Enhanced visualization of nuclear staining and cell cycle analysis for the human commensal Malassezia.

Malassezia is a lipophilic commensal yeast that resides mainly on the mammalian skin and is also found to associate with the internal organs. Dysbiosis of Malassezia is related to several diseases and often escapes detection as it is difficult to culture and maintain. Malassezia cell wall differs from other budding yeasts like S. cerevisiae due to the difference in the lipid content and is difficult to transform. In this study, we present a methodology to stain Malassezia's nucleus and perform cell cycle studies. However, staining presents a challenge due to its exceptionally thick cell wall with high lipid content, hindering conventional methods. Our novel methodology addresses this challenge and enables the staining of the Malassezia nucleus with a low background. This would allow researchers to visualize the overall nuclear health specifically nuclear morphology and analyze DNA content, crucial for cell cycle progression. By employing DNA-specific dyes like DAPI or Hoechst, we can observe the nuclear structure, and using PI we can differentiate cells in distinct cell cycle phases using techniques like flow cytometry. This novel staining methodology unlocks the door for in-depth cell cycle analysis in Malassezia which has challenged us through ages being refractory to genetic manipulations, paving the way for a deeper understanding of this commensal fungus and its potential role in human health.

SUMOylation of nuclear receptor Nor1/NR4A3 coordinates microtubule cytoskeletal dynamics and stability in neuronal cells.

BACKGROUND: Nor1/NR4A3 is a member of the NR4A subfamily of nuclear receptors that play essential roles in regulating gene expression related to development, cell homeostasis and neurological functions. However, Nor1 is still considered an orphan receptor, as its natural ligand remains unclear for mediating transcriptional activation. Yet other activation signals may modulate Nor1 activity, although their precise role in the development and maintenance of the nervous system remains elusive. METHODS: We used transcriptional reporter assays, gene expression profiling, protein turnover measurement, and cell growth assays to assess the functional relevance of Nor1 and SUMO-defective variants in neuronal cells. SUMO1 and SUMO2 conjugation to Nor1 were assessed by immunoprecipitation. Tubulin stability was determined by acetylation and polymerization assays, and live-cell fluorescent microscopy. RESULTS: Here, we demonstrate that Nor1 undergoes SUMO1 conjugation at Lys-89 within a canonical ψKxE SUMOylation motif, contributing to the complex pattern of Nor1 SUMOylation, which also includes Lys-137. Disruption of Lys-89, thereby preventing SUMO1 conjugation, led to reduced Nor1 transcriptional competence and protein stability, as well as the downregulation of genes involved in cell growth and metabolism, such as ENO3, EN1, and CFLAR, and in microtubule cytoskeleton dynamics, including MAP2 and MAPT, which resulted in reduced survival of neuronal cells. Interestingly, Lys-89 SUMOylation was potentiated in response to nocodazole, a microtubule depolymerizing drug, although this was insufficient to rescue cells from microtubule disruption despite enhanced Nor1 gene expression. Instead, Lys-89 deSUMOylation reduced the expression of microtubule-severing genes like KATNA1, SPAST, and FIGN, and enhanced α-tubulin cellular levels, acetylation, and microfilament organization, promoting microtubule stability and resistance to nocodazole. These effects contrasted with Lys-137 SUMOylation, suggesting distinct regulatory mechanisms based on specific Nor1 input SUMOylation signals. CONCLUSIONS: Our study provides novel insights into Nor1 transcriptional signaling competence and identifies a hierarchical mechanism whereby selective Nor1 SUMOylation may govern neuronal cytoskeleton network dynamics and resistance against microtubule disturbances, a condition strongly associated with neurodegenerative diseases.

Functional genetics reveals modulators of anti-microtubule drug sensitivity.

