Earlier, we demonstrated that cytoplasm, mitochon- dria, vacuoles and nuclei of S. cerevisiae cells possesses PolyP by subcellular fractionation . However the methods for obtaining of pure fractions of the organelles need long time. The in vivo DAPI staining is a simple method for vital observing of PolyP. The data obtained suggest, that PolyP localization at the initial stage of its synthesis in S. cerevisiae cells depends on the carbon source. PolyP appears first of all in the cytoplasm and mitochondria under cultivation in glucose-containing medium and in the cytoplasm and vacuoles in the me- dium with ethanol. In the mutant with inactivated genes encoding two exopolyphosphatases (PPX1 and PPN1) PolyP accumulates in the cytoplasm and vacuoles. The role of mitochondria in PolyP synthesis needs further investigation. Comparison of chemical extraction data (Table 2) and DAPI staining (Figures 1 and 2) shows that staining character actually depends on PolyP content. However this dependence is not simple. Nevertheless, DAPI staining is useful for visualization of PolyP local- ization in living cells. The method of confocal micros- copy in vivo should be accompanied with chemical ex- traction of PolyP. The results show that at the early stage of PolyP biosynthesis after P i starvation the polymer is
Results: Viability studies of TD treated Huh7 cells showed an inhibition in cell growth in time and dose dependent manner. Chromatin condensation, DNA fragmentation and apoptotic bodies, which are structural changes characteristic of apoptosis, were found following TD treatment of Huh7 cells. DAPI staining and agarose gel electrophoresis confirmed the induction of apoptosis by TD. Cell cycle analysis of Huh7 cells treated with TD exhibited a marked accumulation of cells in the sub-G1 phase of the cell cycle in a dose dependent manner. Immunofluorescent staining for Ki-67 showed a higher level of expression in untreated cells as compared to TD treated cells. We observed a significant loss in the mitochondrial membrane potential and the release of cytochrome c into the cytosol in TD treated cells. Down regulation of Bcl-2, up regulation of Bax and Bad as well as activation of caspases-3 and 9 were also observed. The p53 gene expression was found to be unaltered in TD treated cells.
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the roots at multiple time points following infection and examined the seam cells in both live and DAPI-stained fixed animals. Using DIC optics, we found that following infection the posterior eight seam cells appear in contact with each other (Fig. 6b). Within 3 days following infec- tion, the nine posterior seam cells undergo a symmetrical cell division to produce two seam cells (Figs. 6c, 7a, b). The resulting 18 seam cells undergo divisions (Fig. 7b, c), to produce four epidermal daughter cells (Fig. 7d), ori- ented dorsoventrally during late J2. By the time of the J2/ J3 molt, we observed only elliptical-shaped nuclei (Fig. 7e arrow) along the lateral ridge suggesting an apparent dor- soventral migration of seam daughter nuclei (Fig. 7e). To confirm this migration of seam cell daughter cells from the lateral ridge, we examined the seam cell division and migration in post-infective J2 live animals dissected from the roots (Fig. 8). While plant-parasitic nematodes will not feed following removal from their host, a lim- ited amount of development will occur. Consistent with our DAPI staining, we observed cell divisions along the lateral ridge of four dpi animals (Fig. 8). Examination of subsequent time points showed that the number of cells along the lateral line increases with time (Fig. 8, 4.5 h). As the J2 approaches the J2/J3 molt (Fig. 8, 25 h), dorsoven- trally oriented cells migrated dorsoventrally (Fig. 8, 34 h). Adult female-12 dpi
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Methods: We firstly constructed the multi-domain recombinant chimeric proteins based on recombinant proteins [G (yeast GAL4), NG (none GAL4), TG (GAL4 + Tat protein) and TNG (Tat protein)] and pUAS-Apoptin plasmid, and transfected them into human HepG-2 cells. The antitumor effect of this multi-domain recombinant chimeric proteins to HCC cells were detected by MTT assay, AO/EB staining, DAPI staining and Annexin V assay. In order to find the path- way of cell apoptosis, the Caspase (1, 3, 6 and 8) activity was detected. We then constructed the H22 liver cancer mice model and analyzed the anti-tumor rate and mice survival rate after treated with G/pUAS-Apoptin NG/pUAS-Apoptin TG/pUAS-Apoptin, and TNG/pUAS-Apoptin.
