Spatiotemporal analysis of multi-scale cell structure in spheroid culture reveals hypertrophic chondrocyte differentiation.

3D cell culture has emerged as a promising approach to replicate the complex behaviors of cells within living organisms. This study aims to analyze spatiotemporal behavior of the morphological characteristics of cell structure at multiscale in 3D scaffold-free spheroids using chondrogenic progenitor ATDC5 cells. Over a 14-day culture period, it exhibited cell hypertrophy in the spheroids regarding cellular and nuclear size as well as changes in morphology. Moreover, biological analysis indicated a signification up-regulation of normal chondrocyte as well as hypertrophic chondrocyte markers, suggesting early hypertrophic chondrocyte differentiation. Cell nuclei underwent changes in volume, sphericity, and distribution in spheroid over time, indicating alterations in chromatin organization. The ratio of chromatin condensation volume to cell nuclear volume decreased as the cell nuclei enlarged, potentially signifying changes in chromatin state during hypertrophic chondrocyte differentiation. Our image analysis techniques in this present study enabled detailed morphological measurement of cell structure at multi-scale, which can be applied to various 3D culture models for in-depth investigation.

Poor Sperm Chromatin Condensation Is Associated with Cryopreservation-Induced DNA Fragmentation and Cell Death in Human Spermatozoa.

Background/Objectives: Semen cryopreservation is routinely performed in fertility clinics for a variety of reasons, including fertility preservation and storage of donor sperm, yet the freeze-thaw process leads to cellular damage via ice crystal formation, osmotic shock, and supraphysiological levels of oxidative stress. Sperm resistance to damage during the freeze-thaw process varies widely, yet the intrinsic factors associated with sperm cryotolerance are largely unknown. The study aimed to investigate whether poor chromatin condensation renders sperm vulnerable to DNA fragmentation and cell death induced by the freeze-thaw process. Methods: Participants (n = 51) from the general community who met the inclusion criteria collected a semen sample after 3-8 days of abstinence. Neat semen samples underwent traditional semen analysis, aniline blue (AB)-eosin staining for chromatin condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay for DNA fragmentation, and the Annexin V assay for apoptosis/necrosis, prior to being cryopreserved using the liquid nitrogen vapour method and stored at -196 °C. Stored samples were later thawed at room temperature and processed using density gradient centrifugation. Motile sperm concentration, DNA fragmentation and apoptosis/necrosis were analysed in post-thaw samples. Results: As indicated by a significant interaction effect in linear mixed models, an increased proportion of AB-positive sperm in the pre-freeze sample exacerbated the adverse effect of freezing on sperm DNA fragmentation (p = 0.004), late apoptosis (p = 0.007), and necrosis (p = 0.007). AB-staining was positively correlated with all three parameters in the post-thaw sample (all rs ≥ 0.424, all p < 0.01) and remained significant after adjusting for neat sperm concentration (all partial rs ≥ 0.493, all p < 0.01). Similarly, AB-staining was significantly correlated with the percentage point change in sperm DNA fragmentation (rs = 0.366, p = 0.014) and necrosis (rs = 0.403, p = 0.009), both of which remained significant after adjusting for neat sperm concentration (both partial rs ≥ 0.404, both p < 0.01), and borderline significantly correlated with percentage point change in late apoptosis (rs = 0.307, p = 0.051). Conclusions: Sperm with poorly condensed chromatin may be more susceptible to cellular damage during the freeze-thaw process, independent of pre-freeze sperm concentration. These findings may help to explain the intrinsic variation in sperm resistance to cryodamage within and between individuals that is poorly understood.

Regulation of Oocyte mRNA Metabolism: A Key Determinant of Oocyte Developmental Competence.

The regulation of mRNA transcription and translation is uncoupled during oogenesis. The reason for this uncoupling is two-fold. Chromatin is only accessible to the transcriptional machinery during the growth phase as it condenses prior to resumption of meiosis to ensure faithful segregation of chromosomes during meiotic maturation. Thus, transcription rates are high during this time period in order to produce all of the transcripts needed for meiosis, fertilization, and embryo cleavage until the newly formed embryonic genome becomes transcriptionally active. To ensure appropriate timing of key developmental milestones including chromatin condensation, resumption of meiosis, segregation of chromosomes, and polar body extrusion, the translation of protein from transcripts synthesized during oocyte growth must be temporally regulated. This is achieved by the regulation of mRNA interaction with RNA binding proteins and shortening and lengthening of the poly(A) tail. This chapter details the essential factors that regulate the dynamic changes in mRNA synthesis, storage, translation, and degradation during oocyte growth and maturation.