Microtubules play essential roles in diverse cellular processes and are important pharmacological targets for treating human disease. Here, we sought to identify cellular factors that modulate the sensitivity of cells to anti-microtubule drugs. We conducted a genome-wide CRISPR/Cas9-based functional genetics screen in human cells treated with the microtubule-destabilizing drug nocodazole or the microtubule-stabilizing drug taxol. We further conducted a focused secondary screen to test drug sensitivity for ~1400 gene targets across two distinct human cell lines and to additionally test sensitivity to the Kif11-inhibitor, STLC. These screens defined gene targets whose loss enhances or suppresses sensitivity to anti-microtubule drugs. In addition to gene targets whose loss sensitized cells to multiple compounds, we observed cases of differential sensitivity to specific compounds and differing requirements between cell lines. Our downstream molecular analysis further revealed additional roles for established microtubule-associated proteins and identified new players in microtubule function.

The microtubule cytoskeleton: A validated target for the development of 2-Aryl-1H-benzo[d]imidazole derivatives as potential anticancer agents.

In this study, a series of 2-Aryl-1H-benzo[d]imidazole derivatives were developed to target intra- and extracellular microtubule networks. Compounds O-7 and O-10 showed impressive anti-proliferative activity across various tested cell lines, demonstrating selectivity indexes of 151.7 and 61.9, respectively. O-7 achieved an IC50 value of 0.236 ± 0.096 µM, while O-10 showed an IC50 value of 0.622 ± 0.13 µM against A549 cell lines. The induction of early-stage apoptosis in a dose-dependent manner further underscored the potential of O-7 and O-10 as effective anti-proliferative agents. O-7 and O-10 exhibited substantial inhibition of wound closure, with wound closure percentages decreasing from 23% at 0 µM to 0.43% and 2.62% at 20 µM, respectively. Colony formation reduction rates were impressive, with O-7 at 74.2% and O-10 at 81.2%. These results indicate that the O-7 and O-10 can impede cancer cell migration and have a high potential to curtail colony formation. The mode of action investigations for O-7 and O-10 revealed that O-7 could inhibit in vitro tubulin polymerization and disrupt the intracellular microtubule cytoskeleton. This disruption led to cell cycle arrest in the G2/M phase, indicating that O-7 exerts its anticancer activity through microtubule destabilization. However, O-10 shows a different mode of action than O-7 and requires further investigation. Overall, our study showcases the potential of the synthesized benzimidazole derivatives as novel and selective anticancer agents, motivating further exploration of their pharmacological properties and therapeutic applications.

Microtubule association of TRIM3 revealed by differential extraction proteomics.

The microtubule network is formed from polymerised tubulin subunits and associating proteins, which govern microtubule dynamics and a diverse array of functions. To identify novel microtubule-binding proteins, we have developed an unbiased biochemical assay, which relies on the selective extraction of cytosolic proteins from U2OS cells, while leaving behind the microtubule network. Candidate proteins are linked to microtubules by their sensitivities to the depolymerising drug nocodazole or the microtubule-stabilising drug taxol, which is quantitated by mass spectrometry. Our approach is benchmarked by co-segregation of tubulin and previously established microtubule-binding proteins. We then identify several novel candidate microtubule-binding proteins, from which we have selected the ubiquitin E3 ligase tripartite motif-containing protein 3 (TRIM3) for further characterisation. We map TRIM3 microtubule binding to its C-terminal NHL-repeat region. We show that TRIM3 is required for the accumulation of acetylated tubulin, following treatment with taxol. Furthermore, loss of TRIM3 partially recapitulates the reduction in nocodazole-resistant microtubules characteristic of α-tubulin acetyltransferase 1 (ATAT1) depletion. These results can be explained by a decrease in ATAT1 following depletion of TRIM3 that is independent of transcription.

Not all benzimidazole derivatives are microtubule destabilizing agents.

