Cell lines and treatments
Melanoma cell line SK-MEL-28 was purchased at ATCC (ATCC® CRL-1772™, ATCC, Manassas, VI, USA) and SK-MEL-103 cell line was provided by Dr. Marisol Soengas. Patient-derived cell line M16 was kindly provided by Dr. Susana Puig from Hospital Clínic de Barcelona. SK-MEL-28 cell line was cultured in RPMI 1640 medium (31870, Thermo Fisher, Gibco, Paisley, Scotland) supplemented with 10% heat-inactivated fetal bovine serum (10270, Thermo Fisher, Gibco), 1% of l-glutamine (25030, Thermo Fisher, Gibco), and 1% of penicillin and streptomycin (15140, Thermo Fisher, Gibco). SK-MEL-103 and M16 cells were cultured in DMEM high glucose medium (41965, Thermo Fisher, Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 1% of penicillin and streptomycin and 1% of l-glutamine. All cells were tested for mycoplasma and maintained at 37°C in a humidified atmosphere of 5% CO2.
BH3 mimetics ABT-199 (venetoclax), A-1331852 (A-133), S63845 and ABT-263 (navitoclax) were purchased at Selleckchem (Munich, Germany) and the PROTAC BCL-xL degrader DT2216 was purchased at AbMole BioScience (Houston, Texas, USA). Treatments were performed directly in the culture media at the indicated concentrations.
Palbociclib-induced senescence
For palbociclib-induced senescence, cells were seeded in a 6 well-plate and treated with palbociclib (S1116, Selleckchem, Munich, Germany) at 1µM dose. After 3 days cells were replenished with fresh media and palbociclib. Seven days after the treatment, senescent cells were used for further analysis.
Irradiation-induced senescence
For irradiated induced senescence, cells were seeded in a 6 well-plate and irradiated with a gamma irradiator MARK I 30 (J.L. Shepherd & Associates, San Fernando, CA, USA) at 10Gy. After 3 days, the media was changed and cells were maintained in culture till day 7 after irradiation and used for further analysis.
Senescence associated β-galactosidase staining
Cells were stained using CellEvent™ Senescence Green Detection Kit (C10851, Thermo Fisher). Briefly, cells were washed with PBS, tripsinized, fixed with 4% formaldehyde during 10 minutes at room temperature, washed with PBS + 1% BSA and incubated with the working solution at 37ºC without CO2 during 2 hours. After incubation, cells were washed with PBS and then imaged using a spectral flow cytometry Cytek AURORA instrument (Cytek Biosciences, California, USA) from the Scientific and Technological Centers of the University of Barcelona.
BH3 profiling
BH3 profiling was developed at the Letai laboratory, and the experiments were performed as previously described 20, 22, 25. After inducing senescence for 7 days cells were stained with the viability marker Zombie Violet (423113, BioLegend, Koblenz, Germany) and then washed with PBS and resuspended in MEB (150 mM mannitol, 10 mM hepes–KOH pH 7.5, 150 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% BSA, 5 mM succinate) in a final volume of 25 µL. Afterwards, cells were exposed to the following peptides: 25 µL of BIM BH3 peptide (final concentration of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM), 25 µL of BAD BH3 peptide (final concentration of 10 µM), 25 µL of HRK BH3 peptide (final concentration of 100 µM), and 25 µL of MS1 BH3 peptide (final concentration of 10 µM) in MEB with 0.002% digitonin in a 96-well plate (3795, Corning, Madrid, Spain) for 1h following fixation with formaldehyde. Cells were finally stained with cytochrome c antibody (Alexa Fluor® 647 anti-Cytochrome c—6H2.B4, 612310, BioLegend), p21 antibody (sc-6246 PE, Santa Cruz Biotechnology, Texas, USA) and p16 antibody (Alexa Fluor® 488 CDKN2A/p16INK4a, bs-4592R-A488, Bioss Antibodies, Woburn, Massachusetts, USA). Individual BH3 profiling analyses were performed using triplicates for DMSO, alamethecin (BML-A150-0005, Enzo Life Sciences, Lorrach, Germany), the different BIM BH3 concentrations used, BAD, HRK, and MS1 BH3 peptides. The expressed values stand for the average of three different readings performed with a high-throughput spectral flow cytometry Cytek AURORA instrument (Cytek Biosciences, California, USA) from the Scientific and Technological Centers of the University of Barcelona. % of cytochrome c released is used to calculate Δ% priming which represents the difference between treated cells minus non-treated cells for a given peptide.
Flow cytometry analysis
Due to the difference of mitochondrial content between proliferating and senescence cells and therefore a major cytochrome c retained signalling was detected in senescent cells versus control cells, we adjusted the gating for cytochrome c retention with the control condition of each experimental setting (proliferating and senescent). Analyses were performed using FlowJo software.