Kalanchoe pinnata (Lam.) Pers. Leaf ethanolic extract exerts selective anticancer activity through ROS-induced apoptotic cell death in human cancer cell lines.

BACKGROUND: The leaves of Kalanchoe pinnata (Lam.) Pers. (K. pinnata), a succulent plant native to tropical regions, are used as a medicinal alternative against cancer in several countries worldwide; however, its therapeutic potential to fight cancer has been little addressed. In this study, we analyzed the phytochemical content, antioxidant capacity, and selectivity of K. pinnata leaf ethanolic extract against different human cancer cell lines in vitro. METHODOLOGY: This study subjected the ethanolic extract to enzymatic assays to quantify the phytochemical content (phenolics, flavonoids, and anthraquinones) and its radical scavenging and iron-reducing capacities. Also, the phytoconstituents and major phenolic compounds present in the extract's subfractions were identified by GC-MS, HPLC, and NMR. Human cancer (MCF-7, PC-3, HT-29) and normal colon (CoN) cell lines were treated with different concentrations of K. pinnata leaf ethanolic extract, and the changes in cell proliferation (sulforhodamine B assay), caspases activity (FITC-VAD-FMK reporter), mitochondrial membrane potential (MMP, rhodamine 123 assay), chromatin condensation/fragmentation (Hoechst 33342 stain), and ROS generation (DCFH2 probe assay) were assessed. RESULTS: The results showed that the K. pinnata leaf ethanolic extract is rich in phytoconstituents with therapeutic potential, including phenols (quercetin and kaempferol), flavonoids, fatty acid esters (34.6% of the total composition), 1- triacontanol and sterols (ergosterol and stigmasterol, 15.4% of the total composition); however, it presents a poor content of antioxidant molecules (IC50 = 27.6 mg/mL for H2O2 scavenging activity vs. 2.86 mg/mL in the case of Trolox). Notably, the extract inhibited cell proliferation and reduced MMP in all human cell lines tested but showed selectivity for HT-29 colon cancer cells compared to CoN normal cells (SI = 8.4). Furthermore, ROS generation, caspase activity, and chromatin condensation/fragmentation were augmented significantly in cancer-derived cell lines, indicating a selective cytotoxic effect. CONCLUSION: These findings reveal that the K. pinnata leaf ethanolic extract contains several bioactive molecules with therapeutic potential, capable of displaying selective cytotoxicity in different human cancer cell lines.

When Chromatin Decondensation Affects Nuclear γH2AX Foci Pattern and Kinetics and Biases the Assessment of DNA Double-Strand Breaks by Immunofluorescence.

Immunofluorescence with antibodies against phosphorylated forms of H2AX (γH2AX) is revolutionizing our understanding of repair and signaling of DNA double-strand breaks (DSBs). Unfortunately, the pattern of γH2AX foci depends upon a number of parameters (nature of stress, number of foci, radiation dose, repair time, cell cycle phase, gene mutations, etc…) whose one of the common points is chromatin condensation/decondensation. Here, we endeavored to demonstrate how chromatin conformation affects γH2AX foci pattern and influences immunofluorescence signal. DSBs induced in non-transformed human fibroblasts were analyzed by γH2AX immunofluorescence with sodium butyrate treatment of chromatin applied after the irradiation that decondenses chromatin but does not induce DNA breaks. Our data showed that the pattern of γH2AX foci may drastically change with the experimental protocols in terms of size and brightness. Notably, some γH2AX minifoci resulting from the dispersion of the main signal due to chromatin decondensation may bias the quantification of the number of DSBs. We proposed a model called "Christmas light models" to tentatively explain this diversity of γH2AX foci pattern that may also be considered for any DNA damage marker that relocalizes as nuclear foci.

Corrigendum: Tpr deficiency disrupts erythroid maturation with impaired chromatin condensation in zebrafish embryogenesis.

[This corrects the article DOI: 10.3389/fcell.2021.709923.].

Impact of heat and cold shock on epigenetics and chromatin structure.