In recent years, microtubule-targeting agents (MTAs) have gained considerable interest in developing novel small-molecule anticancer drugs. MTAs demonstrate anticancer activity either as microtubule-stabilizing agents (paclitaxel) or microtubule-destabilizing agents (nocodazole). FDA-approved drugs containing a benzimidazole ring (nocodazole, albendazole, mebendazole, etc.) are well-known microtubule-destabilizing agents. Thus, most recent research on benzimidazole scaffold-based MTAs focuses on developing microtubule-destabilizing agents. However, there is no report on the benzimidazole scaffold-based microtubule-stabilizing agent. Here, we present the benzimidazole derivatives NI-11 and NI-18 that showed a profound anticancer activity as microtubule-stabilization agents. About twenty benzimidazole analogues were synthesized with excellent yield (80.0% ∼ 98.0%) and tested for their anticancer activity using two cancer cell lines (A549, MCF-7) and one normal cell line (MRC-5). NI-11 showed IC50 values of 2.90, 7.17, and 16.9 µM in A549, MCF-7, and MRC-5 cell lines. NI-18 showed IC50 values of 2.33, 6.10, and 12.1 µM in A549, MCF-7, and MRC-5 cell lines. Thus, NI-11 and NI-18 demonstrated selectivity indexes of 5.81 and 5.20, respectively, which are much higher than the currently available anticancer agents. NI-11 and NI-18 inhibited the cancer cell motility and migration, induced the early phase apoptosis. Both of these comounds were found to show an upregulation of DeY-α-tubulin and downregulation of Ac-α-tubulin expressions in cancer cells. Eventhough the reported benzimidazole scaffold-based commercially available drugs are known to be microtubule-destabilizing agents, the analogues NI-11 and NI-18 were found to have microtubule-stabilizing activity. The in vitro tubulin polymerization assay and the immunofluorescence assay results indicate that the NI-11 and NI-18 exhibit anticancer activity by stabilizing the microtubule network.

Measurement of Microtubule Stability in Mammalian Cells.

The microtubule cytoskeleton is essential for various biological processes such as the intracellular distribution of molecules and organelles, cell morphogenesis, chromosome segregation, and specification of the location of contractile ring formation. Distinct cell types contain microtubules with different extents of stability. For example, microtubules in neurons are highly stabilized to support organelle (or vesicular) transport over large distances, and microtubules in motile cells are more dynamic. In some cases, such as the mitotic spindle, both dynamic and stable microtubules coexist. Alteration of microtubule stability is connected to disease states, making understanding microtubule stability an important area of research. Methods to measure microtubule stability in mammalian cells are described here. Together, these approaches allow microtubule stability to be measured qualitatively or semiquantitatively following staining for post-translational modifications of tubulin or treating cells with microtubule destabilizing agents such as nocodazole. Microtubule stability can also be measured quantitatively by performing fluorescence recovery after photobleaching or fluorescence photoactivation of tubulin in live cells. These methods should be helpful for those seeking to understand microtubule dynamics and stabilization. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Fixing and staining cells for tubulin post-translational modifications Basic Protocol 2: Evaluating microtubule stability following treatment with nocodazole in live or fixed cells Basic Protocol 3: Measurement of microtubule dynamic turnover by quantification of fluorescence recovery after photobleaching Basic Protocol 4: Measurement of microtubule dynamic turnover by quantification of dissipation of fluorescence after photoactivation.

A myeloid leukemia factor homolog is involved in tolerance to stresses and stress-induced protein metabolism in Giardia lamblia.

