4.1 Antibodies and reagents.
Primary antibodies to β-actin (SC-47778, 1:1000), CaMKK-ß (SC-50341, 1:1000), and AMPKα1/2 (SC-25792, 1:1000) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary antibodies to p-CaMKKβ (12818S, 1:500), mTOR (2983S, 1:1000), S6 ribosomal protein (2217S, 1:1000), and p-S6 ribosomal protein (2211S, 1:1000) were from Cell Signaling (Danvers, MA). Anti-p-AMPKα1/2 (11183, 1:1000) and anti-p16 (41296, 1:1000) antibodies were from Signalway (College Park, MD). Anti-LC3B (NB100-2220, 1:500) and anti-p21 (NBP2-29463, 1:500) antibodies were purchased from Novus (St. Louis, MO). Anti-p-mTOR (ab63552) and anti-Ki67 (ab15580) antibodies were from Abcam (Cambridge, UK). Anti-mouse (31430), anti-rabbit (31460), anti-goat (31402), and Alexa Fluor 488 (A11008) immunoglobin G secondary antibodies (1:5000) were purchased from Invitrogen (Carlsbad, CA). The anti-OR2H2 primary antibody (1:1000) was purchased from Antikoerper-online.de (Aachen, Germany). L-Cis diltiazem was from Abcam (Cambridge, UK). SQ22536 and thapsigargin were purchased from Enzo Life Science (Farmingdale, NY) and U73122 was from Sigma-Aldrich (St. Louis, MO). Aldehyde 13–13 was obtained from Henkel (Düsseldorf, Germany). A769662, an AMPK agonist, was purchased from Cayman (Ann Arbor, MI). Rapamycin was obtained from LC Laboratories (Woburn, MA).
4.2 Cell culture.
VK2/E6E7 cells (ATCC, CRL-2616) were purchased from the American Type Culture Collection (ATCC, VA), and cultured in keratinocyte-serum free medium (Gibco-BRL 17005-042), supplemented with 0.1% human recombinant epidermal growth factor, 0.3% bovine pituitary extract, and 0.4 mM calcium chloride as instructed in the manufacturer’s protocol. Culture medium was renewed every 2–3 days. Cells were grown in 37°C with 5% CO2. All compounds except L-Cis diltiazem (Abcam) and aldehyde 13–13 (Henkel) were prediluted in DMSO. L-Cis diltiazem was prediluted in distilled water and aldehyde 13–13 in ethanol. All compounds were diluted 1:1000 in growth medium or Ringer’s solution and applied to VK2/E6E7 cells. All dilutions of aldehyde 13–13 were prepared fresh before use.
4.3 RNA extraction and RT-PCR.
Total cellular RNA was extracted with Total RNA extraction reagent RNAiso Plus (1 mL, Takara Bio, 9109, Japan) with the manufacturer’s instructions. Complementary DNA was synthesized from 1 ug RNA with ReverTra Ace® RT Master Mix kit (Toyobo, FSQ-301, Japan) according to the manufacturer’s instructions. 1 ug of cDNAs were then used as templates for PCR reactions and specific primers were designed by the Primer-BLAST designer program (NCBI, USA). RT-PCR was performed with EmeraldAmp® GT PCR Master Mix reagent according to the manufacturer’s instructions. The RT-PCR products were analyzed by agarose gel electrophoresis (3%) and image analysis were obtained by using a ChemiDoc touch imaging system with the Image Lab 5.2 software (Bio-Rad, Redmond, WA, USA). L32 was used to normalize each data. Oligonucleotide sequences are given in Supplementary Table S1.
4.4 Immunocytochemical staining.
Immunocytochemical staining was performed via immunofluorescence microscopy. VK2/E6E7 cells were seeded on coverslips in a 24-well plate containing 500 µL of cell culture medium and incubated at 37°C in a 5% CO2 atmosphere. When cells reached 80% confluence, medium was removed, and cells were washed with PBS. After fixation with ice-cold acetone, cells were incubated with primary (1:100, anti-OR2H2; Antikorperonline.de) and secondary (1:1000, goat anti rabbit 546; Thermo Fisher) antibodies. Next, cells were incubated with Alexa Fluor phalloidin 488 (1:200, Thermo Fisher) for 45 min at room temperature with gentle shaking. Cells were coated with Immu-Mount solution (Thermo Fisher) and examined under an LSM 510 Meta Confocal Microscope (Carl Zeiss, Jena, Germany) using a 40× oil immersion objective and the Leica Application Suite Software. The specificity of the antibodies used in the study was previously investigated by rho-tagged transfection of Hana3A cell[45].
