Glass bottom plates

Cells were treated with different concentrations of etoposide (Sigma Aldrich) diluted from an initial stock of 10mM in DMSO or with 100μM CK666 (Millipore) from a 100mM stock. Equivalent volumes of DMSO were used as controls. For RNA-seq experiments, the control condition consisted of regular media, while the treated condition consisted of media in which the etoposide powder was dissolved directly to 5μM. For JMY rescue experiments, to be able to detect cleaved caspase-3 in the JMY^KO cell line, etoposide was dissolved to 10μM. For RNAi experiments, cells were grown in 6-well plates for 24h, transfected with 40nM siRNAs (S2 Table) using RNAiMAX (Invitrogen), incubated in growth media for 24h, reseeded into 6-well plates and 24-well glass-bottom plates (MatTek), and incubated for an additional 24h. Cells cultured in 6-well plates were collected and processed for immunoblotting or RT-PCR, and cells cultured in 24-well plates were used in live fluorescence microscopy assays.

The pPD49.83 plasmid was used to generate two cloning vectors for BiFC analysis [24 (link)]. each construct containing: the heat shock promoter, hsp-16.41, a Myc or hemaglutinin tag to detect BiFC fusion proteins, a multiple cloning site to subclone the gene of interest, a linker sequence, and the N-terminal Venus fragment truncated at residue 173 (VN173) or the C-terminal Venus fragment from residue 155 (VC155) -a generous gift from Dr Chang-Deng Hu, Purdue University, USA. Full length murine NMDAR, σ1R, various GPCRs, HINT1, nNOS and RGSZ2 were all subcloned in frame into pCE-BiFC-VN173 or pCE-BiFC-VC155 plasmids using standard cloning strategies. The fidelity of the constructs was verified by sequencing. Chinese hamster ovary (CHO) cells were transfected (0.3 μg plasmids) using Lipofectamine 2000 (Invitrogen, Madrid, Spain) and incubated for 24 h prior to testing for transgenic expression. Samples were visualized on glass bottom plates (MatTek Co, Ashland, MA, USA) using a Leica DMIII 6000 CS confocal fluorescence microscope (Leica, Barcelona, Spain) equipped with a TCS SP5 scanning laser.

As in our prior studies, we utilized NB2a/d1 mouse neuroblastoma cells, which elaborate axonal neurites containing most if not all axonal constituents and translocate them into and along neurites via kinesin and dynein. Transfection allowed for expression of multiple forms of tau in the presence of active forms of tau kinases (Dubey et al., 2008 (link)). NB2a/d1 cells were maintained in DMEM supplemented with 2 mM glutamine, 10% FBS (Atlanta Biologicals) and antibiotics. Differentiation and elaboration of axonal neurites was induced by treatment with 1 mM dibutyryl-cyclic adenosine monophosphate (dbcAMP).
Cells were cultured on glass-bottom plates (MatTek, Ashland, MA 01721, USA) for live-cell imaging or acid-washed glass coverslips in six-well plates for immunofluorescence analyses. Plates and slides were treated with poly-d-lysine (MW>300,000) for 2 h at room temperature, washed five times with autoclaved double deionized water, and dried overnight under UV exposure. They were then treated with 50 µM laminin for 30 min at 37°C, washed twice with autoclaved double deionized water, and dried for 2 h under UV exposure. For immunoblot analyses, cells were cultured on plastic, tissue culture treated 10 cm plates (CELLTREAT, Pepperell, MA 01463, USA).

H1299 cells were grown to ∼60% confluency on glass-bottom plates (Mat-tek) and then transfected with a CFP-RAD18-WT expression plasmid. Twenty hours after transfection, cells were ultraviolet irradiated (20 J m⁻²) or sham treated and fixed 6 h later for staining with anti-MAGE-A4 and fixed-cell imaging on a Zeiss 710 confocal microscope, in the UNC Microscopy Services Laboratory core facility, as described previously30 (link).