Cell culture, reagents and antibodies
Human embryonic kidney 293 T (HEK293T) and differentiated human neuroblastoma SH-SY5Y cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA). Cells were maintained in 90 mm non-pyrogenic tissue culture dishes with 15 mL growth medium at 37°C in a humidified atmosphere with 95% air and 5% CO2. Heat-inactivated fetal bovine serum (FBS), penicillin-streptomycin antibiotic, Dulbecco’s Modified Eagle Medium (DMEM) containing 4500 mg/L D-glucose and L-glutamine, without sodium pyruvate, OptiMEM reduced serum medium and RPMI-1640 medium, both containing L-Glutamine, were all purchased from Gibco (Grand Island, NY). Mouse monoclonal anti-GAPDH antibody (6C5), goat anti rabbit secondary antibody and goat anti mouse secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rabbit polyclonal anti-calpain-5 antibody was purchased from GeneTex (Irvine, CA); rabbit anti-myc tag antibody was purchased from Abcam (Cambridge, MA).
HEK293T adherent cells were grown in DMEM medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) (complete DMEM medium). The HEK293T cells were transformed into cells that stably express a disease mutant of calpain-5 (p.R243L). SH-SY5Y adherent cells were cultured in a 1:1 mix of RPMI 1640 and F-12 media containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL).
Short hairpin RNA plasmids
Four unique short hairpin RNA (shRNA) sequences that target human CAPN5 and one nontargeting sequence (negative control) were purchased from Qiagen (Catalog# KM40517; Frederick, MD). Each plasmid vector expressed a shRNA under the control of a U1 promoter and contained a green fluorescent protein (GFP) reporter gene. The shRNA nucleotide sequences were: clone one: 5’ - GTACAATGTGAAAGGCATCTT - 3’; clone two: 5’ - CAACCACAAGGACACCTTCTT - 3’; clone three: 5’ - GACTTCACGGGTGGTGTTTCT - 3’; clone four: 5’ - GTCAGAGAAGTTGGTGTTTCT - 3’; negative control: 5’ - GGAATCTCA TTCGATGCATAC - 3’.
Stable cell line creation
HEK293T cells were co-transfected with a pUS2 vector that encodes the puromycin-resistant gene and myc-tagged CAPN5 wild type (WT) or p.R243L (disease-allele) in pCMV6 entry vector (Origene Technologies Inc.; Rockville, MD). The pUS2 vector was modified from pCS2 entry vector by replacing the CMV promoter with UbC promoter. In wells of separate 12-well plates, HEK293T cells at 70% confluence in 600 μL complete culture medium were co-transfected with 900 ng of CAPN5 and 100 ng of puromycin plasmids using the lipid reagent Turbofectin (OriGene Technologies, Inc.; Rockville, MD) per manufacturers’ recommendation. After 24-hours incubation at 37°C, the cells were trypsinized and resuspended in 1 mL selection medium (comprising of DMEM with 10% FBS, 1% L-glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL) and 1.5 ug/mL puromycin). Briefly, 500 μL of the cell suspension was added into 1.5 mL of fresh selection media per well in new 12-well plate and incubated for 24 hours. The transfected cells were trypsinized, serially diluted and re-plated in separate 10 cm dishes with 10 mL selection media. The cell cultures were maintained for 12–14 days until the colonies attained diameters between 2–4 mm. Finally, sterile forceps and cloning rings dipped in silicone grease were used to select twenty-four distinct colonies by trypsinization, followed by incubation at 37°C for 15 minutes, and then resuspended in 300 μL of selection medium. The cells of each colony were cultured in a new 24-well plate containing 700 μL selection medium per well. Once the cells in each well attained 50-70% confluence, they were seeded and cultured in a new 24-well plate for screening and also in four 6-well plates for maintenance. The cells were screened for calpain-5 using immunoblot. Cells were lysed on ice for 10 minutes with HEPES buffer [50 mM HEPES (pH 7.0), 150 mM NaCl, 2 mM EGTA, 2 mM MgCl2, 1% Triton X-100, protease inhibitor cocktail (Roche; Indianapolis, IN)]. Twenty-two cell lysates were collected after centrifugation at 20,000xg for 10 minutes. Equal amounts of protein were blotted and probed with HRP-conjugated rabbit polyclonal antibody to Myc tag (1:5000), mouse monoclonal anti GAPDH (1:400), and secondary goat anti rabbit IgG (1:5000).
Gene suppression by shRNA plasmid transfection
HEK293T-CAPN5-p.R243L cells were transfected using Attractene Transfection Reagent (Qiagen). In each well of 6-well plates, 5.0 × 105 HEK293T-CAPN5-p.R243L cells were grown in 2 mL complete DMEM medium to 50 - 70% confluence. Growth medium was replaced with 2 mL of freshly prepared Opti-MEM medium containing 5% FBS, penicillin (100U/mL) and streptomycin (100 μg/mL) (complete Opti-MEM medium). Cells were then transfected with 3.0 μg shRNA plasmid using 4.5 μL Attractene Reagent. At 6 hours post-transfection, growth medium was replaced with fresh complete Opti-MEM medium. Cells were incubated for 48 hours.
