Journal: Antiviral Research

Article Title: Triggering unfolded protein response by 2-Deoxy- d -glucose inhibits porcine epidemic diarrhea virus propagation

doi: 10.1016/j.antiviral.2014.03.007

Figure Lengend Snippet: 2-DG treatment affects virus packaging. (A) Vero cells were pretreated with 10 mM 2-DG for 24 h, and the cells were analyzed by FACS analysis using anti-CD13 polyclonal antibody. (B, C) 2-DG treatment did not affect virus entry as determined by RT-PCR and Western blot analysis. For RT-PCR analysis, the cells were treated with 10 mM 2-DG before virus infection. Bars represent mean percent copy number of viral RNA levels in cells by RT-PCR using primers specific for PEDV N gene. Cell lysates were analyzed by Western blot using antibody against PEDV N. (D) The Vero cells were infected with PEDV for 2 h (MOI = 1), 2-DG (10 mM) was incubated with the cells. The cells were harvested at different time points. Cell lysates were analyzed by Western blot using antibody against PEDV N. (E, F) The Vero cells was treated with different concentrations 2-DG after infected with PEDV (MOI = 0.01) for 2 h, the ratio of PEDV RNA copy numbers between the supernatants and the cell lysates was detected by Q-PCR, and the ratio of the virus titers in supernatants and cell lysates were determined by plaque assay.

Article Snippet: Detached Vero cells were washed once with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, and stained with rabbit anti-human CD13 protein (Boster Bio-Tech Co. Ltd., Wuhan, China) for 1 h at 4 °C.

Techniques: Virus, Reverse Transcription Polymerase Chain Reaction, Western Blot, Infection, Incubation, Plaque Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Blood–spinal cord barrier pericyte reductions contribute to increased capillary permeability

doi: 10.1038/jcbfm.2012.113

Figure Lengend Snippet: Pericyte number and coverage is reduced along blood–spinal cord barrier. ( A ) Confocal microscopy analysis of CD13-positive pericytes (green) and collagen IV-positive capillary profiles (red) in 2-month-old wild-type mouse cortex, caudate, and hippocampal brain regions and cervical, thoracic, and lumbar spinal cord regions (anterior horns). ( B ) Quantification of regional CD13-positive pericyte coverage of collagen IV-positive brain and spinal cord capillaries. Mean±s.e.m., n =5 to 7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05. ( C ) Quantification of regional CD13-positive pericyte cell number normalized to collagen IV-positive capillary surface area in the brain and spinal cord. Mean±s.e.m., n =5 to 7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05. ( D ) Confocal microscopy analysis of platelet-derived growth factor receptor β (PDGFR β )-positive pericytes (green) and lectin capillary profiles (red) in 2-month-old wild-type mouse cortex, caudate, and hippocampal brain regions and cervical, thoracic, and lumbar spinal cord regions. ( E ) Quantification of regional PDGFR β -positive pericyte coverage of lectin-positive brain and spinal cord capillaries. Mean±s.e.m., n =7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05. ( F ) Quantification of regional PDGFR β -positive pericyte cell number normalized to lectin-positive capillary surface area in the brain and spinal cord. Mean±s.e.m., n =7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05.

Article Snippet: Sections were then incubated in the following primary antibodies: goat α -mouse CD13 (10 μ g/mL, R&D Systems), mouse α -mouse desmin (1:100, Dako, Carpinteria, CA, USA), rabbit α -mouse collagen IV (1:200, EMD Millipore), rabbit α -human fibrin which cross reacts with mouse fibrin (1:500, Dako), goat α -mouse thrombin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse α -mouse zonula occludens-1 (ZO-1) (1:50, Invitrogen), mouse α -mouse occludin (1:50, Invitrogen), mouse α -mouse neuronal-specific antigen A60 (NeuN) (1:100, EMD Millipore) and goat anti-mouse ChAT (choline acetyl transferase) (1:50, EMD Millipore).

