Cell culture and plasmid transfections
HeLa (from ATCC) cells were grown in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1x antibiotic/antimycotic (Sigma). In some experiments, HeLa cells were transfected with plasmids (1 ug per 35 mm dish) for expression of JNK KTR-mRuby2, STMN-FLAG, STMN-TetraA-FLAG, and/or STMN-TetraE-FLAG using XtremeGene HP DNA Transfection Reagent (version 1.0; Roche Diagnostics) according to the manufacturer’s protocol. Cells were transfected approximately 4 h following siRNA transfection and assessed approximately 24–72 h following initial transfection. In experiments requiring plasmid expression of the mRuby2-tagged JNK KTR (1 ug per 35 mm dish), HeLa cells were transferred to phenol-free DMEM supplemented with 10% FBS and 1x antibiotic/antimycotic after two rinses in PBS approximately 4 h post-transfection. All plasmids were constructed by total gene synthesis, custom cloning, and verified by sequencing (performed by Genewiz).
RNA interference and transient transfection
HeLa cells were grown in 35 mm dishes and transfected with siRNAs 1–2 days after plating using GeneSilencer (Genlantis) according to the manufacturer’s protocol. Cells were serum-starved from the time of transfection to 4 h post-transfection. siRNA oligonucleotides were purchased from GE Dharmacon and included: STMN1, 5’- CGUUUGCGAGAGAAGGAUADTDT -3’; 5’UTR targeted-STMN1, 5’- CCCAGUUGAUUGUGCAGAAUU -3’; SMARTpool targeting JNK2. SiGenome non-targeting siRNA sequence was used as control siRNA sequences for these experiments.
Drugs and reagents
Chemical inhibitor to JNK (JNK-IN-8) was purchased from Selleckchem. Activator of JNK, anisomycin, was purchased from Sigma. Drugs were prepared as stocks in DMSO and stored at -20°C.
Indirect immunofluorescence and microscopy
HeLa cells were grown on glass coverslips and treated as described above. Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences)/ 20% glycerol in PEM (100 mM PIPES, 1 mM MgSO4, 2 mM ethylene glycol tetraacetic acid, pH 6.9) for 15 min at room temperature. Cells were permeabilized with methanol at -20°C for 5 min. Fixed cells were incubated with blocking reagent (10% FBS in PBS) for 30 min at 37°C. Cells were then incubated with primary antibody for 45 min at 37°C. Cells were next washed with PBS and incubated with secondary antibody for 45 min at 37°C. Antibodies used were: anti-FLAG (1:100; Sigma cat#F7425) and goat anti-rabbit Alexa Fluor 568 (1:50; Invitrogen cat#A-11036). Coverslips were then washed with PBS and mounted on slides with Vectashield (Vector Laboratories). Cells were imaged by wide-field microscopy using a 40x/1.4 numerical aperture planapo objective on an inverted microscope (TE300; Nikon).
Live cell imaging
To follow cell fates over several days, HeLa cells were plated on glass bottom dishes (CellVis) and imaged using a Nikon Biostation IM. Cells were imaged with phase-contrast and fluorescence (eg., 510–560, em:590; G-2A part#96306, Nikon) using a 20x objective, and images were collected at 5 min intervals for 72 h. Cell fates were tracked from image series; cell death was determined based on changes in cell morphology characterized by cell retraction and blebbing of the plasma membrane.
Soluble cell extracts were prepared and protein concentrations were measured by Bradford assay. Lysates were diluted in PAGE sample buffer; 10–20 ug total protein per lane was typically loaded and resolved in 10% polyacrylamide gels and transferred to PVDF membranes (Trans-Blot Turbo Mini; Bio Rad). Membranes were blocked in 5% milk or 5% BSA (for membranes probed with anti-JNK antibody) in Tris-buffered saline with 0.1% Tween and then probed with primary antibodies, including anti-STMN1 (1:2000; EMD Millipore cat#AB2967), anti-JNK (1:1000; Cell Signaling cat#9252), and anti-FLAG (1:1000; Sigma cat#F7425). Membranes were then probed with horseradish peroxidase-linked secondary antibodies, including anti-mouse (1:10,000; Sigma cat#A4416) or anti-rabbit (1:5000; Sigma cat#A0545) IgG. Immunoreactive bands were developed using enhanced chemiluminescence (GE Amersham). Membranes were reprobed with anti-α-tubulin (1:10,000; Sigma cat#T5168) or anti-GAPDH (1:1000; Abcam cat#ab9483) as a loading control.
Cell viability measurements
Cells were allowed to grow for 2 days following treatment with the JNK inhibitor, JNK-IN-8. On the day of measurement, cells were trypsinized and resuspended in PBS with 0.2% Trypan Blue and counted using a hemocytometer to determine viability via Trypan Blue exclusion. Alternatively, cell viability was assessed by the CellTox Green Cytotoxicity Assay (Promega) by the addition of CellTox Green Dye (prepared according to manufacturer’s protocol) approximately 8 h following initial transfection. Cells were allowed to grow for up to 3 days following transfection with siRNA and/or plasmids. Wide-field phase contrast and fluorescence images were acquired at 24 h time points following initial transfection using a 20x/1.4 numerical aperture planapo objective as described above. Approximately 200 cells were counted per condition per experiment. Each experiment was repeated at least three times.
Images were acquired and stored as 12-bit files using MetaMorph or NIS Elements AR 3.2 (Nikon). JNK activity was assessed by the measurement of the integrated intensity ratio of cytoplasmic to nuclear regions (10×10 px) representing active and inactive JNK, respectively. Cytoplasmic regions were measured just outside the nuclear envelope to ensure measurements of consistent cellular volumes.
Statistical analysis of integrated fluorescence intensity and cell viability were performed using unpaired t-tests with either GraphPad Software or Kaleidagraph.