Standard cell recycling is inactive in stem cells and early nerve cells after DNA damage caused by BPDE or etoposide

In this study, we made use of the iPSC lines iPS11 (derived from human foreskin fibroblasts) and iPS12 (human mesenchymal stromal cells; both lines obtained from ALSTEM, INC., Richmond, CA 94806, USA). Both iPSC lines ectopically express OCT4, SOX2, KLF4, and L-MYC. OCT4 expression was confirmed by immunoblotting (Suppl. Figure S1A) and by immunofluorescence microscopy (Suppl. Figure S1B). Differentiation of iPSCs to neural progenitor cells (NPCs) was done as previously described¹⁴ and as depicted in Suppl. Figure S1C, and the resulting cell line was designated niPS11 or niPS12. Expression of NPC marker proteins Pax6 and Nestin was confirmed in niPS11 by immunoblotting and immunofluorescence, respectively (Suppl. Figure S1A and S1B).

In order to evaluate genotoxin-induced autophagy in iPSCs and thereof differentiated NPCs, we first investigated the general starvation-inducible autophagic capacity of these two cell models. For that, we starved the cells in the absence or presence of bafilomycin A₁, which is a vacuolar-type H⁺-translocating ATPase (V-ATPase) inhibitor blocking autolysosomal degradation, and analyzed autophagy by immunoblotting for the autophagy markers phospho-ULK1 (Ser758), LC3B, and p62/SQSTM1. The autophagy-inducing kinase ULK1 is phosphorylated at Ser758 by mTOR complex 1 (mTORC1) and thus kept in an inhibited state. Dephosphorylation of this site correlates to the induction of autophagy^15–17. Lipidated LC3B (LC3B-II) decorates the inner and outer surfaces of autophagic membranes and recruits both components of the autophagic machinery and cargo to be degraded. p62 is an autophagy receptor mediating the recruitment of autophagic cargo. In both cell lines and in both differentiation states, starvation induced autophagic flux as determined by these three markers, i.e., reduced phosphorylation of ULK1 at Ser758, increased LC3B-II turnover (difference in LC3B-II levels with and without bafilomycin A₁), and increased p62 turnover (difference in p62 levels with and without bafilomycin A₁) (Fig. 1A-D). Collectively, these data indicate the “general” autophagic capacity of both iPSCs and NPCs.

In order to determine suitable concentrations of the genotoxins BPDE and etoposide for our autophagy assays, we first determined IC₂₀ values. In the following analyses, we deliberately chose to use IC₂₀ concentrations in order to remain within the sublethal range and avoid cell death. We performed MTT assays in iPS11 and niPS11, and analyzed viability after 24, 48 and 72 h, respectively (Suppl. Figure S2). As treatment scheme, cells were exposed to etoposide for 24 h and subsequent medium exchanges were without etoposide. In contrast, BPDE was freshly supplemented to the cells every 24 h. As control, we used the colon carcinoma cell line HCT116. For both genotoxins, IC₂₀ values were lower in iPS11 as compared to niPS11 over all time points, indicating a higher sensitivity of iPS11 for DNA-damaging agents (Suppl. Figure S2). Next, we assessed DNA damage by immunoblotting for phospho-p53 (Ser15) and phospho-H2AX (Ser139; γH2AX). Ser15 of p53 can become phosphorylated by ATM, ATR, and DNA-PK, and this phosphorylation prevents p53 from associating with MDM2, ultimately leading to the accumulation and activation of p53 following DNA damage^18,19. Similarly, phosphorylation of H2AX at Ser139 is also mediated by the mentioned kinases upon DNA damage^20,21. In iPS11 and iPS12, both phospho-substrates were detectable upon both treatments (Suppl. Figures S3A and S4A). Of note, γH2AX was also clearly induced by starvation (EBSS) in both iPSC lines. This was also the case for niPS11, niPS12 and HCT116 (Suppl. Figures S3B/S3C and S4B), and this observation might be attributed to a p38-dependent phosphorylation of H2AX²². In iPSCs and NPCs, γH2AX appeared to be partially sensitive to bafilomycin A₁ treatment (Suppl. Figures S3A/S3B and S4A/S4B). Generally, the DNA damage-induced phosphorylation of p53 and H2AX was clearly observable in HCT116, and also more distinct in comparison to untreated controls (Suppl. Figure S3C).