Techniques: Confocal Microscopy, Derivative Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Blood–spinal cord barrier pericyte reductions contribute to increased capillary permeability

doi: 10.1038/jcbfm.2012.113

Figure Lengend Snippet: Subregional variation in spinal cord pericyte number and coverage between anterior horn gray matter and lateral and dorsal funiculi. ( A ) Confocal microscopy analysis of CD13-positive pericytes (green) and collagen IV-positive capillary profiles (red) in 2-month-old wild-type mouse anterior horn gray matter or dorsal columns for cervical thoracic and lumbar spinal cord. ( B ) Quantification of CD13-positive pericyte coverage of collagen IV-positive capillaries in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =5 to 7 animals per group; * P <0.05. ( C ) Quantification of CD13-positive pericyte cell number normalized to lectin-positive capillary surface area in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =5 to 7 animals per group; * P <0.05. ( D ) Quantification of platelet-derived growth factor receptor β (PDGFR β )-positive pericyte coverage of lectin-positive capillaries in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =7 animals per group; * P <0.05. ( E ) Quantification of PDGFR β -positive pericyte cell number normalized to lectin-positive capillary surface area in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =7 animals per group; * P <0.05.

Article Snippet: Sections were then incubated in the following primary antibodies: goat α -mouse CD13 (10 μ g/mL, R&D Systems), mouse α -mouse desmin (1:100, Dako, Carpinteria, CA, USA), rabbit α -mouse collagen IV (1:200, EMD Millipore), rabbit α -human fibrin which cross reacts with mouse fibrin (1:500, Dako), goat α -mouse thrombin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse α -mouse zonula occludens-1 (ZO-1) (1:50, Invitrogen), mouse α -mouse occludin (1:50, Invitrogen), mouse α -mouse neuronal-specific antigen A60 (NeuN) (1:100, EMD Millipore) and goat anti-mouse ChAT (choline acetyl transferase) (1:50, EMD Millipore).

Techniques: Confocal Microscopy, Derivative Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Blood–spinal cord barrier pericyte reductions contribute to increased capillary permeability

doi: 10.1038/jcbfm.2012.113

Figure Lengend Snippet: Exacerbation of pericyte deficiency leads to overt blood–spinal cord barrier disruption and leakage of endogenous plasma proteins. ( A ) Confocal microscopy analysis of CD13-positive pericytes (green) and collagen IV-positive capillary profiles (red) in 6-month-old Pdgfrβ +/+ and Pdgfrβ F7/F7 mouse cervical, thoracic, and lumbar spinal cord anterior horn. ( B ) Quantification of regional CD13-positive pericyte coverage of collagen IV-positive anterior horn spinal cord capillaries. Mean±s.e.m., n =3 animals per group; * P <0.05. ( C ) Representative confocal microscopy analysis of IgG (green), fibrin (red), and lectin-positive capillaries (blue) in 6-month-old Pdgfrβ +/+ mouse lumbar anterior horn and anterior horn from Pdgfrβ F7/F7 cervical, thoracic, and lumbar spinal cord. ( D , E ) Quantification of IgG ( D ) and fibrin ( E ) extravascular deposits in the spinal cord regions in tissue sections from 2-month-old B6SJL/F1 Pdgfrβ +/+ , 6-month-old Pdgfrβ +/+ 129S1/SvlmJ, and 6-month-old Pdgfrβ F7/F7 mice. Mean±s.e.m., n =3 animals per group; * P <0.05. ( F , G ) Confocal microscopy analysis of thrombin (white) ( F ) or fibrin (red) ( G ) and SMI-311-positive neurons (blue) in 6-month-old Pdgfrβ +/+ and Pdgfrβ F7/F7 lumbar spinal cord. Arrows indicate neuronal accumulation.

