All three networks can be biochemically targeted in a specific manner (Determine 1a and Table S2). Open in a separate window Figure 1 Effect of cytoskeleton on lobular structure maintenance. differentiation is usually Mouse monoclonal to CD105 equally associated with chromatin reorganization, with deposition of nuclear envelope-limited chromatin linens at NE bending points in human cells [25,26] and wide-spread chromosomal supercontraction in murine cells [27]. In this study, we exploit the suspended nature of myeloid cells to isolate the cellular system from extracellular causes and substrate-anchoring points, and we take lobulation and segmentation of granulocyte nuclei as a model for cell-intrinsic nuclear remodeling. In vivo, remodeling of the spherical myeloid nucleus is usually a three-stage process across bean-shaped nuclei in metamyelocytes, proto-lobulation in band cells and final nuclear segmentation in granulocytes when nuclear lobules individual, linked by thin DNA-containing L-(-)-α-Methyldopa (hydrate) filaments [28,29]. Here, we show that cytosolic cytoskeleton does not contribute to maintenance or generation of nuclear lobules and nuclear segments. In vivo, differentiation is usually uncoupled from nuclear remodeling, as shown by L-(-)-α-Methyldopa (hydrate) functionally mature granulocytes displaying round or non-lobulated nuclei upon mutations in laminB-receptor (LBR) gene [30,31,32]. Given this concurrent but not necessarily causative relationship, we temporally profile transcriptomic changes in differentiating granulocytes and identify a metabolic pathway involving the enzymatic activity of LBR as temporally concurrent with nuclear remodeling. Ultimately, targeted biochemical challenging of several enzymes participating in this pathway reveals a putative contribution of the enzymatic activity of LBR in nuclear lobulation and the essential role of protein prenylation in both lobulation and nuclear segmentation. 2. Materials and Methods All experimental procedures are further detailed in the Extended Materials and Methods section in the Supplementary Materials. 2.1. Cell Cultures HL60 cells were from ECACC (Sigma-Aldrich, St. Louis, MI, USA, cat#98070106) and managed in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA) + 10% FBS (Thermo Fisher Scientific). Granulocytic differentiation was induced by 5 M all-trans-retinoic acid (Sigma-Aldrich, St. Louis, MO, USA) at Day 0 to 2 105 cell/mL cultures. For RNA collection, at Day 2 iHL60 cultures were diluted 1:5 with new medium. Biological replicates are impartial differentiation protocols of subsequent culture passages. 2.2. RNA Processing Total RNA was isolated at 0, 48, and 96 h of ATRA treatment from 107 cells with TRIzol Reagent (Thermo Fisher Scientific) followed by purification with RNeasy Mini Kit (Qiagen, Hilden, Germany). 5 g of total RNA were further processed at GeneWiz, Suzhou, China. For real time PCR, High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific) and PowerUp? SYBR? Green Grasp Mix (Thermo Fisher Scientific) were used. Primer sequences are reported in Table S1. 2.3. Bioinformatics Analyses RNA data were processed as previously reported [33]. For updated software versions and detailed description of data filtering, observe Supplementary Information. Gene expression data are publicly available on Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under the GEO IDs: GSE134922. 2.4. Drug Treatments Targets, suppliers and recommendations for each drug are reported in Table S2. Length of treatment and drug concentration vary and are reported in the text. In double treatment experiments, all compounds were administered simultaneously, with the exception of 3-day long experiments, where cells were pre-treated for 1 h with either latrunculin A or Y-27632 before vincristine sulfate supplementation. 2.5. Live-Cell Imaging Cell nuclei were stained with 1 g/mL Hoechst L-(-)-α-Methyldopa (hydrate) 33,342 (Cell Signaling Technologies, Danvers, MA, USA). The endoplasmic reticulum was stained with 2 mM ER-Tracker? Blue-White DPX (Thermo Fisher Scientific). Imaging was performed with an inverted Zeiss LSM710 laser-scanning confocal microscope, 100 oil-immersion objective, 405 nm excitation wavelength and a 0.5 m step. 2.6. Image Analyses For volume and surface quantifications, images of nuclei stained with ER-Tracker? were processed with the Image Processing Toolbox of MATLAB software (R2015b). 2.7. Qualitative Evaluation of Nuclear Lobulation The Number of lobules was manually derived for each nucleus from Hoechst33342 staining images and plotted as count distribution for quantity of lobules. The Maximum quantity of sections was manually derived from ER-Tracker staining images by considering the maximum number of nuclear sections in a cell for any given focal plane in the z-stack, and plotted as count distribution for quantity of sections. For qualitative analyses, the three groups were defined as Round/Ovoid, according to geometry, Segmented if the nucleus offered at least 2 well defined separated volumes, and Deformed when neither of the previous two applied. Qualitative evaluation is usually offered as percentage on total populace.