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Figure 1.—Staining of chro- matin associated with tomato SC spreads. (A) Early pachy- tene SC set stained with DAPI only. Several SCs possess a re- gion where DAPI staining is not visible, i.e., a DAPI-negative band (arrows). No other chromatin- based features are visible. (B) Early pachytene SC spread stained with PI only. Hetero- chromatic regions (e.g., arrow- heads) appear wider and stain a bit more intensely than the distal euchromatic regions (e.g., arrows). (C) CPD staining of an early pachytene SC spread. Pericentromeric heterochro- matin fluoresces white while distal euchromatin fluoresces blue. A red-fluorescing chro- matin band (CPD band) is visi- ble within the pericentromeric heterochromatin of 5 of toma- to’s 12 SCs (arrows). These bands most likely correspond to the DAPI-negative bands seen in DAPI-stained tomato SC spreads. The NOR near the terminus of the short arm of SC 2 fluoresces red and usually exhibits partial or complete asynapsis (e.g., diamond-head arrows). (D) Late pachytene CPD-stained SC spread. Kinet- ochores are visible as thick- enings along SCs (e.g., white boxes). On two CPD-banded SCs, the kinetochores are lo- cated directly over the CPD band (arrowheads). However, on the remaining three CPD- banded SCs a red band is visi- ble outside of the region en- compassed by the kinetochore (arrows). The elongate, asyn- apsed NOR fluoresces red (dia- mond-head arrows). (E) Phase- contrast image of a complete spread of late pachytene to- mato SCs. A kinetochore is visi- ble on each SC (black arrows). One SC is broken into two pieces (white arrow). Likewise, the NOR region has been lost from the end of SC 2 (white arrowhead). (F) CPD staining of the same SC spread shown in E. CPD bands are visible on five of the SCs (arrows). (G) Diagram showing the relative position of CPD bands and kinetochores. Blue lines represent SCs, yellow dots represent kinetochores, and red bars represent CPD bands. The identity (chromosome number) of each CPD-banded chromosome is shown next to an arrow pointing to its kinetochore. (H) Idiogram of the tomato SC complement showing the distribution of CPD bands. SCs 1-12 are shown in consecutive order from left to right. Euchromatin is blue, heterochromatin is white, kinetochores are orange, and CPD bands are red. The NOR in the short arm of SC 2, which appears red after CPD staining, is colored pink in this idiogram. A constricted area within the NOR marks the general region where SC formation does not occur. Frames that share a common bar: A and B; C and D; and E–G. Bars, 10 mm.
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Background: Hsp90 proteins are important therapeutic targets for many anti-cancer drugs in clinical trials. Geldanamycin (GA) was identified as the first natural inhibitor of Hsp90, increasing evidence suggests that GA was not a good choice for clinical trials. In this study, we investigated two new non-benzoquinone geldanamycin analogs of Hsp90 inhibitors, DHQ3 and 17-demethoxy-reblastatin (17-DR), to explore the molecular mechanisms of their anti-cancer activity in vivo and vitro. Methods: MTT and colony formation assays were used to measure cell viability. Flow cytometry, DAPI staining, ATP assay, electron microscopy, western blots, siRNAs transfection and immunofluorescence were used to determine the molecular mechanism of DHQ3- or 17-DR-induced different forms of death in human breast cancer MDA-MB-231 cells. Malachite green reagent was used to measure ATPase activity of the analogs.
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Many neuropathologists have shown electron micro- graphs of RFs, but there is still little known about the formation of these inclusions. To gain insights into this question, we analyzed RFs during the progression of AxD in mouse models. We have used four murine models of AxD: 1) one based on the insertion of additional copies of human GFAP (transgenic AxD mice, TG); 2) one based on replacement of one copy of GFAP with a mutant (R236H mutation, the mouse homolog of the most common human AxD mutation) mouse GFAP (heterozygous knock-in AxD mice, heterozygous KI); 3) homozygous knock-in (KI) AxD mice, with replacement of both copies of GFAP with R236H mutant mouse GFAP; 4) the offspring of transgenic (TG) and homozygous knock-in (KI) mice (double mutant) [7, 16]. The latter are severely affected, dying within 5 weeks with seizures . All of these mice are characterized by an abundance of RFs. We performed electron microscopic examinations of RFs and found evidence that RFs form from small accu- mulations of electron dense material, containing GFAP and alphaB-crystallin, on intermediate filaments. These small RFs appear to merge with the edges of larger RFs. We also found that in addition to Fluoro-Jade B that DAPI staining is a sensitive marker of RFs, one that can be combined with immunofluorescence.