Cells are continuously exposed to various sources of insults, among which temperature variations are extremely common. Epigenetic mechanisms, critical players in gene expression regulation, undergo alterations due to these stressors, potentially leading to health issues. Despite the significance of DNA methylation and histone modifications in gene expression regulation, their changes following heat and cold shock in human cells remain poorly understood. In this study, we investigated the epigenetic profiles of human cells subjected to hyperthermia and hypothermia, revealing significant variations. Heat shock primarily led to DNA methylation increments and epigenetic modifications associated with gene expression silencing. In contrast, cold shock presented a complex scenario, with both methylation and demethylation levels increasing, indicating different epigenetic responses to the opposite thermal stresses. These temperature-induced alterations in the epigenome, particularly their impact on chromatin structural organization, represent an understudied area that could offer important insights into genome function and potential prospects for therapeutic targets.

Helical chromonema coiling is conserved in eukaryotes.

Efficient chromatin condensation is required to transport chromosomes during mitosis and meiosis, forming daughter cells. While it is well accepted that these processes follow fundamental rules, there has been a controversial debate for more than 140 years on whether the higher-order chromatin organization in chromosomes is evolutionarily conserved. Here, we summarize historical and recent investigations based on classical and modern methods. In particular, classical light microscopy observations based on living, fixed, and treated chromosomes covering a wide range of plant and animal species, and even in single-cell eukaryotes suggest that the chromatids of large chromosomes are formed by a coiled chromatin thread, named the chromonema. More recently, these findings were confirmed by electron and super-resolution microscopy, oligo-FISH, molecular interaction data, and polymer simulation. Altogether, we describe common and divergent features of coiled chromonemata in different species. We hypothesize that chromonema coiling in large chromosomes is a fundamental feature established early during the evolution of eukaryotes to handle increasing genome sizes.

Nuclear sperm integrity and ICSI prognosis in Tunisian patients with MMAF syndrome (multiple morphological abnormalities of the sperm flagella).

Multiple Morphological Abnormalities of the Sperm Flagella (MMAF) is a severe form of teratozoospermia associated with several sperm flagellar abnormalities. The study included 52 patients with MMAF syndrome and a control group of 25 fertile men. The impact of nuclear sperm quality on intracytoplasmic sperm injection (ICSI) results was studied in 20 couples. TUNEL assay was used to assess sperm DNA fragmentation and aniline-blue staining was used to assess chromatin condensation. To investigate chromosomal meiotic segregation, we used fluorescence in situ hybridization (FISH). Semen morphology analysis revealed a mosaic of multiple flagella morphological abnormalities, including 46.73% short flagella, 16.22% bent flagella, 22.07% coiled flagella, and 10.90% absent flagella, all of which were associated with a high percentage of sperm head abnormalities. The mean DNA fragmentation index was substantially higher in patients compared to controls (p = 0.001), whereas the rate of aniline blue-reacted spermatozoa was not significantly different. There was a significant difference in aneuploidy frequencies between the two groups (p < 0.05). Infertile males with MMAF syndrome had lower sperm nuclear quality, which affected ICSI results. As a result, better sperm selection procedures are being employed to increase the success rate of assisted reproductive technologies (ART).

Catalytic and non-catalytic mechanisms of histone H4 lysine 20 methyltransferase SUV420H1.

SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/H4K20me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation. It is dysregulated in several cancers. Many of these processes were linked to its catalytic activity. However, deletion and inhibition of SUV420H1 have shown distinct phenotypes, suggesting that the enzyme likely has uncharacterized non-catalytic activities. Our cryoelectron microscopy (cryo-EM), biochemical, biophysical, and cellular analyses reveal how SUV420H1 recognizes its nucleosome substrates, and how histone variant H2A.Z stimulates its catalytic activity. SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from the histone octamer, which is a non-catalytic activity. We hypothesize that this regulates the accessibility of large macromolecular complexes to chromatin. We show that SUV420H1 can promote chromatin condensation, another non-catalytic activity that we speculate is needed for its heterochromatin functions. Together, our studies uncover and characterize the catalytic and non-catalytic mechanisms of SUV420H1, a key histone methyltransferase that plays an essential role in genomic stability.

Di-2-ethylhexyl phthalate-induced neurobehavioural transformation is associated with altered glutathione biosynthesis and neurodegeneration in zebrafish brain.