The optimization protocols (varying pulse voltage, pulse width and pulse, as recommended by manufacturer) for transfecting adherent and suspension cells with Neon™electroporation system (Invitrogen; Carlsbad, CA) were performed with SH-SY5Y cells. Briefly, 1.0 × 106 SH-SY5Y cells in 100 μL resuspension buffer were electroporated with 12.0 μg shRNA plasmids into 150 × 20 mm tissue culture dishes containing 15 mL Opti-MEM medium containing 5% FBS. After optimization, transfection of SY-SY5Y cells were performed in triplicate for each shRNA plasmid and incubated for 72 hours.
Lactose dehydrogenase (LDH) assay
Toxicity of CAPN5 gene suppression on transfected cells was measured by LDH release assay (Cytotoxicity Detection Kit, Roche Applied Science, Mannheim, Germany). LDH activity in the culture medium indicated LDH release from cells. Culture medium was collected from transfected cells after 48 hours incubation. For low cytotoxicity control, cells were cultured without plasmid transfection. For high cytotoxicity control, cells were treated with 1% Triton X-100 in assay medium. Culture media from all conditions was centrifuged at 250xg to remove cell contaminants. The background controls were prepared by mixing transfection medium with Attractene or electroporation buffer. An aliquot (100 μL) of the supernatants and the background control medium were collected into a 96-well plate and incubated with 100 μL of freshly prepared LDH assay reaction mixture. Incubation was performed in the dark for 30 minutes at room temperature. Absorbance was read at 490 nm using an Infinite M200 PRO microplate reader (Tecan Systems Inc.; San Jose, CA). Cytotoxicity was calculated using the equation: Cytotoxicity (%) = [(test sample – low control)/(high control ‒ low control)] × 100.
Calpain-5 levels were measured by immunoblot densitometry. Cells were rinsed with 1X PBS and homogenized in RIPA extraction buffer (1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10 mM Tris–HCl, pH 8.0, 0.15 M NaCl, 0.047 M NaF and 5.0 mM EDTA) containing protease inhibitor cocktail from Roche, Indianapolis, IN. Homogenates were sonicated on ice for 10 seconds, two times and then centrifuged at 1000xg for 10 minutes at 4°C. Total cellular protein was measured using the modified Lowry method (DC Protein Assay; Bio-Rad), and equal amounts of protein were used. Samples were denatured at 85°C for 5 minutes in LDS-sample buffer with reducing agent (Invitrogen; Carlsbad, CA), and electrophoretically separated using 4-12% NuPAGE Bis-Tris gels. HEK293T-R243L proteins were transferred onto PVDF membranes while SH- SY5Y proteins were transferred onto nitrocellulose membrane. Membranes were blocked at 4°C with 5.0% non-fat dry milk in 1x TBS 0.1% tween-20. The proteins were immunostained with rabbit polyclonal antibody to calpain-5 (GeneTex; 1:1000) and mouse monoclonal antibody to GAPDH (Santa Cruz; 1:400). Goat anti-rabbit-HRP and goat anti-mouse-HRP antibodies (both were used at 1:10,000 dilution ratios) were used as the secondary antibody. The immunoreactive bands were detected with Supersignal West Dura Extended Duration Substrate (Thermo Scientific; Rockford, IL). Visualization and quantification of band intensities were performed with MYECL imager and software (Thermo Scientific). We quantified Calpain-5 expression in three independent experiments by normalizing the calpain-5 to GAPDH and calculating the mean expression in experimental groups (shRNA clones) relative to the control group (negative control shRNA).
Quantitative real time PCR (qPCR)
CAPN5 mRNA levels were measured by qPCR. RNA was isolated from the cells using Qiagen RNeasy mini kit. cDNA was prepared from 1 μg of total RNA using High Capacity cDNA Reverse Transcription kit with RNAse Inhibitor (Applied Biosystems Inc., Foster City, CA). Total RNA and cDNA concentrations were measured at 260 and 280 nm using Nanodrop 2000c spectrophotometer (Thermo Scientific). Quantitative PCR was performed with 100 ng of first-strand cDNA in 25 μL reaction mix using Power SYBR Green PCR Master Mix (Applied Biosystems). The primers used were: CAPN5 forward, 5’-TGATCAACACATCCCACCTG-3’; reverse, 5’-ACGCTGCGTGAGTTGATGTA-3’; GAPDH forward, 5’-CGAGATCCCTCCAAAATCAA-3’; reverse, 5’-GTCTTCTGGGTGGCAGTGAT-3’; Beta-actin forward, 5’-ACCCTGAAGTACCCCATTG-3’; reverse, 5’- TACGACCAGAGGCATACAG-3’. Data were normalized to GAPDH mRNA for HEK293T-R243L cells and Beta actin mRNA levels for SH-SY5Y cells. The qPCR cycles were: 95°C for 10 min; (95°C for15 s; 60°C for 1 min) × 40 cycles. Comparative threshold cycle (C
) method was used to measure relative gene expression in response to shRNA plasmid treatments. All data analyses were conducted using Prism 6.0 software (GraphPad; La Jolla, CA).