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Fig. 3. High-salt exposure limits ventricular wall development in E4.5 chick embryos. Fertilized chick eggs were exposed to 0.7% NaCl (control) and 300 mOsm l −1 NaCl (high salt) for 3 days as described above. Transverse sections of hearts were stained with H&E, and representative images from control (A) and high salt-exposed (B) hearts were taken at the same spatial position. (C,D) High- magnification images were taken from the sites indicated by boxed regions in A and B, respectively. (E,F) MF20 immunofluorescent staining was performed on serial slices as shown in C and D, respectively. (E1,F1) Merged images of MF20 immunofluorescence and DAPI staining. (G) Bar graph comparing the ventricular wall thickness of control and high-salt-exposed hearts. (H) Bar graph comparing the trabecular muscle layers. (I) Semi-quantitative RT-PCR detection of BMP2, Nkx2.5, VMHC and GATA4 mRNA expression in developing chick hearts after high osmolarity treatment. At, atrium; Ve, ventricle. Scale bars: 250 μ m (A,B), 100 μ m in (C,D), 100 μ m in (E – F1).
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A DAPI staining assay was performed to identify the nuclear morphological changes. The cells were seeded in 6- well plates in the medium and incubated for 24 hours. The cells were then treated at IC50 concentration of crude, nano extract of Alphonsea sclerocarpa and Gyrocarpus asiaticus for 24hours. Untreated cells were used as controls which contains only the complete medium and cells. After treatment, the cells were washed with 1X PBS once and harvested using centrifugation at 4000 rpm at 4°C for 5 minutes. The treated cells were fixed at -20°C for 10minutes with 50 μl of methanol and water (1:1). The 100 μl (1 μg/ml) of DAPI dye was added to the frozen cells and the mixture kept at 37°C for 30 minutes for staining protected from light. Excess DAPI was then removed with the supernatant by centrifugation at 4000 rpm at 4°C for 5 minutes and then observed under 40 × magnification using fluorescent microscope [13-15].
an important role in the last stage of apoptosis as it acts as a marker to be engulfed by phagocytes. Therefore, PS can be used as a quantitative model to study apoptosis through counting the amount of Annexin V-FITC antibodies bound to PS. The quadrant graph in Figure 6 showed that PTZ induced apoptosis toward A2780 cells from 76.05% at 0.3 µ M to 82.24% at 0. 9 µ M, associated with slightly increase of per- centage of necrotic cells at highest concentration. To a larger extent, it is suggested that PTZ induces cell death on A2780 cells via apoptosis based on the consistent findings in AO/PI staining, DAPI staining, and Annexin V-FTIC assay.
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Abstract: Objective: This study aimed to investigate the sensitivity of periodic acid-Schiff (PAS) staining, Grocott’s silver staining (GSS) and calcofluor white (CFW) staining in the diagnosis of sporotrichosis. Methods: Paraffin em- bedded tissues (n = 100) which were diagnosed with sporotrichosis by fungal culture were subjected to PAS, GSS, and CFW staining, and the detection rate of sporotrichosis was determined. Results: The sensitivity of PAS, GSS, and CFW staining was 31%, 40% and 74%, respectively, in the diagnosis of sporotrichosis. Conclusion: CFW staining has a high sensitivity in the diagnosis of sporotrichosis, and sections are easily observed and can be repeatedly stained after CFW staining. For patients suspected to have sporotrichosis, CFW staining may be employed for early diagnosis before a fungal culture.