Cell death assay
Cells were trypsinized and stained with fluorescent conjugates of Annexin V (Alexa Fluor® 647 Annexin V, 640912, BioLegend) and DAPI (62248, Thermo Fisher) and analyzed on a flow cytometry Gallios instrument (Beckman Coulter, Nyon, Switzerland) from the Scientific and Technological Centers of the University of Barcelona. Viable cells are Annexin V DAPI negative, and cell death is expressed as 100%-viable cells.
Protein extraction and quantification
Cells were lysed using RIPA buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris–HCl pH = 8, 1% Triton X-100, 0.1% SDS, EDTA-free Protease Inhibitor Cocktail (4693159001 Roche, Mannkin, Germany)) for 30 min at 4°C followed by a centrifugation at 16,100 × g for 10 min. The supernatant was stored at − 20°C for protein quantification performed using PierceTM BCA Protein Assay Kit (23227, Thermo Fisher).
Immunoprecipitation
Cells were lysed using Immunoprecipitation buffer (150 mM NaCl, 10 mM Hepes, 2 mM EDTA, 1% Triton, 1.5 mM MgCl2, 10% glycerol, and EDTA-free Protease Inhibitor Cocktail (4693159001 Roche)) and centrifuged at 16,100 × g, 15 min at 4°C. Supernatants were incubated with magnetic beads (Dynabeads 10003D, Thermo Fisher) conjugated to 5 µg of rabbit anti-BCL-xL antibody (CST2764, Cell Signaling, Leiden, The Netherlands) or 5 µg of rabbit IgG antibody (CST2729, Cell Signaling) at 4°C overnight. After magnetization, a part of the supernatant was mixed with half volume of 4× SDS–PAGE sample buffer, heated at 96°C for 5 min and stored at − 80°C as unbound fractions. The rest of the supernatant was discarded. The resulting pellet was washed and mixed with 60 µL 4× SDS–PAGE sample buffer and heated for 10 min at 70°C following by magnetization and collection of the supernatant for further immunoblotting analyses.
Immunoblotting
Proteins were separated by SDS–PAGE (Mini-Protean TGX Precast Gel 12%, 456–1045, Bio-Rad) and transferred to PVDF membranes (10600023, Amersham Hybond, Pittsburgh, PA, USA). Membranes were blocked with dry milk dissolved in Tris buffer saline with 1% Tween 20 (TBST) for 1 h and probed overnight at 4°C with the primary antibodies of interest directed against: rabbit anti-BCL-xL (CST2764, Cell Signaling), rabbit anti-BIM (CST2933, Cell Signaling), rabbit anti-BAK (CST12105, Cell Signaling), rabbit anti-BAX (CST2772, Cell Signaling), rabbit anti-HRK (PRS3771, Sigma-Aldrich, San Luis, Missouri, USA), mouse anti-γ−tubulin (T6557, Sigma-Aldrich) followed by anti-rabbit or anti-mouse IgG HRP-linked secondary antibody (CST7074 or CST7076 Cell Signaling) in 3% BSA in TBST for 1 h at room temperature. Immunoblots were developed using Clarity ECL Western substrate (1705060, Bio-Rad), visualized with LAS4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ImageJ was then used to measure the integrated optical density of bands.
Transmission electron microscopy procedure for flat embedding preparation of cell monolayers
For transmission electron microscopy (TEM) preparation and analysis of melanoma cells (proliferating and senescence) growing on coverslips, the cells were first washed with 0.1 M phosphate buffer (PB) to remove the excess of culture medium and fixed with a freshly made 3% glutaraldehyde solution in 0.1M PB for 1 hour at room temperature. Subsequently, the fixative was removed, and the samples were maintained at 4º C in a fresh fixative solution until processing.
The post-fixation procedure was conducted using 1% OsO4 for a period of 90 minutes at 4°C. Then, samples were dehydrated in increasing ethanol solutions (in accordance with the standard procedures). Coverslips with cells were then inverted (with the cells facing downwards) and placed on top of BEEM® capsules filled with Spurr resin (Electron Microscopy Sciences, Hatfield, PA, USA). The polymerization of the resin was carried out at a temperature of 60 ºC in a stove for a period of three days. At this point, the glass is removed by thermal contrast switch while the cell monolayer remains at the top of the polymerized block. A Leica ultramicrotome EM UC7 (Leica Microsystems, Wetzlar, Germany) was employed for sectioning. Ultra-thin sections (60–70 nm) were mounted on cooper grids and stained with 2% Uranyl-less solution for 10 minutes and with a lead-staining solution for 5 minutes. The sections were observed using a JEOL JEM-1010 transmission electron microscope (JEOL Ltd., Akishima, Tokyo, Japan) coupled with an Orius SC1000 CCD camera (model 832) (Gatan Inc., Pleasanton, CA, USA) at the Unit of Electron Microscopy, Scientific and Technological Centers of the University of Barcelona, School of Medicine and Health Sciences (Barcelona, Spain).
