Journal: Integrative biology : quantitative biosciences from nano to macro

Article Title: Monitoring of cell oxygenation and responses to metabolic stimulation by intracellular oxygen sensing technique.

doi: 10.1039/c0ib00021c

Figure Lengend Snippet: Fig. 2 O2 levels within the layer of resting dPC12 cells. A. Under external normoxia (200 mM O2), adding more PC12 cells on top of the monolayer (indicated as numbers from 0 to 6.75 105) decreases Cc. B. Cc in the monolayer of dPC12 cell has a linear dependence on the number of added cells. C. At 10% O2 (100 mM O2), steady-state Cc levels in galactose are lower than in glucose medium due to more active respiration. D. At 6% O2 (60 mM O2), the drop in Cc becomes greater and in galactose the cells become anoxic. E. At reduced O2 relative deoxygenation of the cell layer increases and reaches anoxia. F. Increased viscosity of the medium by Ficoll addition decreases Cc (measured at 20.9% O2). Sample volume 100 mL for all the experiments.

Article Snippet: Rat pheochromocytoma PC12 cells (ATCC) were maintained in RPMI 1640 medium supplemented with NaHCO3, 2 mM L-glutamine, 10% horse serum (HS), 5% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (P/S), in 5%CO2 at 37 1C.

Techniques: Viscosity

Journal: Integrative biology : quantitative biosciences from nano to macro

Article Title: Monitoring of cell oxygenation and responses to metabolic stimulation by intracellular oxygen sensing technique.

doi: 10.1039/c0ib00021c

Figure Lengend Snippet: Fig. 3 Dynamics of O2 upon cell stimulation. A. The duration and magnitude of the response (Cc) to 5 mM EGTA (depletion in extracellular Ca2+) is modulated by the sample volume/height. B. At higher O2 barrier (200 mL of medium) even minor respiratory responses becomes more visible: the responses to 0.5oEGTAo1 mM is significant in 200 mL (p o 0.01), but not seen in 100 mL samples. C, D. The shape of changes in Cs during cell stimulation are similar to those in Cc; but the amplitude is smaller. E. Addition of more PC12 cells on top of the monolayer (100 mL of medium) increases the response to stimulation. Dotted lines show the time of temperature equilibration of samples. All the experiments, except E, were conducted in galactose medium.

Article Snippet: Rat pheochromocytoma PC12 cells (ATCC) were maintained in RPMI 1640 medium supplemented with NaHCO3, 2 mM L-glutamine, 10% horse serum (HS), 5% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (P/S), in 5%CO2 at 37 1C.

Techniques: Cell Stimulation

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors.

doi: 10.1159/000488270

Figure Lengend Snippet: Fig. 5. Inhibition of MRP4 expression by siRNA reduces cAMP extrusion and PKA substrate motif phosphorylation in response to CTx and PACAP. PC12 cells were treated with siRNA directed against MRP4 or with a control sequence for three days. Cells pre-treated with MRP4- siRNA or control siRNA were incubated with adenosine (10 µM, 10 min), CGS 21680 (100 nM, 10 min), CTx (200 U/ml, 40 min), or PACAP (100 nM, 15 min), fol lowed by determination of PKA substrate motif phosphorylation or β-actin by im munoblotting (n=3). Band intensities of entire lanes were quantified densitro metrically. Con-siRNA; control-siRNA.

Article Snippet: Materials and Methods Cell culture and stimulation Neuroendocrine PC12 (American Type Culture Collection) and PC3 prostate carcinoma cells (European Collection of Authenticated Cell Cultures) were grown in DMEM containing 5% horse serum and 5% fetal calf serum supplemented with antibiotics [18].

