Centromere Protein (CENP)-W Interacts with Heterogeneous Nuclear Ribonucleoprotein (hnRNP) U and May Contribute to Kinetochore-Microtubule Attachment in Mitotic Cells

<div><p>Background</p><p>Recent studies have shown that heterogeneous nuclear ribonucleoprotein U (hnRNP U), a component of the hnRNP complex, contributes to stabilize the kinetochore-microtubule interaction during mitosis. CENP-W was identified as an inner centromere component that plays crucial roles in the formation of a functional kinetochore complex.</p><p>Results</p><p>We report that hnRNP U interacts with CENP-W, and the interaction between hnRNP U and CENP-W mutually increased each other’s protein stability by inhibiting the proteasome-mediated degradation. Further, their co-localization was observed chiefly in the nuclear matrix region and at the microtubule-kinetochore interface during interphase and mitosis, respectively. Both microtubule-stabilizing and microtubule-destabilizing agents significantly decreased the protein stability of CENP-W. Furthermore, loss of microtubules and defects in microtubule organization were observed in CENP-W-depleted cells.</p><p>Conclusion</p><p>Our data imply that CENP-W plays an important role in the attachment and interaction between microtubules and kinetochore during mitosis.</p></div

Supplemental Material - How and When May Leader Influence Tactics Affect Followers’ Organizational Citizenship Behavior? A Social Cognitive Approach

Supplemental Material for How and When May Leader Influence Tactics Affect Followers’ Organizational Citizenship Behavior? A Social Cognitive Approach by Ui Young Sun, Soojin Lee, and Seokhwa Yun in Group & Organization Management</p

The role of CENP-W in kinetochore-microtubule interaction.

<p>(A) Microtubule regrowth assay. After pre-incubation with siRNAs for 60 h, HeLa-CENP-W cells were placed at 0°C for 30 min. Cells were then incubated at 37°C for 10 min prior to fixation. The bar graph represents microtubule intensity in cells (N > 100 for each sample). Error bars indicate SDs. *P < 0.01. (B) Cold-induced depolymerization assay. Following siRNA treatment (200 nM) for 60 h, HeLa-CENP-W cells were placed at 4°C for 10 min. After fixation, cells were immunostained with anti-α-tubulin antibody and ACA. Images of prometaphase cells were captured by Olympus IX70 fluorescence microscope. The bar graph shows microtubule intensity of each sample normalized to that of control siRNA-treated cells. (N > 100 for each sample). Error bars indicate SDs. *P < 0.01. (C) Kinetochore-derived microtubule outgrowth. Forty-eight hours post siRNA transfection, HeLa CENP-W cells were incubated with nocodazole (100 ng/mL) for 6 h. After thorough washing, cells were incubated in fresh media for short time (1, 7, or 15 min) prior to fixation. The samples were then immunostained with anti-α-tubulin and ACA. The bar graph shows the percentage of cells showing normal or deficient phenotype of chromosome-dependent nucleation after 7-min release (N > 100 for each sample).</p

hnRNP U-CENP-W association increased their protein stability.

<p>(A) After increasing amount of GST-CENP-W plasmids were transfected to 293T cells, the protein level of co-expressed hnRNP U was analyzed by immunoblotting. (B) HeLa-CENP-W cells were incubated with CENP-W siRNAs (200 nM) for a predetermined period. For the rescue experiment, FLAG-CENP-W was transfected at 48 h after siRNA treatment and cells were incubated for another 24 h. (C) In vivo ubiquitination assay. 293T cells transfected with HA-ubiquitin and FLAG-hnRNP U were subjected to immunoprecipitation with anti-FLAG antibody. (D) 293T cells were transfected with GST-hnRNP U along with increasing amount of FLAG-CENP-W. (E) HeLa-CENP-W cells were incubated with 200 nM of hnRNP U siRNAs, followed by immunoblot analysis. If necessary, the siRNA-treated cells were transfected with GST-hnRNP U, and incubated for another 24 h. (F) GST-pulldown was performed using 293T cells transfected with HA-ubiquitin and GST-CENP-W. (G) In situ immunofluorescence staining. Following treatment with siRNAs (200 nM) specific for hnRNP U or CENP-W for 60 h, HeLa-CENP-W cells were double-immunostained with anti-hnRNP U (green) and -FLAG (red) antibodies. Scale bars = 10μm. The bar graph shows the average fluorescence intensity of hnRNP U or CENP-W in siRNA-treated cells relative to the mean fluorescence intensity of control siRNA-treated cells (N > 50 for each sample). Error bars indicate SDs. The three scatter plots are depicted and the fluorescence intensity of CENP-W (X) is plotted against that of hnRNP U in siControl-, sihnRNP U-, or siCENP-W-treated cells. The coefficient if determination (R²) was calculated with the linear regression (black line).</p

Determination of crucial domains for hnRNP U-CENP-W interaction.

<p>(A) For domain mapping of hnRNP U, GST-fused hnRNP U deletion mutants were constructed and co-transfected into 293T cells with FLAG-CENP-W. (B) GST-pulldown was performed after various FLAG-CENP-W deletion mutants were co-expressed with GST-hnRNP U. (C) Effect of RNase treatment on hnRNP U-CENP-W interaction. After HeLa-CENP-W cells were incubated with RNase A at indicated concentrations at 30°C for 20 min, immunoprecipitation was conducted with anti-FLAG antibody. (D) After cells were pre-incubated with RNase A (200 μg/mL) at 30°C for 20 min, total RNA purified from 293T cells was added before immunoprecipitation. (E) After eliminating cellular RNA with RNase A treatment (200 μg/mL), various kinds of RNAs (1 μg) were added to the samples prior to immunoprecipitation.</p

hnRNP U interacts with CENP-W.