Article Snippet: Sections were then incubated in the following primary antibodies: goat α -mouse CD13 (10 μ g/mL, R&D Systems), mouse α -mouse desmin (1:100, Dako, Carpinteria, CA, USA), rabbit α -mouse collagen IV (1:200, EMD Millipore), rabbit α -human fibrin which cross reacts with mouse fibrin (1:500, Dako), goat α -mouse thrombin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse α -mouse zonula occludens-1 (ZO-1) (1:50, Invitrogen), mouse α -mouse occludin (1:50, Invitrogen), mouse α -mouse neuronal-specific antigen A60 (NeuN) (1:100, EMD Millipore) and goat anti-mouse ChAT (choline acetyl transferase) (1:50, EMD Millipore).

Techniques: Disruption, Clinical Proteomics, Confocal Microscopy

Journal: Journal of neuropathology and experimental neurology

Article Title: Cerebrospinal Fluid Particles in Alzheimer Disease and Parkinson Disease

doi: 10.1097/NEN.0000000000000207

Figure Lengend Snippet: Measurement of cerebrospinal fluid (CSF) particles using Apogee A50 flow cytometry. (A) Calibration Beadmix was used to assess light scatter and fluorescence. Shown are typical data from the BeadMix analyzed on the A50-Micro flow cytometer. The left cytogram shows that all populations included, 2 green fluorescent (488 nm laser) polystyrene beads with diameters of 0.11 μm and 0.5 μm, and 6 silica beads with diameters of 0.18, 0.24, 0.30, 0.59, 0.88 and 1.30 μm, were resolved from each other and from instrument noise. The right cytogram was gated to exclude the fluorescent latex beads so that the non-fluorescent beads can be evaluated more clearly. (B) Histograms show side scattering (left) and forward scattering (middle) from 0.18, 0.24, 0.30, 0.59, 0.88 and 1.3 μm silica microspheres (blue peaks) and the noise threshold limit (dotted line). Representative dot plot (right) of a CSF sample showing “smaller” particle gate based on forward angle light scatter from 0.30 μm silica microspheres and “larger” particle gate based on forward angle light scatter from 0.30 to 0.59 μm silica microspheres. (C) Flow cytometric analysis of CSF apolipoprotein-bearing particles in human CSF. Fluorescence analysis of CSF samples labeled with apoE-Alexa Fluor 488, apoJ-Alexa Fluor 488, or apoAI-FITC (right panels). Density plots show background from phosphate-buffered saline (PBS) labeled with the same antibodies (left panels) used as background control. Isotype control of corresponding species of IgG was used to label the same CSF samples (middle panels) as negative controls and results used to set gating for positive events. (D) Fluorescence analysis of a CSF sample labeled with annexin V-FITC with binding buffer containing Ca2+ (right). The same labeling procedure but using buffer without Ca2+ was used as negative control (middle). PBS labeled with annexin V-FITC was used as background control (left). (E) Flow cytometric analysis of β-amyloid (Aβ42)-positive particles in human CSF. PBS (upper, left) and isotype control (upper, right) was used as controls. A representative fluorescence plot shows Aβ42-positive events in CSF (lower, left) that were nearly completely removed following pull down of Aβ42 with 6E10 antibody coupled to agarose beads (lower, right). (F) The protein retrieved from the 6E10 pull down pellet was analyzed by Western blot using 6E10 (right) or Aβ42 (left) antibody to reveal expected smear of Aβ42-immunoreactive molecular masses (and immunoglobulin bands). PBS was substituted for CSF as the negative control.

Article Snippet: The following antibodies were employed: rabbit anti-Aβ 1-42 monoclonal antibody (clone H31L21, Invitrogen, Carlsbad, CA), which recognizes human and mouse Aβ 1-42 ; cross reactivity to Aβ 1-40 is not observed in sandwich ELISA according to the manufacturer’s instructions; mouse anti-human Aβ monoclonal antibody (clone 6E10, Covance, Princeton, NJ), mouse anti-human apoE monoclonal antibody (clone 1H4, Abcam, Cambridge, MA); mouse anti-human apoJ monoclonal antibody (clone 3R3/2, Lifespan Biosciences, Seattle, WA); rabbit anti-mouse apoJ polyclonal antibody (Lifespan Biosciences); fluorescein isothiocyanate (FITC)-conjugated mouse anti-human apoAI monoclonal antibody (clone APO-1-1, Fitzgerald Industries, Acton, MA); rabbit anti-mouse apoAI polyclonal antibody (Lifespan Biosciences); FITC-conjugated annexin V (Beckman Coulter, Pasadena, CA); and mouse anti-human tau monoclonal antibody (clone TAU-5, Abcam).