Calcium imaging and calcium inhibitor treatments
Intracellular calcium concentration changes were monitored using the fluorescent dye Fura-2-acetoxylmethyl ester (Fura-2/AM; Invitrogen). VK2/E6E7 cells were cultured in 35 mm dishes and incubated in growth medium containing 3 µM Fura-2/AM for 30 min at 37°C, protected from light. Next, growth medium was replaced with Ringer’s solution. Aldehyde 13–13 and calcium inhibitors were dissolved in Ringer’s solution (1:1000) and applied directly to the cells. In desensitization experiments, the cells were stimulated repetitively with aldehyde 13–13 (300 µM) for two minutes while control cells were treated with vehicle (ethanol in Ringer's solution) repeatedly for two minutes. In experiments under calcium free conditions, cells were stimulated with aldehyde 13–13 (500 µM) for 3 minutes, then were washed for 3 minutes with Ringer’s solution. Cells were then treated with calcium-free Ringer’s solution for 7 minutes, then added aldehyde 13–13 for 3 minutes. A third application of aldehyde 13–13 was performed for 3 minutes. In experiments with calcium channel inhibitors, cells were stimulated with aldehyde 13–13 (500 µM) for 3 minutes. After two minutes washing with Ringer's solution, the cells were stimulated with the calcium channel inhibitors. Subsequently, aldehyde 13–13 and inhibitors were co-applied for three minutes. A third application was also made under normal conditions after a 2-minute wash-out step. Calcium channel inhibitors were incubated by following conditions: SQ 22536 (10 µM) for 10 minutes; L-cis diltiazem (100 µM) for 3 minutes; U 73122 (5 µM) for 5 minutes; thapsigargin (5 µM) for 25 minutes, respectively. Intracellular calcium flux was observed using a DMI, 6000CS inverse microscope (Leica, Wetzlar, Germany), and calcium-dependent fluorescence changes were detected using a 10-fold fluorescence objective. Fluorescence emission intensities (340 and 380 nm) were detected by a DFX 360FX CCD camera (Leica). Regions of interest (ROIs) were analyzed using Advanced Fluorescence software (LAS AF; Leica).
4.5 Phosphokinase array
The human phosphokinase array kit by RnD systems was applied to investigate the phosphorylation of 43 different kinases under influence of aldehyde 13–13 stimulation for an incubation time of 30 minutes. Before starting the array according to the manufacturer`s protocol cells were cultivated in 75 cm2 culture flasks until a confluence of 90% was achieved. Medium was exchanged with fresh medium containing the diluted aldehyde 13–13 in a concentration of 500 µM. The control was treated with ethanol (< 0.1%). After incubation, medium was aspirated, cells were covered with PBS and manually detached with a cell scraper. The detached cells were then subsequently taken up in the PBS and centrifuged for 10 minutes at 1000 rpm and 4°C. The cell pellets thus obtained were then lysed in RIPA buffer and their protein concentration determined using the Pierce™ BCA protein assay kit. The total amount of protein was 250 µg for the treated and untreated sample. The evaluation of the membrane was performed by the chemiluminescence System Fusion SL 3500-WL. The intensity of the individual array dots was analyzed with the program Image J and a microarray plugin and normalized to the control.
4.6 OR2H2 knockdown.
For mRNA interference of OR2H2, VK2 cells (4 × 105) were seeded in six-well plates and transfected with an siRNA against OR2H2 (Cat No. sc-95450; Santa Cruz Biotechnology, Inc.) using Lipofectamine 2000 according to the manufacturer’s instructions. Briefly, cells were cultured with siRNA and Lipofectamine 2000 in Opti-MEM reduced-serum medium (Cat No. 32985070; Gibco). After incubation for 5 h at 37°C in a CO2 atmosphere, medium was removed, and cells were incubated for 19 h in serum-containing medium at 37°C. To verify transfection, VK2 cells transfected with fluorescein-conjugated control siRNA (Cat No. SN1021; Bioneer) were harvested. Intracellular fluorescence intensity was measured using a flow cytometer equipped with an FL1 detector (BD Biosciences).
4.7 cAMP assay.