Techniques: Inhibition, Expressing, Phospho-proteomics, Control, Sequencing, Incubation

Journal: Journal of Inflammation Research

Article Title: Nrf2 Inhibits the Progression of Parkinson’s Disease by Upregulating AABR07032261.5 to Repress Pyroptosis

doi: 10.2147/JIR.S345895

Figure Lengend Snippet: Elevated oxidative stress and pyroptosis in the cellular PD model. ( A ) Outgrowth of neurites from PC12 cells treated with NGF-β. Morphological alterations of PC12 cells were observed by microscopy following NGF-β treatment for 7 d. ( B ) Relative TH mRNA levels in PC12 cells treated with NGF-β. Quantitative RT-PCR was performed to detect TH mRNA levels. ( C ) In situ expression of TH proteins in PC12 cells following NGF-β treatment. TH protein contents in PC12 cells were analyzed by immunocytochemistry. ( D ) Proliferation of PC12 cells treated with NGF-β and exposed to 6-OHDA (100 μM). Proliferation was assessed by MTS assay. ( E ) Alteration of apoptosis in PC12 cells following NGF-β induction and 6-OHDA exposure (100 μM). Apoptosis of PC12 cells was evaluated by flow cytometry. ( F ) Elevated ROS contents in the cellular PD model. Flow cytometry was performed to detect ROS levels in cells. ( G and H ) Increased IL-1β and LDH release in the cellular PD model. IL-1β ( G ) and LDH release ( H ) were detected by ELISA. ( I ) Alterations of α-synuclein, Nrf2, and pyroptosis marker proteins in the cellular PD model. Protein abundance in cultured PC12 cells was analyzed by Western blot. Relative protein expression was calculated based on the gray values. GAPDH was used as an internal standard. **P < 0.01.

Article Snippet: Permeabilization was achieved by incubation with PBS solution containing 0.2% Triton X-100 for 8 min. After washing with PBS, PC12 cell slides were blocked by incubation with 1% bovine serum albumin (BSA) for 35 min, followed by incubation with diluted antibodies recognizing TH (25859-1-AP; Proteintech, Rosemont, IL, USA) for 1 h at room temperature.

Techniques: Microscopy, Quantitative RT-PCR, In Situ, Expressing, Immunocytochemistry, MTS Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Marker, Quantitative Proteomics, Cell Culture, Western Blot

Journal: Journal of Inflammation Research

Article Title: Nrf2 Inhibits the Progression of Parkinson’s Disease by Upregulating AABR07032261.5 to Repress Pyroptosis

doi: 10.2147/JIR.S345895

Figure Lengend Snippet: Nrf2 overexpression suppressed pyroptosis in the cellular PD model. ( A ) Proliferation rates of Nrf2-overexpressing PC12 cells exposed to 6-OHDA. Cell proliferation was assessed by MTS assay. ( B ) Alterations in apoptotic PC12 cells overexpressing Nrf2 following 6-OHDA exposure. Apoptosis was determined by flow cytometry. ( C ) Differential expression of Nrf2 and pyroptosis marker proteins in Nrf2-overexpressing PC12 cells exposed to 6-OHDA. Protein levels in PC12 cells were analyzed by Western blotting. ( D ) Relative protein expression was counted in Western blot. ( E and F ) Changes of IL-1β and LDH release in the cellular PD model with Nrf2 overexpression. Marker protein contents were detected by Western blotting. *P < 0.05; **P < 0.01.

Article Snippet: Permeabilization was achieved by incubation with PBS solution containing 0.2% Triton X-100 for 8 min. After washing with PBS, PC12 cell slides were blocked by incubation with 1% bovine serum albumin (BSA) for 35 min, followed by incubation with diluted antibodies recognizing TH (25859-1-AP; Proteintech, Rosemont, IL, USA) for 1 h at room temperature.

Techniques: Over Expression, MTS Assay, Flow Cytometry, Quantitative Proteomics, Marker, Western Blot, Expressing

Journal: The Journal of biological chemistry

Article Title: Nerve growth factor-stimulated B-Raf catalytic activity is refractory to inhibition by cAMP-dependent protein kinase.

doi: 10.1074/jbc.274.19.13193

Figure Lengend Snippet: FIG. 3. The catalytic activity of B-Rafcat is inhibited by co- transfection of PKAcat in PC12 cells. A, a plasmid encoding B- Rafcat-GST was expressed in PC12 cells in the absence (2PKA) or presence (1PKA) of co-transfected PKAcat. MEK kinase activity of the glutathione-Sepharose-purified proteins was determined as described in Fig. 2. An anti-B-Raf immunoblot analysis of the samples used for the MEK kinase assay indicates that an equal amount of B-Rafcat-GST fusion protein was present in each reaction. B, a histogram summariz- ing the inhibitory effect of PKAcat on B-Rafcat activity in transfected PC12 cells from four independent experiments. The error bar indicates the standard deviation of the mean.