Techniques: Flow Cytometry, Fluorescence, Labeling, Saline, Control, Binding Assay, Negative Control, Western Blot

Journal: Journal of neuropathology and experimental neurology

Article Title: Cerebrospinal Fluid Particles in Alzheimer Disease and Parkinson Disease

doi: 10.1097/NEN.0000000000000207

Figure Lengend Snippet: Cerebrospinal fluid (CSF) particles were classified by the presence of apoE, apoJ, apoAI, or β-amyloid (Aβ42), as detected by labeled antibodies or by binding of annexin V, and stratified by size into larger (A, 300 to 590 nm) or smaller (B, < 300 nm) particles for each sample. One-way ANOVA for each molecularly-defined particle concentration (events/μL) in these 2 size ranges each had p < 0.0001. Corrected multiple comparisons for CSF particle concentration had p < 0.01 for all paired comparisons except for apoE-positive vs. apoJ-positive, which were not significantly different for either size range.

Article Snippet: The following antibodies were employed: rabbit anti-Aβ 1-42 monoclonal antibody (clone H31L21, Invitrogen, Carlsbad, CA), which recognizes human and mouse Aβ 1-42 ; cross reactivity to Aβ 1-40 is not observed in sandwich ELISA according to the manufacturer’s instructions; mouse anti-human Aβ monoclonal antibody (clone 6E10, Covance, Princeton, NJ), mouse anti-human apoE monoclonal antibody (clone 1H4, Abcam, Cambridge, MA); mouse anti-human apoJ monoclonal antibody (clone 3R3/2, Lifespan Biosciences, Seattle, WA); rabbit anti-mouse apoJ polyclonal antibody (Lifespan Biosciences); fluorescein isothiocyanate (FITC)-conjugated mouse anti-human apoAI monoclonal antibody (clone APO-1-1, Fitzgerald Industries, Acton, MA); rabbit anti-mouse apoAI polyclonal antibody (Lifespan Biosciences); FITC-conjugated annexin V (Beckman Coulter, Pasadena, CA); and mouse anti-human tau monoclonal antibody (clone TAU-5, Abcam).

Techniques: Labeling, Binding Assay, Concentration Assay

Journal: Journal of neuropathology and experimental neurology

Article Title: Cerebrospinal Fluid Particles in Alzheimer Disease and Parkinson Disease

doi: 10.1097/NEN.0000000000000207

Figure Lengend Snippet: Flow cytometric analysis of the association of β-amyloid (Aβ42) with other cerebrospinal fluid (CSF) particles. (A) Dual fluorescence analysis of a representative CSF sample showing Aβ42 co-labeled with apoE-positive (upper left) or apoJ-positive (upper right) particles but not apoAI-positive (lower left) or annexin V-positive (lower right) particles. (B) Scatter plot with best-fit line for apoE associated Aβ42 particles vs. apoJ associated Aβ42 particles for all 131 participants.

Article Snippet: The following antibodies were employed: rabbit anti-Aβ 1-42 monoclonal antibody (clone H31L21, Invitrogen, Carlsbad, CA), which recognizes human and mouse Aβ 1-42 ; cross reactivity to Aβ 1-40 is not observed in sandwich ELISA according to the manufacturer’s instructions; mouse anti-human Aβ monoclonal antibody (clone 6E10, Covance, Princeton, NJ), mouse anti-human apoE monoclonal antibody (clone 1H4, Abcam, Cambridge, MA); mouse anti-human apoJ monoclonal antibody (clone 3R3/2, Lifespan Biosciences, Seattle, WA); rabbit anti-mouse apoJ polyclonal antibody (Lifespan Biosciences); fluorescein isothiocyanate (FITC)-conjugated mouse anti-human apoAI monoclonal antibody (clone APO-1-1, Fitzgerald Industries, Acton, MA); rabbit anti-mouse apoAI polyclonal antibody (Lifespan Biosciences); FITC-conjugated annexin V (Beckman Coulter, Pasadena, CA); and mouse anti-human tau monoclonal antibody (clone TAU-5, Abcam).

