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(2011) Rab5 GTPase controls chromosome alignment through lamin disassembly and relocation of the NuMA-like protein Mud to the poles during mitosis
(2011) Rab5 GTPase controls chromosome alignment through lamin disassembly and relocation of the NuMA-like protein Mud to the poles during mitosis. connection with nuclear lamina, modulate CENPF localization and levels at centromeres, as a result ensuring appropriate spindle size and kinetochore-microtubule attachment in meiotic oocytes.Ma, R., Hou, X., Zhang, L., Sun, S.-C., Schedl, T., Moley, K., Wang, Q. Rab5a is required for spindle size control and kinetochore-microtubule attachment during meiosis in oocytes. (7) exposed that vesicles positive for the Rab11a modulate an actin network for asymmetric spindle placement in oocytes. Rab5, as the expert regulator of the endocytic trafficking, has been well recognized to involve in membrane tethering and docking (8,C11). Three isoforms of Rab5 (a, b, and c) share 90% of sequence identity yet can be functionally different (12). Of notice, recent findings possess suggested that Rab5 GTPase participates in chromosome congression in and human being mitotic cells (13, 14). However, the potential practical involvement of Rab5 in meiosis has not been addressed yet. In this study, we set out to investigate the part of Rab5a during mouse oocyte meiosis. We found out a novel function of Rab5a-containing vesicles: control of the spindle size and chromosome positioning through modulation of centromere protein F (CENPF) localization to the centromere, as reported below. MATERIALS AND METHODS All chemicals and culture press were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. ICR mice were used in this study. All experiments were approved by the Animal Care and Use Committee of Nanjing Medical University or college and were performed in accordance with institutional recommendations. Antibodies Rabbit polyclonal anti-Rab5a (cat no. ab18211), rabbit polyclonal anti–actin (ab5441), and rabbit polyclonal anti-CENPF (ab5) antibodies were purchased from Abcam (Cambridge, MA, USA); mouse monoclonal anti–tubulin-FITC antibody was purchased from Sigma (76074); human being anti-centromere CREST antibody (09C-CS1058) was purchased from Fitzgerald Industries International (Concord, MA, USA); mouse monoclonal anti-NuMA antibody (610562) was purchased from BD Transduction Laboratories (Lexington, KY, USA); goat polyclonal anti-lamin A/C antibody (SC-6215) was from Santa Cruz Biotechnology (San Jose, CA, USA); FITC-conjugated goat anti-rabbit IgG, FITC-conjugated donkey anti-goat IgG, and TRITC-conjugated goat anti-rabbit IgG were purchased from Thermo Fisher Scientific (Rockford, IL, USA); and Cy5-conjugated goat anti-human IgG and Cy5-conjugated goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratory (Western Grove, PA, USA). Oocyte collection and tradition Six- to 8-wk-old female mice were utilized for oocyte collection. To collect fully cultivated germinal vesicle (GV) oocytes, mice were superovulated with 5 IU pregnant mare serum gonadotropin (PMSG) by intraperitoneal injection, and 48 h later on, cumulus-enclosed oocytes were acquired by manual rupturing of antral ovarian follicles. Cumulus cells were eliminated Ispinesib (SB-715992) by repeatedly pipetting. For maturation, GV oocytes were cultured in M2 medium under mineral oil at 37C inside a 5% CO2 incubator. Morpholino (MO) knockdown Microinjection of MO, having a Narishige microinjector (Narishige Group, Tokyo, Japan), was used to knock down Rab5a and CENPF in mouse oocytes. Rab5a-MO 5-TTGTTGCTCCTCGATTAGCCATGTC-3 and CENPF-MO 5-GGCCCAGCTCATCTTGTTTTATTTT-3 (Gene Tools, Philomath, Ispinesib (SB-715992) OR, USA) focusing on initiation of translation were diluted with water to give a stock concentration of 1 1 mM, and then a 2.5 pl MO solution was injected into oocytes. A MO standard control was injected as control. After injections, oocytes were arrested in the GV stage in M2 medium supplemented Ispinesib (SB-715992) with 2.5 CD163L1 M milrinone for 20 h to facilitate knockdown of mRNA translation, then washed 3 times in milrinone-free M2 medium, and cultured for different times. European blotting A pool of 100 oocytes was lysed in Laemmli sample buffer comprising protease inhibitor and then subjected to 10% SDS-PAGE. The separated proteins were transferred to a PVDF membrane. Membranes were clogged in TBS comprising 0.1% Tween 20 and 5% low-fat dry milk for 1 h and then incubated with primary antibodies as follows: rabbit anti-Rab5a antibody (1:1000) or rabbit anti-CENPF antibody (1:1500). After multiple washes in TBS comprising 0.1% Tween 20 and incubation with horseradish peroxidase-conjugated secondary antibodies, the protein bands were visualized using an ECL In addition European Blotting Detection System (GE Healthcare, Piscataway, NJ, USA). The membrane was then washed.