Article Snippet: PC12 B-Raf was immunopurified from NGF-stimulated PC12 cells by incubating 100 mg of total cell lysate protein with 2 mg of anti-B-Raf IgG (Santa Cruz Biotechnology) and 30 ml of a 1:1 slurry of protein G Plus-Agarose (Santa Cruz Biotechnology) at 4 °C for 1 h. The immune complex was washed as described above.

Techniques: Activity Assay, Cotransfection, Plasmid Preparation, Transfection, Purification, Western Blot, Kinase Assay, Standard Deviation

Journal: The Journal of biological chemistry

Article Title: Nerve growth factor-stimulated B-Raf catalytic activity is refractory to inhibition by cAMP-dependent protein kinase.

doi: 10.1074/jbc.274.19.13193

Figure Lengend Snippet: FIG. 4. Activated B-Raf is not inhibited by PKA in vitro. A, activated B-Raf was immunoprecipitated from NGF-stimulated PC12 cells and incubated in the absence (2PKA) or presence (1PKA) of PKA in vitro. Raf-1 was isolated from immature Xenopus oocytes, activated with purified PKC, and subsequently treated in the presence or absence of PKA. MEK kinase activity of the B-Raf and Raf-1 proteins was measured as described in the legend to Fig. 2. In this experiment, B-Raf was activated by NGF to levels 4-fold higher than the activity of B-Raf in unstimulated PC12 cells. Raf-1 was activated by PKC 12.9-fold higher than the level of Raf-1 in immature Xenopus oocytes. KN-MEK, kinase-negative MEK. B, histograms summarizing the inhibitory effect of PKA on NGF-stimulated B-Raf activity and PKC-stimulated Raf-1 activity from three independent experiments. The error bars indicate the standard errors of the mean.

Article Snippet: PC12 B-Raf was immunopurified from NGF-stimulated PC12 cells by incubating 100 mg of total cell lysate protein with 2 mg of anti-B-Raf IgG (Santa Cruz Biotechnology) and 30 ml of a 1:1 slurry of protein G Plus-Agarose (Santa Cruz Biotechnology) at 4 °C for 1 h. The immune complex was washed as described above.

Techniques: In Vitro, Immunoprecipitation, Incubation, Isolation, Purification, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Nerve growth factor-stimulated B-Raf catalytic activity is refractory to inhibition by cAMP-dependent protein kinase.

doi: 10.1074/jbc.274.19.13193

Figure Lengend Snippet: FIG. 5. Full-length B-Raf is activated by co-expressed PKAcat in PC12 cells. Full-length B-Raf-GST was expressed in PC12 cells in the absence (2PKA) or presence (1PKA) of co-transfected PKAcat. A, B-Raf immunoblot analysis of glutathione-Sepharose affinity-purified lysates performed in the absence of bacterially expressed kinase-nega- tive MEK. B, MEK kinase activity of glutathione-Sepharose-purified proteins was measured as described in the legend to Fig. 2. The histo- gram summarizes the stimulatory effect of PKAcat on B-Raf-GST from three independent experiments. The error bar indicates the standard error of the mean.

Article Snippet: PC12 B-Raf was immunopurified from NGF-stimulated PC12 cells by incubating 100 mg of total cell lysate protein with 2 mg of anti-B-Raf IgG (Santa Cruz Biotechnology) and 30 ml of a 1:1 slurry of protein G Plus-Agarose (Santa Cruz Biotechnology) at 4 °C for 1 h. The immune complex was washed as described above.

Techniques: Transfection, Western Blot, Affinity Purification, Activity Assay, Purification