BACKGROUND: The eukaryotic membrane vesicles contain specific sets of proteins that determine vesicle function and shuttle with specific destination. Giardia lamblia contains unknown cytosolic vesicles that are related to the identification of a homolog of human myeloid leukemia factor (MLF) named MLF vesicles (MLFVs). Previous studies suggest that MLF also colocalized with two autophagy machineries, FYVE and ATG8-like protein, and that MLFVs are stress-induced compartments for substrates of the proteasome or autophagy in response to rapamycin, MG132, and chloroquine treatment. A mutant protein of cyclin-dependent kinase 2, CDK2m3, was used to understand whether the aberrant proteins are targeted to degradative compratments. Interestingly, MLF was upregulated by CDK2m3 and they both colocalized within the same vesicles. Autophagy is a self-digestion process that is activated to remove damaged proteins for preventing cell death in response to various stresses. Because of the absence of some autophagy machineries, the mechanism of autophagy is unclear in G. lamblia. RESULTS: In this study, we tested the six autophagosome and stress inducers in mammalian cells, including MG132, rapamycin, chloroquine, nocodazole, DTT, and G418, and found that their treatment increased reactive oxygen species production and vesicle number and level of MLF, FYVE, and ATG8-like protein in G. lamblia. Five stress inducers also increased the CDK2m3 protein levels and vesicles. Using stress inducers and knockdown system for MLF, we identified that stress induction of CDK2m3 was positively regulated by MLF. An autophagosome-reducing agent, 3-methyl adenine, can reduce MLF and CDK2m3 vesicles and proteins. In addition, knockdown of MLF with CRISPR/Cas9 system reduced cell survival upon treatment with stress inducers. Our newly developed complementation system for CRISPR/Cas9 indicated that complementation of MLF restored cell survival in response to stress inducers. Furthermore, human MLF2, like Giardia MLF, can increase cyst wall protein expression and cyst formation in G. lamblia, and it can colocalize with MLFVs and interact with MLF. CONCLUSIONS: Our results suggest that MLF family proteins are functionally conserved in evolution. Our results also suggest an important role of MLF in survival in stress conditions and that MLFVs share similar stress-induced characteristics with autophagy compartments.

Cell Synchronization Techniques for Studying Mitosis.

Cell synchronization allows the examination of cell cycle progression. Nocodazole and other microtubule poisons have been used extensively to interfere with microtubule function and arrest cells in mitosis. Since microtubules are important for many cellular functions, alternative cell cycle synchronization techniques independent of microtubule inhibition are also used for synchronizing cells in mitosis. Here we describe using nocodazole, STLC, and combining thymidine block with MG132 to synchronize cells in mitosis. These inhibitors are reversible and mitotic cells can be released into the G1 phase synchronously. These techniques can be applied to both Western blot and timelapse imaging to study mitotic progression.

Synchronization of Saccharomyces cerevisiae Cells for Analysis of Progression Through the Cell Cycle.

The cell division cycle is a fundamental process required for proliferation of all living organisms. The eukaryotic cell cycle follows a basic template with an ordered series of events beginning with G1 (Gap1) phase, followed successively by S (Synthesis) phase, G2 (Gap 2) phase, and M-phase (Mitosis). The process is tightly regulated in response to signals from both the internal and external milieu. The budding yeast S. cerevisiae is an outstanding model for the study of the cell cycle and its regulatory process. The basic events and regulatory processes of the S. cerevisiae cell cycle are highly conserved with other eukaryotes. The organism grows rapidly in simple medium, has a sequenced annotated genome, well-established genetics, and is amenable to analysis by proteomics and microscopy. Additionally, a range of tools and techniques are available to generate cultures of S. cerevisiae that are homogenously arrested or captured at specific phases of the cell cycle and upon release from that arrest these can be used to monitor cell cycle events as the cells synchronously proceed through a division cycle. In this chapter, we describe a series of commonly used techniques that are used to generate synchronized populations of S. cerevisiae and provide an overview of methods that can be used to monitor the progression of the cells through the cell division cycle.

The Role of Cytoskeletal Proteins in the Formation of a Functional In Vitro Blood-Brain Barrier Model.

The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain's microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB.

Synthesis and evaluation of 2-aryl-1H-benzo[d]imidazole derivatives as potential microtubule targeting agents.