Techniques: Fluorescence, Labeling

Journal: Molecular Biology of the Cell

Article Title: A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

doi: 10.1091/mbc.e14-05-1029

Figure Lengend Snippet: FIGURE 1: Nucleolar associations of human p150. (A) Novel nucleolus-related p150-interacting proteins identified by mass spectrometry. Proteins listed were not previously known to interact with p150 and had multiple peptides detected in two different experiments (E1 and E2). Full lists are in Supplemental Table S1. Number of peptides (#Pep) and spectral counts (SpC) are indicated. (B) Purified p150-associated proteins prepared for mass spectrometry. Affinity- purified samples from untagged HeLa S3-Trex (lane 1) and HeLa S3-Trex-NTAP-p150 (lane 2) cells were analyzed on a silver-stained 5–20% SDS–PAGE gel. CAF-1 subunits p150, p60, and p48 are indicated. (C) Confocal microscopy analysis of p150 (red) and Ki67 (green) in HeLa S3 cells detects partial colocalization at nucleoli in the merged image (yellow). Fields of cells were photographed with a 63× objective. Cells had either been blocked in G1 phase by double- thymidine treatment or released into S phase for 2 h. Scale bars, 10 μm. (D) Location of ChIP primer pairs on the human 47S rRNA–encoding rDNA locus. (E) p150 occupancy at rDNA genes in asynchronous HeLa S3-Trex-shRNA cells. Also assayed were the Pol II gene RPS20 and a gene desert from chromosome 16. Cells expressing either a control shRNA (sh-Luc) or the shRNA targeting p150 (sh-p150) were compared. Chromatin was precipitated with either nonimmune rabbit serum (IgG) or anti-p150 serum (α-p150) as indicated. The mean percentages of input chromatin precipitated from three biological replicates are presented, with error bars representing SDs. Asterisks indicate loci at which sh-p150 caused a statistically significant decrease in p150 occupancy (p < 0.05). Dotted line shows p150 signal at the gene desert. (F) p150 occupancy in asynchronous HeLa cells compared with cells blocked with double- thymidine treatment. Asterisks indicate loci at which thymidine arrest caused a statistically significant increase in p150 occupancy (p < 0.05).

Article Snippet: In addition, rabbit polyclonal anti-p150 antibodies (sc-10772, Santa Cruz Biotechnology, Santa Cruz, CA; and sera specifically generated for this study) were used for immunoblotting and ChIP.

Techniques: Mass Spectrometry, Purification, Affinity Purification, Staining, SDS Page, Confocal Microscopy, shRNA, Expressing, Control

Journal: Molecular Biology of the Cell

Article Title: A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

doi: 10.1091/mbc.e14-05-1029

Figure Lengend Snippet: FIGURE 2: p150 depletion disrupts localization of multiple nucleolar proteins. (A) HeLa S3-Trex-p150-shRNA1 (sh-p150) or luciferase-shRNA cells (sh-Luc) were treated with doxycycline for 72 h to induce shRNA expression and then prepared for indirect immunofluorescence, staining up to two nucleolar proteins simultaneously in each experiment as indicated. Fields of cells photographed with a 63× objective. Scale bar, 20 μm. (B) Representative individual cells from A were enlarged. Scale bar, 10 μm. (C) As in A and B, showing the localization of fibrillarin, which remains unchanged upon p150 depletion. (D) ChIP analysis of TTF-1 in HeLa S3 cells expressing the indicated shRNAs. Chromatin was precipitated with either nonimmune rabbit serum (IgG) or anti–TTF-1 antibodies as indicated and occupancy at the rDNA terminator R1 and the glyceraldehyde-3-phosphate dehydrogenase gene was measured. The mean amounts of precipitated chromatin from three biological replicates are presented as percentages of input. Error bars represent SDs. *p < 0.01 comparing TTF-1 occupancy at R1 in the sh-Luc and sh-p150 samples; p ≥ 0.25 for all other pairs shown.