some patterns were studied at various times after release of the HU block. Figure 7A shows an example of the staining pat- terns of cells 10 h after the release of the HU block, and the data shown in Table 2 summarize the patterns from viewing many fields of cells. The 10-h time point was chosen because this was when the maximum numbers of duplicated and sepa- rated centrosomes were found in control cell populations. As shown in Table 2, centrosomes were clearly visible in nearly all Ad-CMV-infected cells, with 18% showing clearly separated centrosomes that were at or moving toward the poles of the cells. In contrast, none of the Ad-ST-infected cells showed duplicated centrosomes and only a few had distinct single cen- trosomes. The vast majority showed diffuse, disorganized stain- ing with anti-␥-tubulin, suggesting that centrosomal matura- tion had not occurred and thickened centrosomes of detectable size and structure were not present in these cells. These find-
We proved that somatostatin receptor subtypes are fre- quently expressed in pathologically altered thyrocytes, in contrast to normal thyroid follicular epithelium. SSTR 1 protein was expressed in 77,7% of investigated cases, SSTR 2A and 2B both in 44,4%, SSTR 3 in 55,5%, SSTR 4 in 11,2% and SSTR 5 in 33,3%. SSTR 1 is the dominant form in the thyroid gland tumor and hyperplasia. A good correlation with SSTR 1 receptor gene expression was observed in 77,7% of investigated cases. Immunohisto- chemical estimation of SSTR 2A and 3 was agree with RT- PCR method in 77,7%, only 22,2% of results of SSTR 4 mRNA estimation were correlated with immunohisto- chemical staining and SSTR 5 mRNA was good correlated with IHC staining in 100% tissues. It suggests that soma- tostatin multiligand analogs or selective SSTR 1 agonists may represent a further useful approach for the thyroid tumors treatment. The expression of somatostatin recep- tor subtypes in thyroid tumors needs father studies con- cerning greater and more differentiated group of patients. Immunohistochemical staining of SSTR 2A in poorly differen-
normal horse serum (Vector Labs, Burlingame, CA, USA) to block nonspecific reactivity. Sections were incubated with rabbit-anti-mouse MPO (ab9535, AbCam, Cam- bridge, UK; 1:100, 4°C overnight) and with a peroxidase conjugated anti-rabbit as secondary antibody (ImmPRESS, MP-7401, Vector labs, Burlingame, CA, USA; 30 min, RT). After washing, color was developed by adding AEC (3-amino-9-ethylcarbazole) chromogen for 10 min (SK- 4200, Vector Labs, Burlingame, CA, USA). Finally, slides were counterstained with hematoxylin and mounted using faramount (DAKO, Glostrup, Denmark). Negative control sections were treated in the same way but primary antibodies were omitted. Sections were also double- stained with rabbit-anti-mouse MPO (ab9535, AbCam, Cambridge, UK; 1:100, 4°C ON) primary antibody and pri- mary human-polyclonal anti-T. gondii antibodies (1:500, WHO’s standard, Statens Serum Institute, Copenhagen, Denmark) followed by a secondary anti-rabbit Alexa 594 (1:400, Molecular Probes, Eugene, OR, USA) and Alexa Fluor 488 goat anti-human IgG (1:400, Molecular Probes, Eugene, OR, USA), respectively, to detect co-localization of MPO positive neutrophils and parasites. Sections were mounted in medium for fluorescence including DAPI (Vector labs, Burlingame, CA, USA). Stained sections were examined using light microscope and images were capture at 20x or higher magnifications.
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Methods: In adult male Sprague-Dawley rats, the focal cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO) models for 1.5 h. The expression of NLRP3 inflammasome in the penumbral tissue following reperfusion was assessed by western blotting and immunoflourescent staining. The infarct size, neurological deficit score, TUNEL staining and the expression of proinflammatory factors or anti-inflammatory cytokines were evaluated at 72 h after reperfusion in the presence or absence of either α 7nAChR antagonist ( α -BGT) or agonist (PHA-543,613). Results: The contents of inflammasome proteins were gradually increased after cerebral ischemia/reperfusion (I/R). EA stimulus attenuated NLRP3 inflammasome mediated inflammatory reaction and regulated the balance between proinflammatory factors and anti-inflammatory cytokines. The agonist of α 7nAChR induced similar neuroprotective effects as EA stimulus. In contrast, α 7nAChR antagonist reversed not only the neuroprotective effects, but also the inhibitory effects of NLRP3 inflammasome and the regulatory effects on the balance between proinflammatory factors and anti-inflammatory cytokines.
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BORG/Cdc42EP family of Cdc42 effectors, which comprises of 5 members (Fig. 5A). 23,24 Noteworthy, Cdc42EP3 was the only BORG protein that presented differential expression between NFs and CAFs; 12 this may be different in other systems. BORG proteins share a Cdc42/Rac interactive binding (CRIB) motif that allows the interaction with active Cdc42 and RhoQ/TC10 but not RhoA or Rac1. 23-25 The deﬁning characteristic of BORG proteins is the presence of 3 BORG speciﬁc domains (BD1-3). The BD3 domain is present in all members and mediates the interaction between BORG proteins and septin complexes (Fig. 5B), 15,26 allowing BORGs to regulate septin orga- nization in mammalian cells. 15,23 In addition, ectopic expression of BORGs leads to changes in cell shape Figure 4. Modulating the activity of Cdc42 in CAFs affects Cdc42EP3 localization and septin and F-actin organization. (A) Panels show GFP (green), F-actin (magenta) and myc (blue) staining of cancer-associated ﬁ broblasts (CAFs) stably expressing Cdc42EP3-GFP following transfection with empty vector, myc-Cdc42-N17 or myc-Cdc42-V12. The grayscale panels show individual channels as indicated. Scale bars, 25 mm. (B) Panels show GFP (green), SEPT2 (magenta) and myc (blue) staining of cancer-associated ﬁbroblasts (CAFs) stably expressing Cdc42EP3-GFP following transfection with empty vector, myc-Cdc42-N17 or myc-Cdc42-V12. The grayscale panels show indi- vidual channels as indicated. Scale bars, 25 mm.