The contamination of life-sustaining environments with synthetic pollutants such as plastic-derived compounds has increased at an alarming rate in recent decades. Among such contaminants, di-2-ethylhexyl phthalate (DEHP) is an extensively used compound in plastics and plastic products to make them flexible. DEHP causes several adverse effects such as reproductive toxicity leading to infertility, miscarriage and litter size reduction, disruption of the thyroid endocrine system, oxidative stress, neurodevelopmental defect and cognitive impairment. An aquatic environment is a fragile site, where the accumulation of DEHP poses a significant threat to living organisms. In this context, the present study focused on whether the neurobehavioural transformation following exposure to DEHP is an outcome of augmented oxidative stress and neuromorphological alteration in the zebrafish brain. Our preliminary findings advocate that DEHP acts as a typical neurotoxicant in inducing neurobehavioural transformation in zebrafish. Furthermore, our study also supports the idea that DEHP itself acts as a potent neurotoxicant by altering the glutathione biosynthetic pathway through the induction of oxidative stress in the zebrafish brain. Similarly, our findings also link the abovementioned neurobehavioural transformation and oxidative stress with augmented neuronal pyknosis and chromatin condensation in the periventricular grey zone of the zebrafish brain following chronic exposure to DEHP. Therefore, the overall conclusion of the present study advocates the potential role of DEHP in inducing neuropathological manifestation in the zebrafish brain. Future research directed towards elucidating the neuroprotective efficacy of natural compounds against DEHP-induced neurotoxicity may provide a new line of intervention.

Lipid droplets are intracellular mechanical stressors that impair hepatocyte function.

Matrix stiffening and external mechanical stress have been linked to disease and cancer development in multiple tissues, including the liver, where cirrhosis (which increases stiffness markedly) is the major risk factor for hepatocellular carcinoma. Patients with nonalcoholic fatty liver disease and lipid droplet-filled hepatocytes, however, can develop cancer in noncirrhotic, relatively soft tissue. Here, by treating primary human hepatocytes with the monounsaturated fatty acid oleate, we show that lipid droplets are intracellular mechanical stressors with similar effects to tissue stiffening, including nuclear deformation, chromatin condensation, and impaired hepatocyte function. Mathematical modeling of lipid droplets as inclusions that have only mechanical interactions with other cellular components generated results consistent with our experiments. These data show that lipid droplets are intracellular sources of mechanical stress and suggest that nuclear membrane tension integrates cell responses to combined internal and external stresses.

Maintaining soluble protein homeostasis between nuclear and cytoplasmic compartments across mitosis.

The nuclear envelope (NE) is central to the architecture of eukaryotic cells, both as a physical barrier separating the nucleus from the cytoplasm and as gatekeeper of selective transport between them. However, in open mitosis, the NE fragments to allow for spindle formation and segregation of chromosomes, resulting in intermixing of nuclear and cytoplasmic soluble fractions. Recent studies have shed new light on the mechanisms driving reinstatement of soluble proteome homeostasis following NE reformation in daughter cells. Here, we provide an overview of how mitotic cells confront this challenge to ensure continuity of basic cellular functions across generations and elaborate on the implications for the proteasome - a macromolecular machine that functions in both cytoplasmic and nuclear compartments.

Suppression of Chronic Unpredictable Stress-Persuaded Increased Monoamine Oxidase Activity by Taurine Promotes Significant Neuroprotection in Zebrafish Brain.

Neuropsychiatric upshots following chronic exposure to unpredictable adverse stressors have been well documented in the literature. Considering the significant impact of chronic unpredictable stress (CUS), the literature is elusive regarding the neuroprotective efficacy of taurine against CUS-induced oxidative stress and chromatin condensation in the zebrafish brain. In this study, to ameliorate CUS-persuaded neurological outcomes, waterborne treatment of taurine as a prophylactic intervention was undertaken. Further, our approach also focused on the gross neurobehavioral response of zebrafish, oxidative stress indices and neuromorphology of the zebrafish brain following CUS exposure with taurine treatment. Because taurine provides significant neuroprotection against oxidative insult, the cytosolic level of monoamine oxidase (MAO) in the zebrafish brain following CUS exposure is worth investigating. Further, as heightened MAO activity is associated with augmented oxidative and chromatin condensation, the focus of this study was on whether taurine provides neuroprotection by downregulating MAO levels in the brain. Our findings show that CUS-persuaded altered neurobehavioral response was significantly rescued by taurine. Moreover, our findings firmly support the hypothesis that taurine acts as a radical neuroprotector by restoring glutathione biosynthesis in the zebrafish brain subsequent to CUS exposure. Additionally, the rising level of brain MAO following chronic exposure to CUS is ameliorated by taurine treatment. These findings strongly advocate the role of taurine as a natural MAO inhibitor through the neuroprotection it provides against CUS-instigated oxidative stress in zebrafish. However, the fundamental neuroprotective mechanism of such natural compounds needs to be elucidated to determine their neuroprotective efficacy against stress regimens.