We next analyzed Xeroderma pigmentosum group C protein (XPC) levels. XPC generally functions as an initiator of global genome nucleotide excision repair (GG-NER), which repairs lesions generated by BPDE²³. Although DNA double strand breaks are the main type of damage induced by etoposide, NER proteins have also been linked to topoisomerase II inhibitors²⁴. XPC levels were clearly increased by both genotoxins in control HCT116 cells. In iPS11/iPS12 and niPS11/niPS12 cells, effects on XPC levels were less prominent, with the exception of BPDE-treated niPS11 and niPS12 cells (Suppl. Figures S3D and S4C).

We also investigated induction of DNA damage by immunofluorescence microscopy (Fig. 2). iPS11, niPS11 and HCT116 cells were treated with genotoxins for 4 and 24 h, and stained for γH2AX and 53BP1 (Fig. 2A-C). Co-localization of these two proteins is indicative for DSB^25,26. Quantification revealed that etoposide induces γH2AX/53BP1-double positive foci in iPS11 and HCT116 cells after 4 h (Fig. 2D and F). This is also the case for HCT116 cells after 24 h (Fig. 2F). Interestingly, in iPS11 cells single and double-positive foci are reduced in etoposide-treated cells after 24 h (Fig. 2D). This might indicate an “overshooting” repair and thus a hormesis effect. Please note that image-based quantification for niPS11 cells is hampered by their overlapping growing behavior. With regard to BPDE treatment, γH2AX/53BP1-double positive foci were observed in iPS11 and HCT116 cells at both time points (Fig. 2E and G).

Collectively, these data indicate that both genotoxins generally induce a DNA damage response in iPS11/iPS12 and niPS11/niPS12 cells.

Since the main goal of our project was to investigate genotoxin-induced autophagy in stem cells and thereof differentiated cells, we next investigated the activation status of the autophagy-inducing kinase ULK1. For that, we analyzed ULK1 phosphorylation at Ser758 and Ser638 (another mTOR/AMPK-dependent phospho-site) upon etoposide or BPDE treatment. In iPS11 and iPS12, no alterations of ULK1 activation status were observable, neither for etoposide nor for BPDE (Figs. 3A and 4A, Suppl. Figures S5A and S6A), although responsiveness towards starvation could be confirmed (see also Fig. 1A and B). In contrast, ULK1 activation (i.e., dephosphorylation at Ser758 and Ser638) was detected in niPS11 and niPS12 (Figs. 3B and 4B, Suppl. Figures S5B and S6B), although the extent of ULK1 activation was not as strong as with starvation. Of note, the effect of etoposide treatment was indifferent in HCT116, with rather decreased Ser758 phosphorylation and increased Ser638 phosphorylation (Fig. 3C). With regard to BPDE, a consistent pattern of ULK1 activation was observed in HCT116 cells (Fig. 4C). To rule out the possibility that we might have overlooked ULK1 activation at earlier time points, we also performed this analysis in iPS11 cells after 3 and 6 h of genotoxic treatment. However, ULK1 phosphorylation at Ser638 or Ser758 remained unchanged also at these early time points (Suppl. Figure S5C). These data indicate that early autophagy events such as the activation of ULK1 do not occur in iPSCs upon genotoxin treatment, whereas this appears to be the case in NPCs.

Next to early ULK1 activation, we also investigated the effect of the two genotoxins on autophagic flux. For that, we monitored again turnover of LC3B and p62. Neither in iPS11/iPS12 nor in niPS11/niPS12 cells autophagic turnover of LC3B or p62 were significantly induced (Fig. 5A and B, Suppl. Figure S8A and S8B). Of note, this was also the case for the cancer cell line HCT116 (Fig. 5C). All three cell models remained responsive to bafilomycin A₁ (indicating basal autophagy) and to starvation, as indicated by an EBSS-dependent reduction of p62. For LC3B turnover, we again performed earlier analyses after 3 and 6 h of treatment. Similar to the longer incubation times, we did not observe etoposide- or BPDE-induced LC3B turnover in iPS11, niPS11, or HCT116 (Suppl. Figure S9A-C). Furthermore, we also analyzed the effect of higher genotoxin concentrations (IC₅₀ values) in iPS11 cells. Again, no LC3B turnover was detectable (Suppl. Figure S10). Next to immunoblot-based detection of LC3B, we again employed immunofluorescence microscopy in order to monitor LC3 puncta formation (Fig. 6A-C). Quantification for iPS11 cells confirmed the lack of genotoxin-induced LC3 puncta formation (Fig. 6D and E). In contrast to the immunoblot data, etoposide treatment significantly increased the number of LC3 puncta in HCT116 cells after 24 h (Fig. 6F). BPDE, however, was also ineffective in this cell line (Fig. 6G). Taken together, these observations lead to the conclusion that low doses (IC₂₀) of the genotoxins BPDE and etoposide do not mount a canonical autophagic response in iPSCs or NPCs.

