expression of the GFP-18E6 fusion protein. GAPDH expression was used as a loading control. Data shown are representative of at least three biological repeats. E-H) C33a cells were transiently transfected with GFP or GFP tagged HPV18 E6 and left untreated or treated with the STAT3 inhibitors as above and RNA was extracted for qRT-PCR analysis of the indicated STAT3 dependent genes. Samples were normalized against U6 mRNA levels. Representative data are presented relative to the GFP transfected control. Bars are the means ± standard deviation from at least three biological repeats. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test).

Article Snippet: Total protein was resolved by SDS-PAGE (10–15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences) and probed with antibodies specific for Phospho-STAT3 (S727) (ab32143, abcam), Phospho-STAT3 (Y705) (9131, Cell Signalling Technology (CST)), Total STAT3 (C-20: sc-482, Santa Cruz Biotechnology), Phospho-STAT5 (Y694) (9314, CST), Phospho-JAK2 (Y1007/1008) (3776, CST), Total JAK2 (3230, CST), involucrin (SY5, Santa Cruz Biotechnology), HPV18 E6 (G-7, Santa Cruz Biotechnology), HPV18 E7 (8E2, Abcam (ab100953), HPV 16/18 E6 (C1P5, Santa Cruz Biotechnology), HPV 16 E7 (ED17, Santa Cruz Biotechnology), Phospho-ERK1/2 (Thr202/Tyr204) (43705, CST), Phospho-JNK (Thr183/Tyr185) 4668, CST), Phospho-p38 (Thr180/Tyr182) (9211, CST), Bcl xL (H-62, Santa Cruz Biotechnology), Cyclin D1 (A-12, Santa Cruz Biotechnology) p53 (FL-393, Santa Cruz Biotechnology), p21 (2947, CST), FLAG (F3165, Sigma), GFP (B-2: sc-9996, Santa Cruz Biotechnology) and GAPDH (G-9, Santa Cruz Biotechnology).

Techniques: Western Blot, Transfection, Activation Assay, Expressing, Luciferase, Plasmid Preparation, Binding Assay, Activity Assay, Standard Deviation, Quantitative RT-PCR

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