RESUMEN
3T3-L1 is a model cell line which can be differentiated from preadipocytes into mature adipocytes. Here, we present a protocol for changing gene expression in 3T3-L1 (pre)adipocytes using small interfering RNA (siRNA)-mediated knockdown. We describe steps to perform the knockdown of a certain gene prior to differentiation (day 4) to analyze the impact on adipogenesis. We then detail procedures for knockdown on day 8 of differentiation to study the role of a certain gene in mature adipocyte function. For complete details on the use and execution of this protocol, please refer to Kaczmarek et al.1.
Asunto(s)
Células 3T3-L1 , Adipocitos , Adipogénesis , Diferenciación Celular , Técnicas de Silenciamiento del Gen , ARN Interferente Pequeño , Animales , Ratones , Adipocitos/metabolismo , Adipocitos/citología , ARN Interferente Pequeño/genética , Técnicas de Silenciamiento del Gen/métodos , Adipogénesis/genética , Diferenciación Celular/genética , Expresión Génica/genéticaRESUMEN
By differentiating into mature adipocytes, 3T3-L1 cells can be utilized as a model cell line to investigate (pre)adipocyte function in vitro. Here, we present a protocol for combining qualitative and quantitative analysis of lipid droplets in mature 3T3-L1 adipocytes using oil red O. We describe steps to differentiate 3T3-L1 preadipocytes to adipocytes and give detailed procedures to determine total lipid amount as well as lipid droplet size and number using microscopic devices and an ImageJ macro. For complete details on the use and execution of this protocol, please refer to Kaczmarek et al.1.
Asunto(s)
Células 3T3-L1 , Adipocitos , Compuestos Azo , Gotas Lipídicas , Animales , Ratones , Adipocitos/metabolismo , Adipocitos/citología , Gotas Lipídicas/metabolismo , Compuestos Azo/química , Diferenciación Celular , Coloración y Etiquetado/métodos , Metabolismo de los LípidosRESUMEN
AIMS: The purpose of this study is to report novel and unusual USP6 fusion partners in aneurysmal bone cysts (ABCs). These findings may be useful in routine diagnostics as well as in studying the biology of USP6-related disorders. METHODS: A cohort of seven patients diagnosed with ABC examined between 2014 and 2023 at Motol University Hospital in Prague was included into this retrospective non-randomised study. All cases were analysed using histopathological evaluation, immunohistochemistry and Anchored multiplex RNA methods. Demographic characteristics and clinical data were also analysed. RESULTS: We identified two novel (ZFX and IP6K2), three unusual (MEF2A, EIF1 and COL1A2) and two common (CDH11) fusion partners with USP6 gene among all seven cases of ABC. CONCLUSIONS: Cases in our study were diagnosed as ABCs due to characteristic clinical and morphological presentation. However, not all cases are as self-evident, and molecular testing is necessary. The identification of these gene alterations can be useful in distinction between true ABC and ABC-like changes among many benign and malignant bone tumours.
RESUMEN
In this study, we provide a comprehensive clinical and molecular biological characterization of radiation-induced gliomas (RIG), including a risk assessment for developing gliomas. A cohort of 12 patients who developed RIG 9.5 years (3-31 years) after previous cranial radiotherapy for brain tumors or T-cell acute lymphoblastic leukemia was established. The derived risk of RIG development based on our consecutive cohort of 371 irradiated patients was 1.6% at 10 years and 3.02% at 15 years. Patients with RIG glioma had a dismal prognosis with a median survival of 7.3 months. We described radiology features that might indicate the suspicion of RIG rather than the primary tumor recurrence. Typical molecular features identified by molecular biology examination included the absence of Histon3 mutation, methylation profile of pedHGG-RTK1 and the presence of recurrent PDGFRA amplification and CDKN2A/B deletion. Of the two long-term surviving patients, one had gliomatosis cerebri, and the other had pleomorphic xanthoastrocytoma with BRAF V600E mutation. In summary, our experience highlights the need for tissue diagnostics to allow detailed molecular biological characterization of the tumor, differentiation of the secondary tumor from the recurrence of the primary disease and potentially finding a therapeutic target.
Asunto(s)
Astrocitoma , Neoplasias Encefálicas , Glioma , Humanos , Proteínas Proto-Oncogénicas B-raf/genética , Glioma/genética , Glioma/radioterapia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Astrocitoma/patología , MutaciónRESUMEN
Obesity is one of the diseases with severe health consequences and rapidly increasing worldwide prevalence. Understanding the complex network of food intake and energy balance regulation is an essential prerequisite for pharmacological intervention with obesity. G protein-coupled receptors (GPCRs) are among the main modulators of metabolism and energy balance. They, for instance, regulate appetite and satiety in certain hypothalamic neurons, as well as glucose and lipid metabolism and hormone secretion from adipocytes. Mutations in some GPCRs, such as the melanocortin receptor type 4 (MC4R), have been associated with early-onset obesity. Here, we identified the adhesion GPCR latrophilin 1 (ADGRL1/LPHN1) as a member of the regulating network governing food intake and the maintenance of energy balance. Deficiency of the highly conserved receptor in mice results in increased food consumption and severe obesity, accompanied by dysregulation of glucose homeostasis. Consistently, we identified a partially inactivating mutation in human ADGRL1/LPHN1 in a patient suffering from obesity. Therefore, we propose that LPHN1 dysfunction is a risk factor for obesity development.