However, in the present experiments, it is clear that cysteine residues lying on the cytosolic surface of the BKCa channel protein(s) are the sites of NEM action
However, in the present experiments, it is clear that cysteine residues lying on the cytosolic surface of the BKCa channel protein(s) are the sites of NEM action. Although positively charged, MTSEA has been reported to cross cell membranes and lipid bilayers (Karlin TH287 & Akabas, 1998). concentration-dependently increased by the concentration of Ca2+ bathing the cytosolic surface of the patch. Each data point represents the average of six to seven experiments. (D) Normalizing individual experiments to the maximum conductance reveals that NEM caused a significant increase in (11.81.5 mV in control 15 nM Ca2+ PSS and 16.91.5 mV after NEM, respectively; were 59.5 and 17 mV, 50.4 and 16.4 mV, 42.3 and 18.8 mV, and 7.4 and 18.7 mV in 15, 150, 300 nM and 1.5 properties of BKCa channels in excised patches of the guinea-pig taenia caeca. BKCa current amplitudes were measured either a 0 mV (ai, plot between C40 TH287 and +80 mV (bii). bii Inset illustrates the current amplitudes in MSET expressed as a fraction of the control channel amplitudes (Rel. Amp.) plotted against plots (plot in the presence of MTSET is markedly reduced such that the slope conductance (at 0 mV) of these BKCa channels was 155 pS in control PSS and 95 pS in MTSET-containing PSS. In addition, the relative amplitude of these BKCa channel currents in MTSET, expressed as a fraction of TH287 their respective control amplitudes (Figure 8bii, inset), decreased at more positive potentials suggesting a voltage-dependent blockade of current flow at positive potentials. Open in a separate window Figure 5 Positively charged MTS reagent MTSET increased BKCa channel activity (at 0 mV) in a manner reversed upon washout. MTSET did not prevent the excitatory actions of NOCys. Application of MTSET (2.5 mM for 5 min) significantly increased subunit and the regulatory subunit. The primary sequence of the subunit obtained from different tissues is almost identical being encoded by a single gene, KCNMA1, previously termed (Butler subunits are encoded by four genes (KCNMB1-4) (Tanaka subunit. In addition to containing the six membrane-spanning domains (S1CS6), the S4 voltage sensor and the pore domain between S5 and S6, which identifies this channel as a members of the S4 superfamily of voltage-gated K+ channels (subunit also contains a Smad7 seventh transmembrane domain (S0) that locates the amino terminus in the extracellular domain. Additional hydrophobic cytoplasmic-located segments (S7CS10) are located within TH287 the long carboxy-terminal domain. A tetramerization domain (BK-T1) has been located in the hydrophilic region between S6 and S7, while Ca2+ binding has been located to a region (calcium bowl’) between S9 and S10, which is highly conserved in all cloned BKCa channels (Orio subunit changes the kinetics and Ca2+ sensitivity of expressed subunit channels. However, larger changes in channel kinetics are generally created when subunits are co-expressed with subunits (Orio subunits (B1CB4) consist of two transmembrane segments connected by an extracellular loop’ so that both termini are in the cytoplasmic domain (Knaus subunit increases the sensitivity of the subunit to Ca2+, particularly at high [Ca]i (McCobb subunit selectivity for K+ ions (McManus subunit is also necessary for the extracellular binding and activation of BKCa channels by 17-estradiol (Valverde ((Figure 3C, D). However, exposure to NEM did not affect the sensitivity of the BKCa channel activation to raising the cytosolic concentration of Ca2+ (Figure 3). These results are consistent with the NEM-evoked decrease in BKCa channel activity in excised patches from the rabbit aorta (Bolotina (channel activity evoked by the NO donor, channel activity may well be arising from differences in the amino-acid sequence and therefore the microenvironment of the native BKCa and the channels under study. In particular, there may well be a number of different spliced variants of the subunit of the BKCa channel being expressed in the smooth muscle cells of the taenia ceca. These differing spliced variants could be combining in varying proportions with a number of differing spliced variants of the subunit to form a variety of BKCa channels, which could explain the increases and decreases in channel gating observed in the presence of oxidizing agents and NEM, respectively. However, in the present experiments, it is clear that cysteine residues lying on the cytosolic surface of the BKCa channel protein(s) are the sites of NEM action. Although positively charged, MTSEA has been reported to cross cell membranes and lipid bilayers (Karlin & Akabas, 1998). Thus, we have assumed that MTSEA will have the greatest access to the cysteine residues of the subunit both within the hydrophobic regions and hydrophilic regions. In our experiments, MTSEA evoked a significant decrease in BKCa channel opening that remained upon washout, suggesting an irreversible binding.
