Culture of iMKTPCs

Generation of iMKTPCs and culture conditions have been described previously (Schmid et al. 2020). In brief, iMKTPCs were immortalized by the insertion of human telomerase (hTERT) by PiggyBag transposon (Schmid et al. 2020). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM) high-glucose media (Gibco, Paisly, UK) supplemented with 10% fetal bovine serum (Capricorn Scientific, Ebsdorfergrund, Germany), 1% penicillin/streptomycin (Biochrom, Berlin, Germany), 1× NEAA (Gibco), 1× Glutamax (Gibco), and 0.3 µg/mL of puromycin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C and 5% (v/v) CO2. For the experiments, iMKTPCs in passages (P) 28–44 were used.

Induction of senescence with bleomycin

iMKTPCs were cultured in cell culture flasks (T75) until they reached approximately 70–80% confluence. Pilot studies were performed to optimize the concentration of bleomycin sulfate (Cayman Chemical, Ann Arbor, MI, USA). These initial studies included beta-galactosidase staining and cell size measurements. They showed that 25 µg/mL bleomycin sulfate added to the cells for 24 h proved suitable and robustly caused senescence. The respective amount of the solvent dimethyl sulfoxide (DMSO; Invitrogen, Carlsbad, CA, USA) was used as control. After the treatment, cells were washed with phosphate-buffered saline (PBS) and cultured in normal cell culture media for 10 days before analysis by β-galactosidase staining, quantitative polymerase chain reaction (qPCR), proteomics, and functional assays, as described in the following. In addition, cells were cultured for a total of 15 days, after which cell number and size were determined, and qPCR and Western blotting experiments were performed.

Cell number and cell size measurement

Both the cell size and number of iMKTPCs were measured using the CASY® Cell Counter (Schärfe Systems, Reutlingen, Germany). The cells were trypsinized, centrifuged, resuspended in PBS, and measured as described earlier (Schell et al. 2010).

Beta-galactosidase staining

iMKTPCs were seeded onto coverslips and staining was performed using a commercial kit (Senescence β-Galactosidase Staining Kit, Cell Signaling Technology #9860, Danvers, MA, USA) according to the manufacturer’s instructions. Evaluation of the staining was done with a light microscope (Zeiss Axiovert; Zeiss GmbH, Oberkochen, Germany).

Isolation of mRNA, reverse transcription, and qPCR

The total messenger RNA (mRNA) of iMKTPCs was isolated using the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. A total of 500 ng mRNA was reverse transcribed using the LunaScript™ RT Super Mix (New England Biolabs, Frankfurt am Main, Germany). A LightCycler® 96 System (Roche, Penzberg, Germany) and Luna® Universal qPCR Master Mix (New England Biolabs), which contains a proprietary double-stranded DNA (dsDNA)-binding intercalating dye-based detection chemistry, were used for the qPCR analysis. Oligonucleotide primers for amplification are listed in Table 1. Quantitative analysis was performed using the 2−ΔΔCq method and normalized to reference genes YWHAZ and PPIA. Primer pairs, especially those for IL1B and IL6, were validated in our previous publications (Schmid et al. 2018, 2020) and in the experiments showed comparable quantification cycle (Cq) shifts between groups. Moreover, single melt peaks were recorded and single amplicon size was verified by gel electrophoresis. The identity of the complementary DNAs (cDNAs) was confirmed upon sequencing. Statistics were obtained using unpaired t-tests (two-tailed) of −ΔΔCq values via GraphPad Prism 6.0 software (GraphPad Software Inc.; San Diego, CA, USA); results are shown as means ± SEM. In total n = 7 samples were analyzed; non-reverse transcription and non-template reactions served as negative controls.

Table 1 Oligonucleotide primer for qPCR studiesWestern blotting

Cells were harvested and used for Western blotting as described previously (Schell et al. 2010). A monoclonal antibody recognizing lamin B1 (1:500; Proteintech, Planegg-Martinsried, Germany; 66095-1-Ig) was used, and an anti-beta actin antibody (1:5,000; Sigma-Aldrich) served as a loading control.