In order to get a global overview of the genotoxin-mediated alterations of the cellular proteome, we performed mass spectrometry. In iPS11, proteome changes with regard to biological relevance and statistical significance remained minimal for both treatments (Fig. 7A). This was also true for a list of autophagy-relevant genes (orange in Fig. 7A). One of the slightly enriched proteins upon BPDE treatment was the transcription factor nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1), which has been implicated in the regulation of both S phase entry and double-strand break repair^27–31. This upregulation was also confirmed by immunoblotting of the samples analyzed by mass spectrometry (Fig. 7B). Similar to iPS11, the proteome alterations with regard to autophagy-relevant proteins remained at low levels in niPS11, possibly except for a downregulation of PRKAR2A upon BPDE treatment and an upregulation of SESN2 upon etoposide treatment (the latter was confirmed by immunoblotting, Suppl. Figure S11). We specifically investigated if proteins linked to stress response (GO:0006950 from String 12.0 database) or metabolic processes (GO:0008152 from String 12.0 database) are altered (Suppl. Figure S12). However, no general left- or right-sided bias is observable in the corresponding volcano plots. What we did observe in niPS11, however, was the upregulation of proteins involved in the regulation of mitosis (Fig. 7C). Again, this was confirmed by immunoblotting for the candidate proteins polo like kinase 1 (PLK1) and aurora kinase A (AURKA) (Fig. 7D). Interestingly, for BPDE treatment, an AURKA downregulation was observed. We next aimed at determining whether these observed changes in protein abundance translated into mitotic alterations. For that we performed immunoblotting (acetylated tubulin), mitotic index assays, and growth curves (Fig. 7D and E). During mitosis, microtubules become acetylated, and this posttranslational modification is important for proper spindle function and chromosome segregation^32,33. We observed increased levels of acetylated tubulin upon BPDE treatment, but this was not the case for etoposide. We also analyzed AURKA, PLK1 and acetylated tublin levels in niPS12, and observed increases for all three proteins upon treatment with both genotoxins except for AURKA upon etoposide treatment (Suppl. Figure S13). Phosphorylation of histone H3 at Ser10 is linked to chromosome condensation during mitosis^34–36. However, we did not detect any differences of H3 Ser10 phosphorylation upon treatment with genotoxins. Finally, proliferation rates appeared to be similar between untreated and treated niPS11 (Fig. 7E).

In summary, our data indicate that the environmental genotoxin BPDE and the topoisomerase II inhibitor etoposide do not activate the autophagic program in iPSCs and NPCs. Although niPS11/12 show a slight activation of ULK1 upon treatment, this does not result in increased autophagic flux. Generally, both cell types mount an autophagic response upon starvation, indicating that a functional autophagy machinery is present in these cells.