Article Snippet: In addition, rabbit polyclonal anti-p150 antibodies (sc-10772, Santa Cruz Biotechnology, Santa Cruz, CA; and sera specifically generated for this study) were used for immunoblotting and ChIP.

Techniques: Luciferase, shRNA, Expressing, Immunofluorescence, Staining

Journal: Molecular Biology of the Cell

Article Title: A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

doi: 10.1091/mbc.e14-05-1029

Figure Lengend Snippet: FIGURE 3: Domain analysis of human p150. (A) Diagram of transgenes introduced into HeLa cells. Colored boxes indicate the V5 epitope tags (yellow), PEST region (red), low-complexity regions KER (blue) and ED (green), dimerization region (D, orange), the N-terminal PCNA interaction peptide (PIP, cyan), and the HP1-binding site (pink). The I99A point mutation that inactivates the SUMO-interaction motif (SIM, red asterisk) and silent mutations rendering the transgenes resistant to RNAi (shR, black asterisks) are indicated. Regions previously shown to be sufficient for in vitro chromatin assembly and p60 interaction are drawn below the top diagram. (B) Immunoblot analysis of V5-tagged transgene-expressing cell lines in two independent experiments (lanes 1–10 and 11–28). Cells were infected for 72 h with lentiviruses expressing either sh-luciferase (L, odd-numbered lanes) or sh-p150 (P, even-numbered lanes) and whole-cell extracts were separated on 10% SDS–PAGE gels. After transfer, blots were probed with anti-p150 to detect endogenous p150, anti-V5 to detect transgene products, and anti-tubulin (TUBA) or anti-fibrillarin (FBL) antibodies as loading controls. Arrowheads indicate full-length p150 protein. (C) Interaction between p150 transgene products and p60. Extracts from the indicated V5-tagged transgene-expressing cell lines were immunoprecipitated with anti-V5 antibodies. Input extracts (In, odd-numbered lanes) and immunoprecipitates (IP, even-numbered lanes) were resolved on 10% SDS–PAGE gels, transferred to membranes, and probed with anti-p150, V5, and p60 antibodies. Arrowhead indicates full-length p150 protein; asterisks indicate anti-V5 IgG bands in IP samples detected by the anti-mouse secondary antibody. Note that the 1-310 transgene comigrates with the IgG heavy chain. (D) V5-tagged, RNAi-resistant p150 transgenes were tested for the ability to maintain cell proliferation in the presence of either sh-luciferase (sh-Luc) or p150-shRNA1 (sh-p150) in HeLa cells. Cells (4500) were plated in six-well dishes and infected with the indicated lentivirus, and selection for infected cells began 48 h post infection. At 7d postinfection, cell proliferation was assessed by crystal violet staining.

Article Snippet: In addition, rabbit polyclonal anti-p150 antibodies (sc-10772, Santa Cruz Biotechnology, Santa Cruz, CA; and sera specifically generated for this study) were used for immunoblotting and ChIP.

Techniques: Binding Assay, Mutagenesis, In Vitro, Western Blot, Expressing, Infection, Luciferase, SDS Page, Immunoprecipitation, Selection, Staining

Journal: Molecular Biology of the Cell

Article Title: A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

doi: 10.1091/mbc.e14-05-1029

Figure Lengend Snippet: FIGURE 4: The p150 SUMO-interaction motif (SIM) is required to maintain nucleolar Ki67 localization. HeLa cell lines expressing the indicated V5-tagged transgenes (A, luciferase; B, 1-938; C, 1-310; D, 1-310-ΔHP1; E, 1-310-I99A; F, 1-310-ΔPIP1; see Figure 3A) were infected with lentiviruses encoding the indicated shRNAs (sh-luciferase or sh-p150) and prepared for indirect immunofluorescence 72 h later. Scale bar, 20 μm.

