Cell culture and treatment
SH-SY5Y human neuroblastoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were grown and maintained as mentioned below. D-MEM (dulbecco’s modified eagle’s medium)/ F-12 supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin were purchased from GIBCO (Thermo Fisher Scientific Inc., Waltham, MA, USA). The condition of cell culture is under a 5% CO2 atmosphere at 37°C. Cells were seeded on a six-well plastic plate at 4.0 x 105 cells/well, and were pre-incubated for 24 h. The cells were harvested 24 h after the subjected to analysis as mentioned below. Cells were incubated in medium without fetal bovine serum containing an appropriate concentration of CdCl2 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 24 h. Salubrinal (Merck Millipore, Burlington, MA, USA) and bafilomycin A1 (Adipogen Corp., San Diego, CA, USA) were dissolved in dimethyl sulfoxide (DMSO). Cells were incubated in serum-free medium containing DMSO (0.1%) and 10 µM salubrinal for 2 h and then treated with 0 to 2.0 µM CdCl2 and 10 µM salubrinal for an additional 24 h.
Cytotoxicity and viabilities were evaluated by using a WST-8 assay (Nacalai Tesque, Kyoto, Japan), which is a modification of the methods for a mitochondrial function of redox potential. A total of 10 µl of 5 mM WST-8 was added to each well of a 96-well plastic culture plate. In each well, the absorbance of redox chemical form of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was determined at 450 nm as measurement wavelength and at 655 nm as a reference wavelength according to the manufacturer’s instructions.
After incubation with CdCl2 and/or salubrinal in medium for 24 h, cells were washed with phosphate-buffered saline (PBS) and lysed by Laemmli sample buffer (BIO-RAD Laboratories, Hercules, CA, USA) supplemented with 5% 2-mercaptoethanol (Nacalai Tesque). The experiments of electrophoresis and electrotransfer were reported previously . The transferred membranes were incubated overnight with primary antibodies against eIF2α (#9722), phospho-eIF2α (Ser51) (#3597), LAMP-1 (#9091), TFEB (#4240), SQSTM1/p62 (#8025), LC3B (#3868) (Cell Signaling Technology, Inc., Beverly, MA, USA), ATF4 (sc-390063), GRP78 (sc-13539), and actin (sc-1616) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) respectively. The membrane was then incubated with the secondary antibodies, namely, donkey anti-goat IgG-HRP (sc-2020) (Santa Cruz Biotechnology, Inc.) against for sc-1616, goat anti-rat IgG-HRP (sc-2006) (Santa Cruz Biotechnology, Inc.) against for sc-13539, anti-rabbit IgG-HRP-linked antibodies (#7074S) (Cell Signaling Technology, Inc.) against for #9722, #3597, #9091, #4240, #8025, #3868 and sc-390063, in TBST containing 5% skimmed milk powder (Nacalai Tesque), and washed three times with TBST. The detection of blots from membrane was detected by chemiluminescent reagents (20X LumiGLO® Reagent and 20X Peroxide, Cell Signaling Technology, Inc.). The intensities of individual bands on the developed films (Amersham Hyperfilm™ ECL, cytiva, Shinjuku, Tokyo, Japan) were quantified using image processing programmed software (ImageJ 1.42, National Institutes of Health, Bethesda, MD, USA), and normalized to the intensity of actin.
Quantitative real-time PCR
Total RNA was extracted and isolated by RNeasy® Plus Mini kit (Qiagen, Venlo, Netherlands) according to the protocol provided by instruction. Aliquots of total RNA (1.0 µg) were reverse-transcribed into cDNA by a PrimeScript™ 1st strand cDNA Synthesis kit (Takara Bio Inc., Kusatsu, Shiga, Japan) according to the protocol provided by instruction. Reverse transcription reaction of cDNA at 42°C for 60 min, denaturation with reverse transcriptase at 95°C for 5 min. Quantitative real-time PCR was performed with a PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific Inc.). Thermal cycler program for 40 cycle was following below. Denaturation of cDNA at 95°C for 3 s. Annealing and extension at 60°C for 30 s. The sequences of gene-specific primers as follows: CHOP, 5’-TGGAAGCCTGGTATGAGGAC-3’ (forward) and 5’-AGTCAGCCAAGCCAGAGAAG-3’ (reverse); GAPDH, 5’-AATCCCATCACCATCTTCCA-3’ (forward) and 5’-TGGACTCCACGACGTACTCA-3’ (reverse). The expression level of CHOP mRNA was normalized to that of GAPDH mRNA. Fluorescence intensity of the amplified PCR products was determined by StepOne™ Real-Time PCR System (Thermo Fisher Scientific Inc.).
Fluorescence imaging of lysosomal pH and autophagosomal formation
LysoTracker® Blue DND-22, a specific imaging fluorescence probe for lysosomal pH, was purchased by Life Technologies Japan Ltd. (Shibaura, Tokyo, Japan). Cyto-ID®/Hoechst® 33342, a specific imaging fluorescence probe for autophagosome with cell nucleus, was purchased by Enzo Life Sciences Inc. (Farmingdale, NY, USA). SH-SY5Y cells were seeded on the glass bottom dish (Matsunami Glass, Ind., Ltd., Wada, Osaka, Japan) for 24 h according to the same maintain protocol as that mentioned above. Cells were incubated under a 5% CO2 atmosphere at 37°C with LysoTracker® Blue DND-22 and Cyto-ID®/Hoechst® 33342 for 30 min, respectively. After 30 min, adherent cells on the glass bottom dish were washed with PBS, and then fluorescence images in living cells were observed by a confocal laser scanning microscope (LSM-710, Carl Zeiss, Jena, Thuringia, Germany).
Results are presented as mean ± standard error of the mean (SEM) of three independent samples. The statistical significance of differences between two groups was calculated by the Student's t-test. A P value of less than 0.05 was considered indicative of a statistically significant.