Rnase a

In vivo interactions studies were carried out following the protocols published previously (18 (link),27 (link)). For immunoprecipitation of GFP-tagged proteins, 10 μl slurry of GFP-Trap_A beads (Chromotek) and for TAP-tagged proteins, 20 μl slurry of IgG-Sepharaose beads (GE Healthcare) were used per reaction and incubated with 200 (high abundant proteins) and up to 2000 μl (for low abundant proteins) of the clarified lysate for 3 h rotating at 4°C. If indicated, the samples were treated with 0.2 mg/ml RNase A (AppliChem) for additional 30 min at 4°C. Finally, the eluted proteins were separated on 10% SDS-polyacrylamide gels and analysed by western blotting.

Twenty-four hours before treatment, HMEC-1 cells were plated in 6-well plates. Propidium iodide (PI) staining and flow cytometry analysis for apoptotic DNA-fragmentation (subG1 population) was performed 48, 72, or 96 h post-treatment. HMEC-1 cells were incubated for 15–30 min at RT with a staining solution (0.1 M Tris, 0.1 M NaCl, 5 mM MgCl2, 0.05% Triton X-100 (all Roth, Karlsruhe, Germany)), 62 µg/mL of RNaseA (AppliChem, Darmstadt, Germany), and 40 µg/mL PI (Sigma-Aldrich, St. Louis, MI, USA) [32 (link)]. Subsequently, samples were analyzed by flow cytometry (FACS Calibur, Becton Dickinson, Heidelberg, Germany; FL-2) [33 (link)]. Cell cycle phase distribution was analyzed with Kaluza software Version 2.1 to identify the subG1 population (apoptotic DNA-fragmentation, whole population), and, in a second step, the living cell population (G1, S, G2/M phase) was investigated for a G2/M arrest [15 (link)]. Statistical analysis was performed in GraphPadPrism Version 8.3.0. Exemplary histograms of flow cytometry are shown in Supplementary Figure S9.

The cells were stained with 50 µg/mL propidium iodide and 1 µg/mL Nonidet P40 (Sigma-Aldrich, Stockholm, Sweden), 10 µg/mL RNAse A (AppliChem, Kongens Lyngby, Denmark) and 0.58 µg/mL NaCl, and the cell cycle distribution was determined using a CytoFLEX analyzer.

In situ hybridization was performed using protocol and digoxigenin (DIG)-labeled probes established by Simmons et al. [37 (link)]. In short, cryo- or paraffin sections were thawed or deparaffinized and rehydrated, respectively. Sections were fixed using 4% paraformaldehyde (Merck, Darmstadt, Hessen, Germany; #818715) and treated with proteinase K (20 μg/mL, Merck, Darmstadt, Hessen, Germany; #1245680500). After another fixation step, sections were blocked. Probes (2 ng/μL) for PL1, PLF, and TPBPA were denatured and incubated with sections overnight, followed by a RNase A (AppliChem, Darmstadt, Hessen, Germany; #A2760) treatment. Sections were blocked, and anti-digoxigenin-AP (1:1000, Sigma-Aldrich, St. Louis, MO, USA; #11093274910) was added for probe detection. Incubation with BM-purple followed overnight; then, sections were counterstained with nuclear fast red (Sigma-Aldrich, St. Louis, MO, USA; #N3020). Sections were dehydrated, treated with xylene (VMP Chemie Kontor GmbH, Siegburg, North Rhine-Westfalia, Germany; #1000649172), and mounted in Entellan^® (Merck, Darmstadt, Hessen, Germany; #107960). Leica DM LB microscope (Wetzlar, Hessen, Germany) was used for analysis of sections.

One million EPP and EPV cells were incubated with 1 µM CN133 and CN147 at indicated times. Pellets were resuspended in staining solution (38 mM Sodium citrate (Sigma-Aldrich), 500 μg/mL Propidium iodide (Thermo Fisher Scientific), 10 mg/mL RNAse A (AppliChem, Darmstadt, Germany) in PBS and incubated overnight at 4 °C. Stained cells were subjected to flow cytometric analysis on FACScan (BD Biosciences, San José, CA, USA) and cells in Sub G1 peak were quantified. Data were analyzed with FACS Diva 6.0 (BD Biosciences) and CellQuest Pro software (BD Biosciences).