Microtubule targeting agents (MTAs) are the potential drug candidates for anticancer drug discovery. Disrupting the microtubule formation or inhibiting the de-polymerization process by a synthetic molecule can lead to an excellent anticancer drug candidate. Here, we present the 2,5-substituted-1H-benzo[d]imidazole derivatives as potential colchicine, nocodazole binding site targeting agents. About 20 benzimidazole derivatives were synthesized with 82.0%-94.0% yield using mild reaction conditions. The synthesized compounds showed moderate to excellent anticancer activity established in three cell lines, including Hela cells, A549 cells, MRC-5 cells. The compounds B15, B16, B19, and B20 are the potential candidates with the IC50 values <15 µM in the three different cell lines. In MTT assay, compounds B15, B16, B19, and B20 showed excellent antiproliferation activity indicated by IC50 values in the range of 5.3 ± 0.21 to 18.1 ± 0.32 µM using HeLa and A549 cell lines. The predicted absorption, distribution, metabolism and excretion (ADME) properties and drug-likeness properties of B15, B16, B19, and B20 indicate that these compounds can be used as lead compounds for further study to develop excellent MTAs.

Potential therapies and diagnosis based on Golgi-targeted nano drug delivery systems.

With the rapid development of nanotechnology, organelle-targeted nano drug delivery systems (NDDSs) have emerged as a potential method which can transport drugs specifically to the subcellular compartments like nucleus, mitochondrion, lysosome, endoplasmic reticulum (ER) and Golgi apparatus (GA). GA not only plays a key role in receiving, modifying, packaging and transporting proteins and lipids, but also contributes to a set of cellular processes. Golgi-targeted NDDSs can alter the morphology of GA and will become a promising strategy with high specificity, low-dose administration and decreased occurrence of side effects. In this review, Golgi-targeted NDDSs and their applications in disease therapies and diagnosis such as cancer, metastasis, fibrosis and neurological diseases are introduced. Meanwhile, modifications of NDDSs to achieve targeting strategies, Golgi-disturbing agents to change the morphology of GA, special endocytosis to achieve endosomal/lysosomal escape strategies are also involved.

Role of microtubule dynamics in Wallerian degeneration and nerve regeneration after peripheral nerve injury.

Wallerian degeneration, the progressive disintegration of distal axons and myelin that occurs after peripheral nerve injury, is essential for creating a permissive microenvironment for nerve regeneration, and involves cytoskeletal reconstruction. However, it is unclear whether microtubule dynamics play a role in this process. To address this, we treated cultured sciatic nerve explants, an in vitro model of Wallerian degeneration, with the microtubule-targeting agents paclitaxel and nocodazole. We found that paclitaxel-induced microtubule stabilization promoted axon and myelin degeneration and Schwann cell dedifferentiation, whereas nocodazole-induced microtubule destabilization inhibited these processes. Evaluation of an in vivo model of peripheral nerve injury showed that treatment with paclitaxel or nocodazole accelerated or attenuated axonal regeneration, as well as functional recovery of nerve conduction and target muscle and motor behavior, respectively. These results suggest that microtubule dynamics participate in peripheral nerve regeneration after injury by affecting Wallerian degeneration. This study was approved by the Animal Care and Use Committee of Southern Medical University, China (approval No. SMU-L2015081) on October 15, 2015.

Differential Expression of PD-L1 during Cell Cycle Progression of Head and Neck Squamous Cell Carcinoma.