Antibodies against ULK1 (Cell Signaling Technology, Danvers, MA, USA, #8054, 1:1000), phospho ULK1 Serine 757 (Cell Signaling Technology, Danvers, MA, USA, #6888, 1:1000), phospho ULK1 Serine 638 (Cell Signaling Technology, Danvers, MA, USA, #14205, 1:1000), GAPDH (abcam, Cambridge, UK, #ab8245, 1:5000), LC3B (MBL, Woburn, MA, USA, #M-152–3, 1:200 for IF and Cell Signaling Technology, Danvers, MA, USA, #2775, 1:1000 for WB), SQSTM1/p62 (PROGEN, Heidelberg, Germany, #GP62-C, 1:1000), Vinculin (Sigma-Aldrich, St. Louis, MO, USA, #V9131, 1:2000), XPC (Cell Signaling Technology, Danvers, MA, USA, #14768, 1:1000), p53 (Cell Signaling Technology, Danvers, MA, USA, #9282, 1:1000), phospho p53 Serine 10 (Cell Signaling Technology, Danvers, MA, USA, #9284, 1:1000), OCT4a (Cell Signaling Technology, Danvers, MA, USA, #2840, 1:1000), Nestin (Merck Millipore, Darmstadt, Germany #MAB5326, 1:200), PAX6 (Biolegend, San Diego, CA, USA, #901302, for WB: 1:1000, for IF: 1:100), NUCKS1 (Proteintech, Chicago, IL, USA, #12023-2-AP, 1:1000), γH2AX (For WB: Cell Signaling Technology, Danvers, MA, USA, #2577, 1:1000 and for IF: Merck Millipore, Darmstadt, Germany #05-636, 1:100), 53BP1 (Novus bio, Centennial, CO, USA, 1:2500), Sestrin-2 (Cell Signaling Technology, Danvers, MA, USA, #8487, 1:1000), Aurora A (Cell Signaling Technology, Danvers, MA, USA, #144755, 1:1000), acetylated Tubulin (Sigma-Aldrich, St. Louis, MO, USA, #312701, 1:20000), PLK1 (abcam, Cambridge, UK, #ab189139, 1:1000), phospho Histone 3 Serine 10 (Cell Signaling Technology, Danvers, MA, USA, #3377, 1:1600) were used. For WB, IRDye 800- or IRDye 680-conjugated secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE, USA, #926-68077, #926-32211 and #926-32210). Secondary antibodies for immunofluorescence analyses and mitotic index assay were purchased from Jackson ImmunoResearch (Alexa Fluor 488-AffiniPure Goat Anti-Mouse IgG, 1:500, #115–545-003; Alexa Fluor 647-AffiniPure Goat Anti-Mouse IgG, 1:500, #115–605-003; Alexa Fluor 647-AffiniPure Goat Anti-Rabbit IgG, 1:500, #111–605 − 144 and Alexa Fluor 488-AffiniPure Goat Anti-Rabbit IgG, 1:500, #111–545-003). Other reagents used were bafilomycin A₁ (Sigma-Aldrich, St. Louis, MO, USA, #B1793), DMSO (PanReac AppliChem, Darmstadt, Germany, #A3672), 70% ethanol (VWR, Radnor, PA, USA, #85825.360), thiazolyl blue tetrazolium bromide (MTT, ROTH, Karlsruhe, Germany, #4022.3), Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA, #23225), DRAQ7™ (abcam, Cambridge, UK, #ab109202, 1:100), Benzo[a]pyrene diol epoxide (Santa Cruz, Dallas, TX, USA, #sc-503767) and etoposide (abcam, Cambridge, UK, #ab120227).

iPS11 and iPS12 were obtained from ALSTEM, INC., Richmond, CA 94,806, USA. iPSCs were cultured in mTeSR Plus (Stemcell Technologies, Vancouver, Canada, #100–0276) supplemented with 100 units/mL Penicillin-Streptomycin (P/S; 10,000 U/ml Penicilin, 10,000 µg/ml Streptomycin) (Thermo Fisher Scientific, Waltham, MA, USA, Gibco, #15140122). Neural progenitor cells were differentiated and cultured in self-prepared medium as previously described¹⁴. Maintenance medium (sm-) consists of Neuralbasal A medium (Thermo Fisher Scientific, Waltham, MA, USA, #10888022), DMEM/F12 HEPES (Thermo Fisher Scientific, Waltham, MA, USA, #31330038), B27 supplement without vitamin A (Thermo Fisher Scientific, Waltham, MA, USA, #12587010), N2 supplement (Thermo Fisher Scientific, Waltham, MA, USA, #17502048) and L-Glutamine (200 mM) (Thermo Fisher Scientific, Waltham, MA, USA, Gibco, #25030081). Medium was stored up to 2 weeks at 4 °C or aliquoted and frozen at -20 °C. Before usage, 3 µM CHIR99021 (Cayman Chemical, Ann Habor, MI, USA, #Cay13122-5), 500 nM Purmorphamine (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-104-465) and 150 µM (+)-Sodium L-ascorbate (Vitamin C) (Merck, Sigma-Aldrich, St. Louis, MO, USA, #A4034) were supplemented to the maintenance medium (sm+). Both cell types were cultivated in 6 well plates coated with Geltrex (Thermo Fisher Scientific, Waltham, MA, USA, #a1413302). Coated plates were incubated at 37 °C for 1 h before usage. After thawing of iPSCs and NPCs, cells were supplemented with 10 µM Rock inhibitor/Y-27,632 (Dihydrochloride) (Stemcell Technologies, #72304) for 24 h. For passaging and seeding, iPSCs were treated with ReLeSR (Stemcell Technologies, Vancouver, Canada, #100–0483) accordingly to manufacturer´s instructions and NPC were passaged by Accutase (Stemcell Technologies, Vancouver, Canada, #07922). HCT116 were cultured in McCoy medium (Thermo Fisher Scientific, Waltham, MA, USA, Gibco, #36600-021) supplemented with 100 units/mL Penicillin-Streptomycin and 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA, #F9665, LOT 0001655439) and passaged with Trypsin/EDTA 0.05% (Thermo Fisher Scientific, Waltham, MA, USA, Invitrogen, #2530096). All cells were cultured and treated at 37 °C and 5% CO₂ in a humidified atmosphere.