Subcellular elements responsive to the biomechanical activity of triple-negative breast cancer-derived small extracellular vesicles.

Triple-negative breast cancer (TNBC) stands out for its aggressive, fast spread, and highly metastatic behavior and for being unresponsive to the classical hormonal therapy. It is considered a disease with a poor prognosis and limited treatment options. Among the mechanisms that contribute to TNBC spreading, attention has been recently paid to small extracellular vesicles (sEVs), nano-sized vesicles that by transferring bioactive molecules to recipient cells play a crucial role in the intercellular communication among cancer, healthy cells, and tumor microenvironment. In particular, TNBC-derived sEVs have been shown to alter proliferation, metastasis, drug resistance, and biomechanical properties of target cells. To shed light on the molecular mechanisms involved in sEVs mediation of cell biomechanics, we investigated the effects of sEVs on the main subcellular players, i.e., cell membrane, cytoskeleton, and nuclear chromatin organization. Our results unveiled that TNBC-derived sEVs are able to promote the formation and elongation of cellular protrusions, soften the cell body, and induce chromatin decondensation in recipient cells. In particular, our data suggest that chromatin decondensation is the main cause of the global cell softening. The present study added new details and unveiled a novel mechanism of activity of the TNBC-derived sEVs, providing information for the efficient translation of sEVs to cancer theranostics.

Functional Aspects of Sperm Chromatin Organization.

Sperm nuclei present a highly organized and condensed chromatin due to the interchange of histones by protamines during spermiogenesis. This high DNA condensation leads to almost inert chromatin, with the impossibility of conducting gene transcription as in most other somatic cells. The major chromosomal structure responsible for DNA condensation is the formation of protamine-DNA toroids containing 25-50 kilobases of DNA. These toroids are connected by toroid linker regions (TLR), which attach them to the nuclear matrix, as matrix attachment regions (MAR) do in somatic cells. Despite this high degree of condensation, evidence shows that sperm chromatin contains vulnerable elements that can be degraded even in fully condensed chromatin, which may correspond to chromatin regions that transfer functionality to the zygote at fertilization. This chapter covers an updated review of our model for sperm chromatin structure and its potential functional elements that affect embryo development.

Determination of Apoptotic Mechanism of Action of Tetrabromobisphenol A and Tetrabromobisphenol S in Human Peripheral Blood Mononuclear Cells: A Comparative Study.

BACKGROUND: Tetrabromobisphenol A (TBBPA) is the most commonly used brominated flame retardant (BFR) in the industry. TBBPA has been determined in environmental samples, food, tap water, dust as well as outdoor and indoor air and in the human body. Studies have also shown the toxic potential of this substance. In search of a better and less toxic BFR, tetrabromobisphenol S (TBBPS) has been developed in order to replace TBBPA in the industry. There is a lack of data on the toxic effects of TBBPS, while no study has explored apoptotic mechanism of action of TBBPA and TBBPS in human leukocytes. METHODS: The cells were separated from leucocyte-platelet buffy coat and were incubated with studied compounds in concentrations ranging from 0.01 to 50 µg/mL for 24 h. In order to explore the apoptotic mechanism of action of tested BFRs, phosphatidylserine externalization at cellular membrane (the number of apoptotic cells), cytosolic calcium ion and transmembrane mitochondrial potential levels, caspase-8, -9 and -3 activation, as well as PARP-1 cleavage, DNA fragmentation and chromatin condensation in PBMCs were determined. RESULTS: TBBPA and TBBPS triggered apoptosis in human PBMCs as they changed all tested parameters in the incubated cells. It was also observed that the mitochondrial pathway was mainly involved in the apoptotic action of studied compounds. CONCLUSIONS: It was found that TBBPS, and more strongly TBBPA, triggered apoptosis in human PBMCs. Generally, the mitochondrial pathway was involved in the apoptotic action of tested compounds; nevertheless, TBBPS more strongly than TBBPA caused intrinsic pathway activation.

CHD5 gene (rs9434741) might be a genetic risk factor for infertility in non-obstructive azoospermia and severe oligozoospermia.