Experiments were performed as previously reported[46]. To measure intracellular cyclic adenosine monophosphate (cAMP) concentration changes, cells were cultivated in a 96-well plate until 90% confluence. Aldehyde 13–13 was diluted in induction buffer, consisting of Ringer’s solution, 500 µM isobutyl-1-methylxanthin, and 100 µM 4-imidazolidin-2-one (Sigma-Aldrich). After incubation for 30 min, intracellular cAMP concentrations were measured using cAMP Glo Assay Kit (Promega) and Fusion α Multi-well Plate Reader (Packard Bioscience, Wellesley, MA). Data were normalized to the solvent control (ethanol). Treatment for 30 min with 10 µM forskolin (Sigma-Aldrich) served as the positive control.
4.8 Cell viability measurement.
Cell viability was examined by Trypan blue staining. VK2/E6E7 cells were seeded in 75 cm2 culture flasks as reported previously[47]. After reaching 80% confluence, cells were treated with aldehyde 13–13 for 24 h. Cells were collected and resuspended in PBS and Trypan blue for 3 min at room temperature. Cells were counted using a Neubauer counting chamber (Brand Tech, Essex, CT) under a light microscope, and the total number of cells and the ratio of nonviable to viable cells were determined. The number of cells per volume unit was calculated according to the formula: total cells per microliter = number of cells ÷ counting area (mm2) × chamber depth (mm) × dilution factor.
4.9 Cell proliferation assay.
Cell proliferation was measured using the CyQuantR Cell Proliferation Assay Kit (Life Technologies, Carlsbad, CA). VK2/E6E7 cells were seeded in a 96-well plate at 1 × 103/well and cultivated for 48 h at 37°C in 5% CO2. Next, cells were treated with aldehyde 13–13 for 24 h; further procedures were according to the manufacturer’s instructions. Proliferation was measured using a Fusion α Multi-well Plate Reader (Packard Bioscience). Values were standardized to the corresponding controls and are presented as percentage changes.
4.10 Immunoblot analysis.
Total cellular proteins were extracted using radioimmunoprecipitation assay buffer supplemented with Halt protease and phosphatase inhibitor reagent (Thermo Fisher Scientific, Carlsbad, CA). Total cellular proteins were centrifuged for 20 min at 12,000 rpm to obtain the soluble protein fraction. Protein concentration was measured using a protein assay dye reagent concentrate (Bio-Rad). Denatured proteins were run on a sodium dodecyl sulfate-polyacrylamide gel, and transferred to nitrocellulose membranes (GVS Filter Technology, Morecambe, UK). The membranes were blocked in TBS with 5% (w/v) non-fat dried milk for 1 h at room temperature. The primary antibodies were diluted in TBS-Tween 20 (0.1%) with 5% (w/v) non-fat dried milk. The secondary antibodies were diluted in TBS with 0.1% Tween-20. Immunoblot images were obtained using the ChemiDoc Touch Imaging System and analyzed with Image Lab 5.2 software (Bio-Rad). Protein levels were normalized with β-actin after stripping the identical blot using stripping buffer (Dyne Bio, CBS3180, Korea).
4.11 mRFP-GFP-LC3 puncta formation assay.
VK2/E6E7 cells (2 × 105) were seeded in a six-well confocal plate. After 24 h, cells were transfected with 1.5 ng of mRFP-GFP-LC3 plasmid using DharmaFECT transfection reagent (Horizon Discovery, Cambridge, UK) as per the manufacturer’s instructions and incubated for 48 h. Next, cells were treated with ethanol (control) or 500 µM aldehyde 13–13 for 30 min. Cells were washed once with PBS and fixed with 4% paraformaldehyde for 10 min. mRFP and GFP fluorescent puncta were observed under an LSM510 META confocal microscope and images were analyzed with LSM700 version 3.2 software (Carl Zeiss, Jena, Germany).
4.12 Monodansylcadaverine staining assay.
VK2/E6E7 cells were seeded in a black or clear 96-well plate at 1 × 104 per well. After 24 h, cells were treated with ethanol, rapamycin (100 nM), or aldehyde 13–13 (500 µM) for 30 min and autophagy vacuoles were stained using a monodansylcadaverine (MDC) staining kit (Cayman, Ann Arbor, MI) as per the manufacturer’s protocol. Autophagy vacuoles were detected under a fluorescence microscope (Nikon Eclipse Ti) and images were analyzed using NIS-elements software (Nikon Instruments, Inc.).
4.13 Autophagy analysis by transmission electron microscopy.
VK2 cells (5 × 106) were cultured in 175T flasks for 24 h and treated with aldehyde 13–13 (500 µM) for 30 min. For TEM, cells were fixed with Karnovsky’s fixative and stored at 4°C overnight. Cells were post-fixed with 1% osmium tetroxide and stained with 0.5% uranyl acetate overnight followed by dehydration in an ethanol gradient. The cells were infiltrated with Spurr’s resin overnight and visualized using a JEM1010 electron microscope at 120 kV (JEOL, Japan).