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We also evaluated the therapeutic feasibility of periure- thral injection of hAFSCs in an SUI animal model. When hAFSCs were injected into the animal, IHC staining with HuNu confirmed that the injected cells were able to sur- vive in the host environment. They integrated into the mouse sphincter muscle layer and survived within these in vivo conditions for 14 days. Real-time PCR gave us valuable information on the interaction between human cells and mouse cells. Human myogenic gene expression gradually decreased over time, while mouse gene expression steadily increased. These results indicate the grafted hAFSCs might have undergone in situ myogenic differentiation and induced host muscle regeneration. These findings are simi- lar to other reports of human stem cell transplantation into animals [17-19]. The details underlying the specific mechanism of action need to be investigated.
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The decrease in blood perfusion caused by CA4P generated areas of significant apoptosis 24 h after treatment. Figure 3A (top row of Figure 3) shows a low magnification view of untreated tumor sections stained in different manners. From left to right, sections are shown with DAPI nuclear stain (blue), TUNEL apoptosis stain (green), CD-31 blood vessel stain (red), a composite of DAPI, TUNEL, and CD-31, and Masson’s trichrome stain. Figure 3B directly below shows a tumor section 24 h after treatment with 100 mg/kg CA4P. The middle of this tumor contains a large amount of apoptosis as well as a loss of blood vessel structure. Figure 3C shows additional sections of tumors treated with CA4P, each with varying degrees of apoptosis in response to the CA4P treatment. High-magnification composite images in Fig 3D and 3E also show a lower cell density and reduced vessel structure in apoptotic regions.
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Figure 4 | The Notch receptor is differentially localized in worker ovaries. Activation of the Notch receptor causes part of the receptor to become cleaved (NICD) where it moves to the nucleus to regulate gene expression. (a) Immunohistochemistry using an antibody against the NICD indicates that in queen-right worker bees the NICD is predominately nuclear-localized in the cells of the terminal ﬁlament and all of the cells of the germarium. The NICD is also present in the nucleus of the anterior terminal ﬁlament cells in queen-less worker bees; but the NICD is essentially absent from the anterior germarium. In the posterior germarium, as oocytes become clearly identiﬁable, the NICD is detectable on cell membranes but not in the nuclei. In queen ovarioles, the NICD is present in the nucleus of cells of the terminal ﬁlament, indicating that these cells are receiving a Notch signal, but there is little immunoreactivity detected throughout the germarium. Ovaries were counter-stained with DAPI and phalloidin to visualize nuclei and cortical actin. (b) We examined the expression of Numb, a gene implicated in regulation and recycling of the Notch receptor, using qRT–PCR and in situ hybridization. qRT–PCR indicates that numb mRNA is induced more than twofold when the queen is removed from the hive before any morphological difference is detectable in the ovaries. qRT–PCR data is the mean of transcript levels (Log 10 ) in ﬁve biological samples for each condition. Boxplot whiskers indicate minimum and
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DAPI-positive objects with extensive DNA unfolding were observed in human neutrophils cultured in hypo- thermic conditions followed by rewarming. Our experi- mental data indicated that such DNA structural altera- tions in neutrophils may be related to NET formation, but can be biochemically and pharmacologically discrimi- nated from NET formation. We also considered that the objects might not represent apoptotic cells, given that apoptotic cells contain condensed DNA enclosed in membrane, which is not observed in the objects. We thus suggest that the hypothermia/rewarming-induced DNA unfolding is regulated in a manner distinct from either canonical NETosis or canonical apoptosis, arguing the existence of a previously unappreciated signaling path- way that alters global genomic DNA structures in eu- karyotic cells. Further, the results indicate that cold- treatment followed by warming may affect NET forma- tion, which is an important consideration because many researchers use hypothermal conditions during the isola- tion and culture of neutrophils.