Cell culture

Normal human dermal fibroblasts (NHDF; C-12300) and normal human epidermal keratinocytes (NHEK; C-12003) were obtained from PromoCell GmbH (Heidelberg, Germany). NHDF were grown and maintained in low-glucose Dulbecco’s Modified Eagle’s Medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific). NHEK were cultured in a growth medium (PromoCell, C-20011) containing bovine pituitary extract, epidermal growth factor, insulin, hydrocortisone, epinephrine, transferrin, and CaCl2.

Replicative senescence (ReS) was defined as lack of cell growth for more than 2 weeks, i.e., cells with a cell population doubling level of 50–55. For ionized radiation-induced senescence, cells were exposed to 10 Gy of X-rays by the X-irradiator CellRad (Faxitron, Tucson, AZ, USA) and analyzed after 10 days. Control (proliferating) cells were mock irradiated by removal from the incubator, transport to the irradiator, and maintenance outside of the irradiator for the same period as the irradiated cells. For doxorubicin-induced senescence, cells were treated twice with 0.1 μM doxorubicin (Sigma-Aldrich, St. Louis, MO, USA) at 2-day intervals and analyzed after 7 days. SA-β-gal activity in cells was assessed using the Senescence β-Galactosidase Staining Kit from Cell Signaling (Danvers, MA, USA).

Assessment of BrdU incorporation in fibroblasts by flow cytometry

Cells were incubated with BrdU at 37 °C for 24 h, collected, and incubated with the BrdU-FITC antibody (BrdUFlowEx FITC Kit; EXBIO Praha, a.s., Vestec, Czech Republic) for 30 min. FlowJo (Ver. 10.2) was used to analyze the data using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Immunocytochemistry

Cells were placed on glass slides, fixed in acetone at room temperature (15–25 °C) for 5 min, and dried completely before staining. The cells were incubated overnight at 4 °C with an anti-cathepsin F antibody (PA5-87,925, Thermo Fisher Scientific) and anti-IL-6 antibody (ab6672, Abcam, Cambridge, UK) diluted 1:100 in phosphate-buffered saline (PBS). To confirm the specificity of the immunostaining, fetal bovine serum diluted to the same concentration was used instead of the primary antibody as a negative control. After washing three times with PBS, the slides were incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody and AlexaFluor555-conjugated donkey anti-goat antibody (Thermo Fisher Scientific) diluted 1:2000 in PBS for 1 h at room temperature. After incubation, nuclei were washed three times with PBS and counterstained for the visualization of nuclei using ProLong Gold Anti-fade Mountant containing 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific).

Immunohistochemistry

Whole skin sample slides of 3- and 89-year-old human trunks were obtained from OriGene Technologies, Inc. (Rockville, MD, USA) under ethical considerations. Samples were collected after obtaining informed consent of anonymous donor patients. The procedures were compliant with American standards and applicable local ethical guidelines. Paraffin was dissolved in a slide heater (ThermoBrite; Leica Biosystems, Nussloch, Germany) at 65 °C for 30 min immediately before use. The slides were then deparaffinized by washing with xylene twice at room temperature (5 min per soak). Slides were soaked twice with 100% ethanol (3 min per soak) and then stepwise with 95%, 70%, and 50% ethanol (3 min each), and then rehydrated at room temperature. After antigen activation by heat, slides were incubated with 2% goat serum in PBS for 30 min at room temperature to block nonspecific binding sites. The slides were then incubated overnight at 4 °C with a 1:100 dilution of the same anti-CTSF antibody (in PBS) used for immunohistochemistry.

After washing three times with PBS, the slides were incubated with a 1:500 dilution of biotinylated rabbit anti-goat antibody (Vector Laboratories, Burlingame, CA, USA) in PBS for 1 h at room temperature.

The signal was amplified by the avidin-biotinylated peroxidase complex (ABC) method using the VECTASTAIN ABC Kit (Vector Laboratories) and 20 mg/dL 3,3′-diaminobenzidine solution (FUJIFILM Wako Pure Chemicals, Co., Osaka, Japan) for 1 to 3 min to develop color.

Sections were then washed once with running tap water for 5 min before nuclear counterstaining with Gill’s hematoxylin solution (Merck Millipore, Billerica, MA, USA) for 6 s at room temperature. Finally, sections were rinsed with tap water for 5 min, dehydrated with ethanol (twice with 95% and twice with 100%, 5 min each), rinsed with xylene three times, and sealed with Mount Quick Sealant (Takara Bio, Shiga, Japan).

