Generation and characterization of p75NTR-native and p75NTR-deficient PC12 cell lines have been described previously [8
]. The p75NTR-deficient PC12 cell line was also transfected with an empty construct or a full-length p75NTR expression construct as we have described [12
]. ATCC PC12 cells were obtained from the American Type Culture Collection (Rockville, MD). Murine primary cortical neurons were the kind gift of Rita Giuliani (Center for Neural Development and Disease, University of Rochester, Rochester, NY).
All PC12-derived cell lines were grown in DMEM (1x) with 4.5 g/L glucose and L-glutamine and without sodium pyruvate (Cellgro, Manassas, VA), supplemented with 10% w/v donor horse serum (Cellgro), 5% w/v fetal bovine serum (Cellgro) and 1% w/v penicillin-streptomycin (Cellgro). This medium was used for all subsequent cell culture experiments. All lines were plated at 10 000 cells/96-well plate or 10 cm plate and were grown in 5% CO2 at 37°C.
Cell pellets containing 3 × 106 cells were used for RNA isolation with the Qiagen RNeasy Mini Kit (Valencia, CA) with Step 3a used for lysis of the homogenate by Qiagen QIAshredder. Genomic DNA was minimized by using DNase I (Invitrogen, Carlsbad, CA) as instructed by the manufacturer. The reverse transcriptase reaction was performed using the SuperScript III First-Strand Synthesis System (Invitrogen) with or without reverse transcriptase in the reaction. Primers specific to necdin (forward: gctggtgcagaaggcgcacga, reverse: gctggtacttcaggtaattc) were used to amplify a 455bp fragment by polymerase chain reaction (Tm= 58, 35 cycles).
Necdin siRNA (Santa Cruz Biotechnology, Santa Cruz, CA), p75NTR siRNA (Integrated DNA Technologies, Coralville, IA) and control scrambled siRNA (Santa Cruz Biotechnology) were thawed in a tissue culture hood, diluted into 50 µL medium, and incubated at room temperature for 5 min. Lipofectamine 2000 (Invitrogen) was added to a total of 50 µL of culture medium as per the manufacturer’s instructions (0.25 µL Lipofectamine/well of a 96-well plate; 30 µL Lipofectamine/10 cm plate) and allowed to sit for 5 min at room temperature in a tissue culture hood. The siRNA and Lipofectamine solutions were then combined, mixed, and allowed to incubate at room temperature together for 20 min. This mixture was then diluted and added to the cells to a final working siRNA concentration of 20 µM (10 µM each when two siRNAs were used simultaneously). The siRNA treatment was performed overnight (18–24 h) at 37°C, at which time some cells were harvested for protein (10 cm plates) and the rest (96-well plates) were treated with various concentrations of 6-OHDA.
Custom fluorescently-tagged p75-Cy5, necdin-Cy3 and control scrambled-Cy3 siRNA were purchased from Qiagen. All siRNAs were used as described above at a final concentration of 20 µM overnight at 37°C. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 1 hour at room temperature co-treated with 10 µg/mL Hoechst 33342 stain (Fluka). A standard mercury lamp was used with appropriate filters to capture an image with a gain of 2x (unless otherwise stated). The intensity (time of exposure) was kept constant for each set of samples. The overlap between the Hoechst 33342 stain and the released fluorescent tags was displayed in yellow and counted vs cells with only the nuclei stained blue expressed as a percent of the total.
JNK phosphorylation inhibitor treatment
Following siRNA treatment, the JNK phosphorylation inhibitor SP600125 (BIOMOL, Plymouth Meeting, PA) was added to the cells at a concentration of 10 µM for 1 h at 37°C immediately prior to 6-OHDA treatment.
The 96-well plates of PC12 cells treated with siRNA were subsequently treated with 0–500 µM 6-OHDA (Sigma-Aldrich, St. Louis, MO). Briefly, 10 mg of 6-OHDA was diluted into 200 µL of saline containing 100 µg/mL L-ascorbate (Sigma-Aldrich) for stabilization of 6-OHDA. This solution was then diluted into culture medium to a final working concentration of between 0 and 500 µM, added to the siRNA-treated cells on 96-well plates, and incubated at 37°C for 24 h.
Alamar blue assay
The metabolic viability of PC12 cells treated with siRNA and then 6-OHDA was determined using the Alamar blue assay (Invitrogen Biosource, Carlsbad, CA). Alamar blue dye was diluted to 10% v/v in cell culture medium and cells were treated for 1 h at 37°C, at which time the fluorescence in each well was quantified with a Molecular Devices SpectraMax M5 plate reader at an excitation wavelength of 530 nM and an emission wavelength of 590 nM. Six measurements were taken per well and the average fluorescence was calculated.
Trypan blue exclusion assay
The Trypan blue exclusion assay was used as a second measurement of cell viability following siRNA and 6-OHDA treatment. Trypan blue (0.4%; Invitrogen Gibco, Carlsbad, CA) was diluted to 50% v/v in cell culture medium and added to the PC12 cells. The stained cells were subsequently counted manually with a minimum of 4 repeats each.