The expression of PD-L1 by tumor cells is mainly associated with its immunosuppressive effect. In fact, PD-1/PD-L1 immune checkpoint inhibitors demonstrated remarkable effects in advanced cancer patients including HNSCC. In this context, irradiation is currently being investigated as a synergistic treatment modality to immunotherapy. However, the majority of HNSCC patients still show little improvement or even hyperprogression. Interestingly, there is increasing evidence for additional cell-intrinsic functions of PD-L1 in tumor cells. In previous studies, we showed that PD-L1 has a strong influence on proliferation, migration, invasion, and survival after irradiation. We demonstrated that cellular expression and localization of PD-L1 differed depending on sensitivity to irradiation. Here, we show that PD-L1 is also differentially expressed during cell cycle progression of HNSCC. Furthermore, cellular localization of PD-L1 also changes depending on a particular cell cycle phase. Moreover, distinct observations occurred depending on the general differentiation status. Overall, the function of PD-L1 cannot be generalized. Rather, it depends on the differentiation status and microenvironment. PD-L1 expression and localization are variable, depending on different factors. These findings may provide insight into why differential response to PD-1/PD-L1 antibody therapy can occur. Detailed understanding of cell-intrinsic PD-L1 functions will further allow antibody-based immunotherapy to be optimized.

Synthesis, Fungicidal Activity, and Molecular Docking of 2-Acylamino and 2-Thioacylamino Derivatives of 1H-benzo[d]imidazoles as Anti-Tubulin Agents.

This work deals with the synthesis and evaluation of fungicidal activity of benzimidazole derivatives, which are structural analogues of commercial anti-tubulin fungicides. A number of N-acyl and N-thioacyl derivatives of 2-amino-1H-benzo[d]imidazole were prepared, and their fungicidal activity against 13 strains of phytopathogenic fungi was studied. The most active compounds against the majority of the studied strains were 3a, 4l, and 4o, and the EC50 values of these compounds were in the range 2.5-20 µg/mL. Compound 3a showed the highest activity against the P. infestans strain, the growth of which is not suppressed by carbendazim. The formation of ligand-receptor complexes of various tautomeric forms of the studied benzimidazoles with homologous models of ß-tubulins of B. cinerea, F. oxysporum, and P. infestans was modeled. Induced fit docking has been used for the simulation. The obtained data suggest the possibility of binding of benzimidazole fungicides to ß-tubulin in the ″nocodazole cavity″ in the tautomeric form bearing a double exocyclic C═N bond. The importance of the formation of hydrogen bonds of benzimidazoles with the amino acid residue Val236 along with the Glu198 residue is also revealed in the present study.

Anti-Tumoral Effects of a (1H-Pyrrol-1-yl)Methyl-1H-Benzoimidazole Carbamate Ester Derivative on Head and Neck Squamous Carcinoma Cell Lines.

Nocodazole is an antineoplastic agent that exerts its effects by depolymerizing microtubules. Herein we report a structural analog of nocodazole, a (1H-pyrrol-1-yl)methyl-1H-benzoimidazole carbamate ester derivative, named RDS 60. We evaluated the antineoplastic properties of RDS 60 in two human head and neck squamous cell carcinoma (HNSCC) cell lines and we found that this compound significantly inhibited replication of both HNSCC cell lines without inducing any important cytotoxic effect on human dermal fibroblasts and human keratinocytes. The treatment of HNSCC cell lines with 1 µM RDS 60 for 24 h stopped development of normal bipolar mitotic spindles and, at the same time, blocked the cell cycle in G2/M phase together with cytoplasmic accumulation of cyclin B1. Consequently, treatment with 2 µM RDS 60 for 24 h induced the activation of apoptosis in both HNSCC cell lines. Additionally, RDS 60 was able to reverse the epithelial-mesenchymal transition and to inhibit cell migration and extracellular matrix infiltration of both HNSCC cell lines. The reported results demonstrate that this compound has a potent effect in blocking cell cycle, inducing apoptosis and inhibiting cell motility and stromal invasion of HNSCC cell lines. Therefore, the ability of RDS 60 to attenuate the malignancy of tumor cells suggests its potential role as an interesting and powerful tool for new approaches in treating HNSCC.

The inhibition of microtubule dynamics instability alters lipid homeostasis in TM4 Sertoli cells.