Slides were observed using an integrated stereomicroscope (BZ-X800; KEYENCE, Osaka, Japan).

RNA extraction and reverse transcription

Total RNA was extracted from cells using a monophasic solution of phenol and guanidine isothiocyanate (ISOGEN; Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. Total RNA was mixed with random primers, reverse transcriptase, and a deoxynucleotide mixture (Takara Bio). The mixtures were incubated in a T100™ thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 25 °C for 5 min for annealing, 55 °C for 10 min for synthesis, and 80 °C for 10 min for thermal inactivation of reverse transcriptase to provide cDNA.

Real-time quantitative polymerase chain reaction (RT-qPCR)

RT-qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). A total of 40 cycles were performed and the fluorescence of each sample was measured at the end of each cycle. The polymerase chain reaction (PCR) was performed in two major steps: (1) 95 °C for 3 s (denaturation) and (2) 60 °C for 30 s (annealing and extension). In the subsequent melting curve analysis, the temperature was increased from 60 to 95 °C and fluorescence was measured continuously. Gene expression was determined using primers for CTSF (assay ID: Hs00186901_m1), Il-6 (Hs00985639_m1), Il-1a (Hs00174092_m1), Il-8 (Hs00174103_m1), p16ink4a (Hs00923894_m1), and CEBPB (Hs00270923_s1) (all from Thermo Fisher Scientific) and PCR master mix (Cat. No. 4352042; Applied Biosystems, Foster City, CA, USA) following the manufacturers’ instructions. GAPDH (Hs02786624_g1) was used as a control gene for normalization. The gene expression level in the proliferating cell population was used as the baseline, and fold change values were determined by the 2−ΔΔCT method.

Western blotting

Total protein was extracted from cells with lysate buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5% Nonidet P40, 0.5% sodium deoxycholate, and phenylmethylsulfonyl fluoride (all from FUJIFILM Wako Pure Chemical Co.)).

Each sample (40 μg) was electrophoresed on 10% polyacrylamide gels (Mini-PROTEAN TGX Precast Gels; Bio-Rad Laboratories, Inc.) and transferred to Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc.) onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA).

After blocking with 3% nonfat milk for 2 h at room temperature, primary antibodies against CTSF (PA5-48,002; Thermo Fisher Scientific, 1:200), p21 (ab220206; Abcam, 1:100), CDKN2A/p16INK4a (EPR1473; Thermo Fisher Scientific, 1:200), SIRT1 (ab32441; Abcam, 1:200), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:2000) diluted with blocking solution were incubated overnight at 4 °C. The next day, the samples were incubated with the following secondary antibodies: donkey anti-goat IgG H&L (HRP) (ab6885; Abcam), goat anti-rabbit IgG H&L (HRP) (ab205718; Abcam), and goat anti-mouse IgG H&L (HRP) (ab205719; Abcam) at a 1:1000 dilution for 2 h at 37 °C. After washing, immunoreactive protein bands were visualized using an Electrochemiluminescence Detection Kit (Pierce Biotechnology, Rockford, IL, USA). Images of bands were obtained using a chemiluminescence imager (ImageQuant LAS4000mini; GE Healthcare, Chicago, IL, USA). The image analysis was performed using ImageJ (Ver. 1.53p, National Institutes of Health, Maryland, USA). Each experiment was repeated three times.

Fluorescence-activated cell sorting (FACS)

Proliferating and senescent human skin fibroblasts were counted using a TC20 Cell Counter (Bio-Rad) and washed using FACS buffer (0.5% bovine serum albumin in PBS). After washing, human TruStain FcX (BioLegend, San Diego, CA, USA) was added to block the Fc receptor and cells were incubated with an anti-CTSF antibody (Thermo Fisher Scientific) for 10 min at 4 °C. Cells were then incubated with an Alexa Fluor 488-labeled rabbit anti-goat antibody (Thermo Fisher Scientific) for 15 min at 4 °C in the dark. Next, 7-amino-actinomycin D (7AAD) (Immunostep, S.L., Salamanca, Spain) was added and incubated at 4 °C for 15 min to label the photoreceptor cells. A FACS analysis was performed using FlowJo (version 10.2). Briefly, the background autofluorescence of the negative population was measured using an unstained control. 7AAD-viable cells were then gated, and the number of Alexafluor488-positive cells among them was counted.

Statistical analysis

Statistical analyses were performed using GraphPad Prism (version 5.0; San Diego, CA, USA) or SPSS 22.0 (Chicago, IL, USA). Single end-point measures were compared using a non-paired sample t-test. Values of P < 0.05 were considered statistically significant.