Inability to conceive is one of the health concerns. Chromodomain helicase DNA-binding protein5 (CHD5) gene is a major regulator in the replacement of histone proteins with protamines, the chromatin remodelling in spermatogenesis process. Thus, functional SNPs in this gene can disrupt sperm development. This study aimed to investigate the relationship between CHD5 polymorphism (rs9434741) and male infertility. This case-control study was conducted on 103 infertile and 121 fertile men. CHD5 polymorphism rs9434741 was tested using T-ARMS-PCR to investigate its association with male infertility. The presence of G allele caused 1.52 fold increase (OR = 1.52, 95% CI = 1.09-2.31 and p = 0.019) in infertility susceptibility in the patient group. GG genotype and (AG+GG) were significantly related to the increased risk of infertility (OR = 3.13, 95% CI = 1.26-7.76, p = 0.013; OR = 2.72, 95% CI = 1.35-5.47), respectively. Significant differences were observed between genotypes in NOA and SO groups compared to the control group (p = 0.029). Sperm count and total motility were significantly different among three genotypes in infertile men and the control group (p < 0.001). Analysis of genotypes and alleles frequency indicated statistically significant differences between the patient and control groups (p < 0.05). This study showed that CHD5 polymorphism (rs9434741) could be associated with the risk of male infertility. It is recommended to conduct further studies on different populations.

Delayed reorganisation of F-actin cytoskeleton and reversible chromatin condensation in scleral fibroblasts under simulated pathological strain.

Mechanical loading regulates the functional capabilities of the ocular system, particularly in the sclera ('white of the eye') - the principal load-bearing tissue of the ocular globe. Resident fibroblasts of the scleral eye wall are continuously subjected to fluctuating mechanical strains arising from eye movements, cerebrospinal fluid pressure and, most influentially, intra-ocular pressure (IOP). Whilst fibroblasts are hypothesised to actively participate in scleral biomechanics, to date limited information has been reported on how the macroscopic stresses and strains are transmitted via their cytoskeletal networks. In this study, the effect of applying either a 'physiological load' (simulating healthy IOP) or a 'pathological load' (simulating an elevated glaucomatous IOP) to bovine scleral fibroblasts, as a model of human glaucoma, was conducted to characterise cytoskeletal organisation, chromatin condensation and cell dimensions using immunofluorescence confocal microscopy. Quantification of cell parameters and cytoskeletal element anisotropy were subsequently performed using FibrilTool, and chromatin condensation parameter assessment through a bespoke MATLAB script. The novel findings suggest that physiological load-induced F-actin rearrangement is transient, whereas pathological load, recapitulating in vivo glaucomatous IOP levels, had a reversible and inhibitory influence on remodelling of the cytoskeletal architecture and, further, induction of chromatin condensation. Ultimately, this could compromise cell behaviour. These findings could provide valuable insight into the mechanism(s) used by scleral fibroblasts to mechanically adapt to support biomechanical tissue integrity, and how it could be potentially modified for therapeutic avenues targeting mechanically mediated ocular pathologies such as glaucoma.

Changes in intra-nuclear mechanics in response to DNA damaging agents revealed by time-domain Brillouin micro-spectroscopy.

How DNA damage and repair processes affect the biomechanical properties of the nucleus interior remains unknown. Here, an opto-acoustic microscope based on time-domain Brillouin spectroscopy (TDBS) was used to investigate the induced regulation of intra-nuclear mechanics. With this ultrafast pump-probe technique, coherent acoustic phonons were tracked along their propagation in the intra-nucleus nanostructure and the complex stiffness moduli and thicknesses were measured with an optical resolution. Osteosarcoma cells were exposed to methyl methanesulfonate (MMS) and the presence of DNA damage was tested using immunodetection targeted against damage signaling proteins. TDBS revealed that the intra-nuclear storage modulus decreased significantly upon exposure to MMS, as a result of the chromatin decondensation and reorganization that favors molecular diffusion within the organelle. When the damaging agent was removed and cells incubated for 2 h in the buffer solution before fixation the intra-nuclear reorganization led to an inverse evolution of the storage modulus, the nucleus stiffened. The same tendency was measured when DNA double-strand breaks were caused by cell exposure to ionizing radiation. TDBS microscopy also revealed changes in acoustic dissipation, another mechanical probe of the intra-nucleus organization at the nano-scale, and changes in nucleus thickness during exposure to MMS and after recovery.