Immunofluorescence
Cells were gently washed once with cold PBS and then fixed for 20 minutes with 4% paraformaldehyde in PBS. Cells were then washed three times with PBS and permeabilized for 10 minutes with 0.1% saponin in PBS. Blocking was performed for 5 minutes with 0.02% saponin and 1% BSA in PBS. For primary antibody incubation, cells were incubated with anti-Tom20 (ab186734, Abcam, Cambridge, UK) diluted in 0.02% saponin and 0.1% BSA solution for 1 hour at room temperature. After primary antibody incubation, cells were washed three times with PBS. Secondary antibody incubation was carried out by incubating cells with anti-Mouse IgG (H + L), Superclonal™ Recombinant Secondary Antibody Alexa Fluor™ 488 (A28175, Thermo Fisher) and anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A21428, Thermo Fisher) for 45 minutes in 0.02% saponin and 0.1% BSA solution at RT. For DAPI staining, cells were incubated with diluted DAPI stock solution (10374168, Thermo Fisher) to a final concentration of 300 nM in PBS for 5 minutes. Following staining, cells were washed three times with PBS, rinsed in water and mounted in Mowiol® 4–88 (17951-500, Polysciences, Warrington, PA, USA).
Bioinformatic analysis of SK-MEL-103 data
In the present study, GEO2R (www.ncbi.nlm.nih.gov/geo/geo2r/) was utilized to download the raw counts of the GSE246690 series to identify genes differentially expressed across experimental conditions of the SK-MEL-103 cell line (control and Palbociclib)34. The data was analyzed using the edgeR (v.4.0.16) package from Bioconductor in the R environment (v.4.3.3) 39. The sva (v. 3.50.0) package was used for removing batch effects and other unwanted variation 40. Subsequently, differential expression analysis was conducted using the quasi-likelihood negative binomial generalized log-linear model (GLM) functions provided by the edgeR package. For visualization purposes Log2CPM expression values were converted to Z-scores and statistical significance was represented as Fold Discovery Rate (FDR) < 0.05.
Patients and samples for molecular validation
Fourteen healthy subjects, comprising 10 female and 4 male, mean age 52.6 years (range 41–66) were recruited in Hospital Clínic de Barcelona (Table 1). Two distinct areas on the forearm were identified for biopsy: one area of photodamaged skin (L01) and one area of less sun-damaged (naturally protected) skin on the inner forearm (L02).
Subsequently, two 3-mm punch biopsies were taken on L01 and L02 areas. Each biopsy was included in RNAlater (RNAprotect Tissue Reagent, Qiagen) and frozen to extract RNA and perform sequencing.
RNA extraction and RNA-sequencing analysis
Biopsies in RNAlater were frozen to extract RNA for sequencing. Total RNA was isolated from fresh tissue with Trizol, and purified with RNeasy Minikit (Trizol-chloroform + RNeasy MinElute Cleanup Kit, Qiagen). RNA quantification was performed using Qubit RNA BR Assay Kit. Integrity was measured via Bioanalyzer RNA 6000 NanoKit (Agilent Technologies, Santa Clara, CA).
RNA samples were sent to “Centro Nacional de Análisis Genómico” to conduct the stranded mRNA library preparation and sequencing on HiSeq 4000 (Illumina).
RNA-Seq libraries were prepared from total RNA using TruSeq Stranded mRNA LibraryPrep Kit (Illumina). mRNA was enriched with oligo-dT magnetic beads from the total RNA (500ng) and fragmented to 80-450nt. The second strand cDNA synthesis was performed in the presence of dUTP to achieve strand specificity. The blunt-ended double-stranded cDNA was 3´adenylated and Illumina platform-compatible adaptors with unique dual indexes and unique molecular identifiers (Integrated DNA Technologies) were ligated. The ligation product was enriched with 15 PCR cycles. The final library was validated on a Bioanalyzer DNA 7500 assay. The libraries were sequenced on NovaSeq6000 (Illumina) in paired-end mode with a read length of 2x101bp, following the manufacturer’s protocol for dual indexing. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real-Time Analysis (RTA 3.4.4) and followed by the generation of FASTQ sequence files.
Statistical analysis
Data was analyzed using GraphPad Prism v.9.3.0 software and was represented as mean ± SEM of independent biological replicates. Statistical significance of the results was analyzed using Student’s t-tail test. *p < 0.05 and **p < 0.01 were considered significant.
Specifically, for RNA-Seq data analysis of patient samples, the R package LIMMA was used for data normalization, specifically employing the Variance Modeling at the Observational Leve (VOOM). After normalization of expression data, the genes of interest for validation (CDKN2A, CDKN1A, and HRK) were selected. Global results of the RNA-sequencing analysis in under revision for their publication. Comparisons between the expression between L01 and L02 samples from the same patient were conducted using a paired Student's t-test. Assumptions of normality and homogeneity of variances were checked and appropriately addressed. All analyses were performed using R version 3.3.0.