PC12 cells were washed with Hank’s Balanced Salt Solution (Cellgro) and then trypsinized (Cellgro) for 5 min at 37°C. The cells were then pipetted into a conical tube, centrifuged at 250 × g for 3 min and the supernatant was removed. The pellet was resuspended in 100 µL RIPA buffer [10 mM Tris, pH 8 (MP Biomedicals, Solon, OH); 150 mM NaCl (EMD Biosciences, Madison, WI); 0.1% v/v Nonidet P-40 (Sigma-Aldrich); 0.5% w/v sodium deoxycholate (Sigma-Aldrich); 0.1% w/v sodium dodecyl sulfate (Bio-Rad, Hercules, CA)] supplemented with 4 µg/mL aprotinin (Sigma-Aldrich), 1mM phenylmethanesulphonylfluoride (G-Biosciences, Maryland Heights, MO) and 1 mM sodium orthovanadate (Sigma-Aldrich). This solution was then sonicated through 10 cycles with a Branson Sonifier 450 on a constant output setting of 2 and incubated at 4°C for 30 min, vortexing every 10 min. The resulting mixture was centrifuged at 10 000 × g for 15 min to pellet the protein and the supernatant was removed. The protein concentration was measured with a protein assay kit (Bio-Rad).
Gradient separating and stacking acrylamide gels were poured [7.5–12% gradient separating gel: 7.5%–12% v/v acrylamide/bis (Bio-Rad); 375 mM Tris, pH 8.8 (MP Biomedicals, Solon, OH); 0.05% w/v ammonium persulfate (VWR, Batavia, IL); 0.05% v/v TEMED (Bio-Rad); 4% stacking gel: 4% v/v acrylamide/bis; 375 mM Tris, pH 6.8; 0.05% w/v ammonium persulfate; 0.1% TEMED]. Protein samples were denatured and loaded onto the gels at 50–150 µg protein/lane and were run at 60 V for 30 min and then 90 V for 2 h. The gel was then transferred onto a nitrocellulose membrane at 90 volts for 1.5 h on ice. The membrane was then blocked for 1 h at room temperature in blocking buffer [5% dry milk (Bio-Rad); 1x PBS (Sigma-Aldrich)] on an orbital shaker. Primary antibodies, all purchased from Santa Cruz, except for anti-p75NTR (Promega, Madison, WI), were added at dilutions ranging from 1:200–1:1000 and the membranes were allowed to incubate at 4°C overnight on an orbital shaker. Membranes were washed twice for 10 min with washing buffer [0.1% v/v Tween-20 (Fisher Scientific, Hampton, NH) diluted in 1x PBS] while shaking. The secondary antibody was added in a ratio of 1:5000 in blocking buffer and the membrane and overlying solution were incubated at room temperature for 1 h on an orbital shaker. The membranes were washed with washing buffer 4 times for 10 min each at room temperature on an orbital shaker. The membranes were then placed on Saran Wrap™ and a chemiluminescent solution (Santa Cruz Biotechnology) was added. The excess solution was wiped away and the membranes were exposed onto Kodak Biomax film (Kodak, Rochester, NY). The films were then digitally scanned as TIFs.
Western blot band quantification and statistical analysis
Scion Imaging Software was used to quantify the TIF images. Background measurements were subtracted from each band density and the results were normalized to an actin loading control. A univariate ANOVA (2-way) with a post hoc Fisher’s LSD test was used to determine the statistical significance of the difference between pairs of samples at each concentration of 6-OHDA (, , and ), or pairs of Western blot bands (, and ). Statistical significance was assigned to differences with p values of no greater than 0.01. LD50 and IC50 values were calculated by using a non-linear regression analysis (Graphpad Prism 5, Graphpad Software Inc., La Jolla, CA). A Student’s T-test was used to determine the statistical significance between pairs of samples at each LD50/IC50 and for transfection efficiency of siRNA.
The effect of necdin siRNA on metabolic viability following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells
The effect of necdin and p75NTR siRNAs on metabolic viability following 6-OHDA treatment of p75NTR-native PC12 cells
The effect of necdin siRNA on cell membrane integrity following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells
The effect of necdin siRNA and a JNK phosphorylation inhibitor, SP600125, on cell metabolic viability following 6-OHDA treatment of p75NTR-native and p75NTR-deficient PC12 cells
Western blotting and quantification of TrkA signaling effectors in p75NTR-native and p75NTR-deficient cell lines treated with scrambled control siRNA (Scr) or necdin siRNA (Ndn)
Western blotting and quantification of pro- and anti-apoptotic proteins in p75NTR-native and p75NTR-deficient cell lines treated with scrambled control siRNA (Scr) or necdin siRNA (Ndn)
Western blotting and quantification of the p75NTR-native and p75NTR-deficient cell lines treated with scrambled control siRNA (Scr) or necdin siRNA (Ndn) +/− SP600125