The lower right quadrant (Q3) represented the early stage apoptotic cells as (FITC+/PI-). NSCLC, we classified patients into resistant and sensitive groups based on their response to chemotherapy. Based on the clinical data of the two groups (Table 1), we found no significant differences in gender and age between the sensitive and resistant groups. However, more patients with smoking history, poor differentiation, lymph node metastasis, and II-IV TNM staging were observed in the resistant group than the sensitive group (< 0.05). According to the qRT-PCR detection of TUG1 expression level in each patient (Figure 1A), the TUG1 expression of the patients in the resistant group was significantly lower than that in the sensitive group (< 0.01). All patients were followed up for a median follow-up of 14 months. Kaplan-Meier method was used to analyze the overall survival of the NSCLC patients. The total survival time of the sensitive group (26.93 1.63 months) was significantly higher than that of the resistant group (13.48 1.17 months) (Figure 1B). Table 1 The clinical data of NSCLC patients in the resistant and sensitive groups. ItemSensitive group (n = 43)Resistant group (n = 65)valueGender0.695?Male2440?Female1925Age (years)0.879?< 602639? 601726Smoking history0.014?Yes1235?No3130Differentiation degree< 0.001?Poor1246?High/Medium3119Tumor node metatstasis0.003?Yes1543?No2822TNM staging0.006?I2418?II-IV1947 Open in a separate window Open in a separate window Figure 1 The TUG1 expression level and the intracellular localization. (A) The TUG1 expression level of NSCLC patients in the resistant and sensitive groups; (B) survival conditions of NSCLC patients in the resistant and sensitive groups; (C) expression level of TUG1 determined by qRT-PCR in NSCLC cells; (D) the intracellular localization by fluorescence hybridization in NSCLC cells. ** < 0.01. Expression level of TUG1and the intracellular localization in NSCLC cells In this study, NSCLC cell lines SPC-A1, NCI-H1650, NCI-H520 and NCI-H1299 in addition to the normal epithelial cell line 16HBE of Rp-8-Br-PET-cGMPS lung mucosa were selected. By comparing the expression of TUG1 in cells, we revealed that, SPC-A1 cells had the highest expression of TUG1 and NCI-H520 had the lowest relative to 16HBE cells (Figure 1C). So we selected these two cell lines for subsequent experiments. The effects lncRNAs exert is closely implicated in its cellular localization. LncRNAs located in the nucleus play a major role in transcriptional regulation, and lncRNAs located in the cytoplasm mainly play a role in post-transcriptional Rp-8-Br-PET-cGMPS regulation. Therefore, we isolated the nucleus and cytoplasm, and observed the intracellular localization of TUG1 by Rp-8-Br-PET-cGMPS fluorescence hybridization. Fluorescence hybridization showed that TUG1 was mainly localized in the nucleus, and a small amount was localized in the cytoplasm. CD207 The fluorescence intensity of TUG1 in drug-resistant cells was significantly weaker than that of the parental cells (Figure 1D). Overexpressed TUG1 promotes sensitivity of NSCLC cells to DDP To investigate the possible effects of lncRNA TUG1 on chemoresistance in NSCLC, SPC-A1 and H520 cells were transfected with si-TUG1 and si-NC, respectively, besides, the SPC-A1/DDP and H520/DDP cells were transfected with pcDNA-TUG1 and pcDNA3.1. The transfection efficiency of TUG1 was detected by qRT-PCR. Compared with the si-NC group, siRNAs significantly down-regulated the expression of TUG1 in SPC-A1 and H520 cell lines, with the efficiency of siTUG1-3 being the most significant. The expression of TUG1 in SPC-A1/DDP and H520/DDP cells transfected with pcDNA-TUG1 was significantly up-regulated (Figure 2A). The effect of TUG1 on the IC50 value of DDP-induced NSCLC cell line was detected by MTT assay. The IC50 values of DDP in SPC-A1/si-TUG1 or H520/si-TUG1 cells were significantly elevated, versus the si-NC group (Figure 2B, < 0.01). Conversely, IC50 values of DDP in SPC-A1/DDP/TUG1 or H520/DDP/TUG1 cells were lower than those in the si-NC group (Figure 2B, < 0.01). The results indicated that TUG1 enhanced the sensitivity to DDP in NSCLC. Through colony formation experiments, we obtained similar results. When exposed to DDP, the SPC-A1/si-TUG1 cells and H520/si-TUG1 cells displayed ability to significantly enhanced ability of forming colonies after overactivation of TUG1. Whereas, the overexpression of TUG1 reduced the ability of TUG1-SPC-A1/DDP cells and H520/DDP cells to form colonies (Figure 2C). Through scratch test and Transwell assay, we demonstrated that cell migration and invasion were enhanced after inactivation of TUG1 in SPC-A1/si-TUG1 cells and H520/si-TUG1.