To circumvent autophagy induction by starvation due to rapid proliferation of the stem cells the medium was exchanged on a daily basis. Thereby, cells were exposed to etoposide for 24 h and subsequent medium exchanges were without etoposide. In comparison, BPDE was freshly supplemented to the cells every day. On the day of sample harvesting, the medium was exchanged 4 h before lysis and 40 nM bafilomycin A₁ were supplemented to the cells 2 h before lysis. Alternatively, bafilomycin A₁ was added to the medium 2 h prior to lysis. For showing autophagic response in the experiments, cells were washed with DPBS and incubated with EBSS (Thermo Fisher Scientific, Waltham, MA, USA, #24010-043) and corresponding medium control for 2 h. A detailed treatment scheme is depicted in supplemental Figure S14.

For 3 and 6 h incubation, cells were incubated with the genotoxins for the corresponding time periods and 40 nM bafilomycin A1 were supplemented to the cells 2 h before lysis.

For the analysis of differential proteome, cells were treated like described above. Thereby, analysis for etoposide treated cells were performed at 24 h and treated with BPDE after 48 h.

For assessment of cytotoxicity the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed. Cells were seeded in 96-well plates (density per well: iPS11: 2500 cells; niPS11: 15000 cells; HCT116: 300–1250 cells). 3–4 days after seeding, cells were treated with different concentrations of BPDE or etoposide, 0.1% DMSO as a solvent control for 24, 48 and 72 h. After the incubation time, 20 µL of a 5 mg/mL MTT stock solution (ROTH, Karlsruhe, Germany, #4022.3) were added to the cells and they were incubated at 37 °C and 5% CO₂ in a humidified atmosphere for 30 min. Upon removal of the MTT-containing medium 100 µL DMSO per well were added for extraction of the formazan. Absorbance was measured at 570 nm and 650 nm (reference) with a microplate reader (SynergyMx, BioTek, Winooski, VT, USA). After subtraction of the reference value, the mean of the absorbance of the solvent control was set as 100% and the relative viability was calculated for each sample.

For SDS PAGE and western blotting, cells were washed with DPBS and lyzed with RIPA buffer (150 mM Sodium cloride, 50 mM Tris-HCl, 1% Nonidet-40, 0.5% Sodium deoxycholate [w/v], 0.1% SDS [w/v] X PhosSTOP [Roche, Basel, Switzerland, #4906837001]), 1X protease inhibitor cocktail [Roche, Basel, Switzerland, #4693132001]) for 20 min on ice and the lysates were cleared by centrifugation at 18000 rcf and 4 °C for 20 min and quickly frozen in liquid nitrogen. Protein concentration was determined by BCA assay and sample buffer was added (62.5 mM Tris, 8.6% [v/v] glycerol, 2% [w/v] SDS, 33.3 µg/mL bromophenol blue, 1% [v/v] β-mercaptoethanol). Samples were heated at 95 °C for 5 min and then equal amounts of protein (25 µg) were subjected to SDS-polyacrylamide gels. After separation by SDS-PAGE, proteins were transferred to PVDF membranes (Merck, Darmstadt, Germany, #IPFL00010), blocked with 5% milk powder in TBST or EveryBlot Blocking Buffer (Bio-Rad, Hercules, CA, USA, #12010020) and analyzed using the indicated primary antibodies followed by appropriate IRDye 800- or IRDye 680-conjugated secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA). Fluorescence signals were detected using an Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, NE, USA) and signals were quantified with Image Studio Lite 5.2 (LI-COR Biosciences, Lincoln, NE, USA).