4.14 Senescence-associated ß-galactosidase staining.
VK2/E6E7 cells were seeded in 24-well plates at 5 × 104/well. Cells were grown for 24 h and treated with ethanol (control) or aldehyde 13–13 (15, 30, or 40 µM) for 1 h before H2O2 (300 µM) treatment. Next, the cells were incubated at 37°C in 5% CO2 for 3 days to induce senescence.
4.15 Immunocytochemistry.
VK2/E6E7 cells (3 × 104) were seeded on glass coverslips for 24 h. Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 min. Fixed cells were incubated with 5% bovine serum albumin (BSA) in TBS-Tween 20 (0.1%) for 45 min and then with Ki-67 antibody (1:500, Abcam, UK) overnight at 4°C. Next, cells were incubated with the Alexa Fluor 488-tagged anti-IgG secondary antibody (1:1000; Invitrogen) for 1 h in the dark. After washing, nuclei were stained with DAPI (Invitrogen) for 5 min. Coverslips were embedded in anti-fade mounting solution (Thermo Fisher Scientific) and slides were visualized using an LSM510 Meta confocal microscope and LSM700 v. 3.2 software (Carl Zeiss).
4.16 Annexin V and propidium iodide staining.
Cells were seeded in a six-well plate at 3 × 105/well and grown for 24 h. Proliferating cells were treated with vehicle and 40 µM aldehyde 13–13. Senescent cells were cotreated with 200 µM H2O2 plus vehicle and aldehyde 13–13. Next, cells were stained incubated with 3 µL Annexin-V-FITC (BD Biosciences, CA) and 10 µL propidium iodide (BD Biosciences, CA) for 20 min and apoptosis analysis was performed using Accuri C6 Plus FACS (BD Biosciences).
4.17 Budding yeast and culture conditions.
Saccharomyces cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0, ura3Δ0) was streaked onto yeast extract peptone-dextrose (YPD) agar containing 2% Bacto agar, 1% Bacto yeast extract, 2% Bacto peptone, and 2% Difco dextrose (BD Biosciences). The plates were incubated at 30°C to obtain isolated single colonies. A few picked colonies were inoculated into 10 mL of YPD medium (1% Bacto yeast extract, 2% Bacto peptone, and 2% Difco dextrose), and cultured overnight to make a seed culture. The seed culture was inoculated into 20 mL of YPD medium to an optical density at 600 nm of 0.2. All yeast cultures were incubated at 30°C in an orbital shaker at 200 rpm.
4.18 Measurement of chronological lifespan by PI staining and flow cytometry.
Yeast cells were harvested by centrifugation, washed by resuspending in 1 mL of phosphate-buffered saline (PBS), and incubated for 20 min at 30℃ after adding 5 µL of PI solution (1 mg/mL, Sigma Aldrich). Stained cells were analyzed using a flow cytometer (FACS Verse; Becton Dickinson) with excitation at 488 nm and emission at 585 nm. Approximately 20,000 cells were analyzed per sample, and data were analyzed using Cell Quest software (Becton Dickinson).
4.19 Colony-forming unit assay of budding yeast.
A colony-forming unit (CFU) assay was performed to determine yeast viability. Yeasts were harvested, serially diluted in PBS, counted using a hemocytometer, and 200 cells were spread onto YPD agar and incubated at 30°C until colony formation.
4.20 Caenorhabditis elegans longevity assay.
Strains were cultured on fresh NGM plates for 2–3 generations without starvation, and lifespan analysis was conducted at 20°C. Worms were synchronized by isolating eggs from gravid adults using hypochlorite and NaOH. When worms reached the L4 stage, 2′-deoxy-5-fluorouridine (FUdR) was added to prevent internal hatching (final concentration 0.6 mM). Wells containing > 15 worms were excluded from the analysis because of the risk of dietary restriction. Worm movement was used to determine whether the animals were alive or dead. Survival curves were analyzed by the log-rank (Mantel-Cox) method.
4.21 Statistical analysis.
Results are means ± standard error of the mean (SEM). To generate dose-response curves, SigmaPlot software was used, and curves were fitted using the Hill three-parameter equation. Significance was examined by two-tailed unpaired t-test. Data were subjected to one-way ANOVA followed by Tukey’s HSD test or Student’s t-test for multiple or two-group comparisons. Different letters indicate a significant difference at P < 0.05 (ANOVA).