Sertoli cells (SC) structurally support and transport nutrients to germ cells during spermatogenesis facilitated by an active cytoskeleton. Chemical perturbation of SC microtubule (MT) dynamics instability leads to premature germ cell exfoliation demonstrating that this process is essential for male fertility, yet the effects of MT damaging drugs on SC lipid metabolism have been less explored. The aim of this study was to advance our understanding of how adequate SC MT dynamicity is needed to finely tune lipid homeostasis. To elucidate the role of MT dynamics instability on the latter, we suppressed MT dynamicity by long-term exposures to 10 nM of nocodazole (NCZ) on TM4-SC cultures. Inhibition of MT dynamics instability affected the distribution of [3H] arachidonate on TM4-SC. Triacylglycerols (TAG) exhibited a higher proportion of the [3H] label, with significantly lower percentages in the mitochondrial phospholipid cardiolipin, and notably, also in phosphatidylethanolamine. A noteworthy and progressive accumulation of lipid droplets during the period of exposure to NCZ was accompanied by increased TAG levels but not cholesterol levels in TM4-SC. NCZ-exposed cells reduced their mitochondrial membrane potential and increased ROS production without triggering apoptosis, had a compromised autophagic flux, and lost their transferrin expression. Although SC morphology was preserved, the NCZ-exposed cells displayed alteration of the normal organization of microfilaments (f-actin) and intermediate filaments (vimentin). Our findings suggest that a preserved MT dynamicity is essential in the maintenance of lipid and fatty acids homeostasis in SC, and thus highlights a novel target in these cells for drugs that impair MT dynamicity.

Optimizing Cell Synchronization Using Nocodazole or Double Thymidine Block.

Cell synchronization is crucial when studying events that take place at specific points of the cell cycle. Several chemical agents can be used to achieve the cell culture synchronization but not all type of cells respond equally to a given concentration of these drugs. Here we describe a simple optimization method to select concentrations and timings for nocodazole or thymidine treatments using fluorescence staining. In addition, we provide detailed protocols to arrest an asynchronous culture of either suspension or adherent cells in G1/S or in G2/M.

TGFBI is involved in the formation of polyploid cancer cells and the response to paclitaxel.

BACKGROUND: Most human solid tumors are aneuploid; at the same time, polyploid cancer cells are found to be resistant to radiotherapy and chemotherapy and have a poor prognosis. The transforming growth factor beta induction (TGFBI) protein plays important roles in the development of tumors, depending on the cancer of origin. METHODS: In this study, we established polyploid clones of breast cancer treated with nocodazole. The drug sensitivity was measured by MTT assay. Western blot analysis was used to detect the expression of TGFBI protein in polyploid clones. The effects of paclitaxel on apoptosis, cell cycle and DNA ploidy were analyzed by flow cytometry. TGFBI protein expression was performed in samples from patients with epithelial ovarian tumors by immunohistochemical staining. RESULTS: We found that compared with the MDA-MB-231 cell line, the expression of TGFBI in the HGF1806 cell line was relatively higher. In addition, compared with its parental cells, TGFBI showed relatively low expression in the polyploid breast cancer cell line T-MDA-MB-231. Compared with the empty vector, under paclitaxel treatment, the over-expression of TGFBI in MDA-MB-231 and T-MDA-MB-231 both showed a higher growth inhibition rate. After nocodazole treatment, the over-expression of TGFBI in MDF-MB-231 cells proved that the expression of tetraploid cells was lower compared to the control. The positive rate of TGFBI expression in ovarian cancer specimens before chemotherapy was 33.3% (5/15), which was higher than the positive rate of TGFBI expression in ovarian cancer specimens matched with relapsed specimens after treatment (0%, 0/15). CONCLUSIONS: TGFBI can increase the sensitivity of paclitaxel in polyploid cancer cells and participate in the formation of polyploidy in MDA-MB-231 induced by nocodazole. This newly recognized role of TGFBI provides further insight into the pathogenesis of polyploid cancer and identifies potential new therapeutic targets.