For immunofluorescence microscopy, cells were seeded on glass coverslips in 24-well plates. Coverslips for iPSCs and NPCs were coated with geltrex and incubated at 37 °C for 1 h. After treatment, cells were fixed in 4% paraformaldehyde for 15 min on room temperature, quenched with 50 mM NH₄Cl for 15 min and permeabilized with either 50 µg/mL digitonin (Sigma-Aldrich, St. Louis, MO, USA, #D141) or 0.5% Triton X-100 for 10 min. Fixed samples were blocked with 3% BSA (Roth, Karlsruhe, Germany, #8076) for 30 min and incubated with primary antibodies diluted in 3% BSA for 1 h at RT. Samples were then washed three times with DPBS, incubated with the appropriate secondary antibodies and 2 µg/mL DAPI (Roth, Karlsruhe, Germany, #6335.1) diluted in 3% BSA for 1 h and washed three times with DPBS. Afterwards, cells were embedded in ProLong Glass Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA, #P36980↗). Images were recorded with an Axio Observer 7 fluorescence microscope (Carl Zeiss Microscopy, Oberkochen, Germany) using a 40x/1,4 Oil DIC M27 Plan-Apochromat objective (Carl Zeiss Microscopy, Oberkochen, Germany) and an ApoTome 2 (Carl Zeiss Microscopy, Oberkochen, Germany).

For determination of the mitotic index, cells were detached and fixed in cold 70% ethanol and stored at 4 °C for up a week. During sample preparation, cells were permeabilized in 0.25% Triton X-100 on ice for 15 min and incubated with rabbit anti-H3 phospho-Ser10 antibody in wash buffer, consisting of 1% BSA in DPBS, overnight at 4˚C in constant rotation. All samples were washed twice in wash buffer and incubated anti-rabbit Alexa 488 diluted in wash buffer for 30 min at RT in the dark. After a final wash in washing buffer each cell pellet was re-suspended in 500 µL DPBS containing 3 µM Draq7 for DNA staining, filtered through a cell strainer, processed in a BD LSR Fortessa flow cytometer with FACSDIVA software and analyzed with FlowJo software.

To investigate the impact on proliferation, niPS11 were treated and cultured as described above and detached by using Accutase. Cells were incubated for 5 min at 37 °C and subsequently centrifuged at 60 x g for 3 min. Cell pellet was resuspended in 250 µL sm+ medium. Afterwards, 20 µL of cell suspension were mixed with 20 µL Trypan Blue Stain (0.4%) (Thermo Fisher Scientific, Waltham, MA, USA, Gibco, #15250-061) and measured by Luna Automated Cell Counter (Biocat, Heidelberg, Germany, Model #L10001↗). Number of daily measured cells were divided by the cell number at 0 h.

All IC₂₀ values were calculated with non-linear regression using GraphPad Prism 8.0.2. For quantification of immunoblotting experiments, the signal of each protein band was divided by the average signal of all protein bands of the respective protein and furthermore normalized to the ratio of the loading control. These normalized ratios were divided by the average normalized ratio of the DMSO controls of all biological replicates to calculate fold changes. The background signal for each membrane was subtracted ahead of quantification. All p-values were calculated with ordinary one-way ANOVA (Tukey´s multiple comparison test) and student´s t-test if not indicated otherwise. For immunofluorescence analyses, puncta, nuclei and co-localization were quantified and analyzed using Biovoxxel ImageJ v1.54p. A punta/foci to nuclei ratio was calculated for each image to determine the average number of punta/foci per cell, and were normalized by dividing through the mean dot number of the solvent control. 10 representative images from three biological replicates per experiment were analyzed. For all immunofluorescence analyses, results are shown in scatter plot diagrams visualized as mean with standard deviation and p-values were determined by student´s t-test and are shown in the diagrams. All p-values are shown as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

The authors declare no competing interests.

iPSC lines iPS11 and iPS12 were obtained from ALSTEM, INC. (Richmond, CA 94806, USA) with material transfer agreement (MTA).

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE⁶⁰ partner repository with the dataset identifier PXD066480 and can be accessed here: https://www.ebi.ac.uk/pride/archive?keyword=PXD066480. The fasta files containing the protein sequence databases (downloaded on 12/21/2023 for the MaxQuant search or on 07/08/2024 for the DIA-NN search; present link: https://www.uniprot.org/uniprotkb?query=(proteome%3AUP000005640)↗) are provided with these proteomics data. Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author, Björn Stork (bjoern.stork@hhu.de).