<p>(A) After transfection of GST-CENP-W into 293T cells, GST-pulldown was performed and the CENP-W-interacting proteins were visualized using silver staining kit (Peptron) after SDS-PAGE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149127#pone.0149127.ref013" target="_blank">13</a>]. A CENP-W-specific band was cut and analyzed by mass spectroscopy (Genomine). (B) Following transfection of GST-hnRNP U and FLAG-CENP-W, the 293T cell lysates were subjected to GST-pulldown. (C) Reciprocal interaction between GST-CENP-W and FLAG-hnRNP U using GST-pulldown. (D) In vitro binding assay; after His-hnRNP U and GST-CENP-W (either wild-type or deletion mutant (1–30 aa)) were separately expressed in <i>E</i>. <i>coli</i> Rosetta (DE3) cells using pET15b-hnRNP U and pGEX-4T-3-CENP-W, GST-pulldown was performed. (E) Binding assay at endogenous level. 293T cell lysates were used for immunoprecipitation using either anti-hnRNP U or -CENP-W antibody. Then, co-fractionated proteins were visualized using specific antibodies. (F) Nuclear matrix extraction. Cells were sequentially extracted to soluble, chromatin-enriched, and the nuclear matrix fraction following high-salt extraction method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149127#pone.0149127.ref013" target="_blank">13</a>]. (G) HeLa-CENP-W cells were lysed and applied to the linear glycerol gradient (10–40%), and fractions collected from the bottom (fraction 1). (H) Size exclusion chromatography was performed using HeLa-CENP-W cell lysate on Sephacryl S-300 size exclusion column. Fifty 1-ml fractions were collected.</p

Cellular localization of hnRNP U and CENP-W.

<p>(A) Localization of CENP-W and hnRNP U during mitosis. After cells were synchronized with nocodazole (100 ng/mL) for 12h, HeLa-CENP-W cells were harvested at 30 min time-intervals and immunostained with anti-hnRNP U and -FLAG antibodies. Scale bar equals 10 μm. (B) Co-localization of hnRNP U and CENP-W during the interphase. HeLa-CENP-W cells grown in coverslips were subjected to double-immunostaining with anti-hnRNP U and -FLAG antibody (left panels). If necessary, cells were treated with either DNase I (1 unit/μL) or RNase A (200 μg/mL) in presence of 0.5% Triton X-100, followed by ammonium sulfate extraction (0.25 M). (C) After cells were synchronized with double-thymidine block, HeLa-CENP-W cells were harvested at 2 h (interphase) or 10 h (Mitosis) after release. Then, cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody. (D) Interaction between CENP-W and SPC25 at endogenous level. 293T cell lysates were subjected to immunoprecipitation with anti-CENP-W antibody, and co-isolated CENP-T and SPC25 were visualized using specific antibodies. (E) After HeLa-CENP-W cells were transfected with siRNAs (200 nM) for 72 h, the protein levels of each sample were determined by immunoblotting. (F) Mislocalization of kinetochore components in hnRNP U-depleted cells. HeLa-CENP-W or HeLa-CENP-T cells were incubated with hnRNP U-specific siRNAs (200 nM) for 60 h, and further incubated with nocodazole for 12 h for cell synchronization. For the rescue, GST-hnRNP U was transfected at 48 h after siRNA treatment. At 30 min post-release, cells in mitotic prophase were immunostained with appropriate antibodies. The bar graphs show relative fluorescence signal intensities of each sample normalized by that of control siRNA-treated cells and presented as a bar graph (N > 100 for each sample). Error bars indicate SDs.</p

CENP-W protein level is closely related to microtubule integrity.

<p>(A) After HeLa-CENP-W cells were incubated with nocodazole or paclitaxel at indicated concentrations for 12 h, protein levels were determined by immunoblot analysis. (B) HeLa-CENP-W cells were incubated with nocodazole (100 ng/mL) or paclitaxel (5 μM) for 12 h, and harvested at indicate time points after media change. (C) HeLa cells were incubated with CENP-W-specific siRNAs (200 nM) for 72 h prior to cell harvest. (D) After HeLa-CENP-W cells were treated with siRNAs (200 nM) for 60 h, cyclohexamide (100 μg/mL) was added to the culture media. Then, cells were harvested at indicated time points and analyzed by immunoblotting. (E) Following treatment of siCENP-W for 48 h, HeLa-CENP-W cells were either continuously incubated with the siRNA for another 24 h or transfected with FLAG-CENP-W.</p

sj-docx-1-taj-10.1177_20406223221128444 – Supplemental material for Development of a medication review tool for residents in Korean long-term care facilities

Supplemental material, sj-docx-1-taj-10.1177_20406223221128444 for Development of a medication review tool for residents in Korean long-term care facilities by Kwanghee Jun, Soojin Lee, Ah young Lee, Young-Mi Ah and Ju-Yeun Lee in Therapeutic Advances in Chronic Disease</p

The distribution of glucose by <i>K, pneumoniae</i> SGSB100, SGSB103, SGSB104 and SGSB105 in batch fermentation at 12hr (carbon conversion ratio (%)).

<p>The distribution of glucose by <i>K, pneumoniae</i> SGSB100, SGSB103, SGSB104 and SGSB105 in batch fermentation at 12hr (carbon conversion ratio (%)).</p