Contractility measurements

Live cell monitoring of cellular contractility was performed as described previously (Nie et al. 2022). Cells were seeded onto ibidi imaging plates (35 mm µ-dishes; ibidi, Gräfelfing, Germany) and serum-starved for 24 h. Plates were then placed in the imaging chamber at 37 °C and 5% CO2 and equilibrated for 30 min. Subsequently, cells were treated with 30% fetal calf serum (FCS, in medium), and the contractile response was monitored for 30 min by acquiring phase-contrast images at 1-min intervals (Zeiss GmbH, Oberkochen, Germany). Cell contractility was quantified by manual measurement of a defined cell area using Fiji (Schindelin et al. 2012). In total, n = 20 cells were analyzed. Statistical analysis was performed using a paired t-test.

Sample preparation for proteomics analysis

For proteomics, treated (25 µg/mL bleomycin) iMKTPCs were compared against controls (DMSO) (both n = 4). Cells in each sample were lysed in a buffer consisting of 8 mol/L urea (Carl Roth, Karlsruhe, Germany) in 50 mmol/L ammonium bicarbonate (Riedel-de Haën, Seelze, Germany) and sonicated in a Bandelin Sonoplus HD 3200 cup resonator (Bandelin, Berlin, Germany). Lysates were centrifuged through QIAshredder devices (QIAGEN, Hilden, Germany). Protein concentration was determined using the Pierce 660 nm assay reagent (Thermo Scientific, Waltham, MA, USA). Cysteine residues were reduced using 1,4-dithiothreitol for 30 min at 56 °C at a concentration of 5 mM and subjected to carbamidomethylation using iodoacetamide at a concentration of 15 mM (30 min in darkness, room temperature). The digestion of proteins was performed in two steps: (i) Lys C (Fujifilm Wako, Neuss, Germany) in a 1:100 enzyme/protein ratio for 3 h 40 min at 37 °C; (ii) dilution of the samples to 1 mol/L urea and overnight digestion with modified porcine trypsin (Promega, Madison, WI, USA) in a 1:50 enzyme/protein ratio at 37 °C. Dried samples were desalted using ZipTips (Merck KGaA, Darmstadt, Germany) with the protocol recommended by the manufacturer.

Nano-LC–MS/MS analysis

Nano-liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis was carried out on an UltiMate 3000 RSLC coupled with a Q Exactive HF-X (both: Thermo Scientific, Waltham, MA, USA). Samples were injected onto a trap column (Acclaim Pepmap™ 100 µm × 2 cm, C18, 5 µm, 100 Å, Thermo Scientific) at a flow rate of 5 μL/min. The LC separation of peptides was performed on an EASY-spray column (Pepmap™ RSLC C18, 2 µm, 100 Å, 75 µm × 50 cm, Thermo Scientific, Waltham, MA, USA) with the flow rate set to 250 nL/min, as follows: a two-step gradient from 3% B (0.1% [v/v] formic acid in acetonitrile) to 25% B in 160 min followed by a ramp to 40% B for 10 min. Peptides were analyzed in data-dependent acquisition (DDA) mode with the top 15 MS/MS scans selected per cycle.

Data analysis and bioinformatics processing

Acquired MS spectra were processed using MaxQuant (1.6.11.0) and the C. jacchus subset of the UniProt database. Statistical evaluation and visualization were carried out with Perseus v1.6.7.0 (Tyanova et al. 2016) and R (R Core Team 2025). For protein annotation and overrepresentation analysis, the STRING database (https://string-db.org/) was used (Szklarczyk et al. 2023). The mass spectrometry data were deposited to the ProteomeXchange Consortium (www.proteomexchange.org, accessed October 3, 2022) via the Proteomics Identification Database (PRIDE) partner repository with the dataset identifier PXD073445 (Perez-Riverol et al. 2025).