Article Snippet: In addition, rabbit polyclonal anti-p150 antibodies (sc-10772, Santa Cruz Biotechnology, Santa Cruz, CA; and sera specifically generated for this study) were used for immunoblotting and ChIP.

Techniques: Expressing, Luciferase, Infection, Immunofluorescence

Journal: Molecular Biology of the Cell

Article Title: A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

doi: 10.1091/mbc.e14-05-1029

Figure Lengend Snippet: FIGURE 5: The p150 SIM is required to maintain nucleolar Nopp140 localization. As in Figure 4, HeLa cell lines expressing the indicated V5-tagged transgenes were infected with lentiviruses encoding the indicated shRNAs (sh-luciferase or sh-p150) and prepared for indirect immunofluorescence 72 h later. Scale bar, 20 μm.

Article Snippet: In addition, rabbit polyclonal anti-p150 antibodies (sc-10772, Santa Cruz Biotechnology, Santa Cruz, CA; and sera specifically generated for this study) were used for immunoblotting and ChIP.

Techniques: Expressing, Infection, Luciferase, Immunofluorescence

Journal: Molecular Biology of the Cell

Article Title: A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

doi: 10.1091/mbc.e14-05-1029

Figure Lengend Snippet: FIGURE 6: Higher-order interactions of rDNA chromatin are altered upon p150 depletion. (A) DNA FISH analysis of MCF10A-Tet-KRAB cells expressing the indicated shRNAs. Three biological replicate experiments were performed, with mean percentage nucleolar association and SDs graphed. Probes analyzed were a chromosome 10q BAC (10q, total alleles assayed [n] = 332 for sh-Luc, 262 for sh-p150), a 5S rDNA–containing BAC (5S, n = 312 for sh-Luc, 352 for sh-p150), α-satellite DNA from chromosome 17 (αSat 17, n = 288 for sh-Luc, 306 for sh-p150), and negative control BAC (− Control, n = 314 for sh-Luc, 306 for sh-p150) that was previously reported to be unassociated with nucleoli (Nemeth et al., 2010). The p values comparing the sh-Luc and sh-p150 samples for each probe are indicated, with p < 0.05 indicated in red, demonstrating statistical significance. (B) Fluorescence microscopy images of representative cells from an experiment from A. rDNA is colored red, the 10q BAC is green, and DAPI is blue. Scale bar, 5 μm. (C) Immunoblot of representative whole-cell extracts from an experiment in A, with tubulin as loading control. (D) Immuno-FISH analysis of primary human foreskin fibroblasts treated either with luciferase control or p150-targeted sh-RNAs. Cells were treated with anti-fibrillarin antibodies, followed by hybridization with a αSat 17 probe. total alleles assayed = 324 for sh-Luc, 308 for sh-p150. (E) Data from D, plotting the percentage of cells displaying the indicated numbers of αSat 17 alleles associated with fibrillarin. The total cells assayed = 162 for sh-Luc, 154 for sh-p150. (F) Fluorescence microscopy images of representative cells from an experiment from D. Fibrillarin is in green, and the αSat 17 probe is red. Scale bar, 5 μm. (G) Immunoblot of representative whole-cell extracts from an experiment in D, with fibrillarin as loading control.

Article Snippet: In addition, rabbit polyclonal anti-p150 antibodies (sc-10772, Santa Cruz Biotechnology, Santa Cruz, CA; and sera specifically generated for this study) were used for immunoblotting and ChIP.

Techniques: Expressing, Negative Control, Control, Fluorescence, Microscopy, Western Blot, Luciferase, Hybridization

Journal: Molecular Biology of the Cell

Article Title: A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

doi: 10.1091/mbc.e14-05-1029

Figure Lengend Snippet: FIGURE 7: The p150 N-terminus is necessary and sufficient for nucleolar interchromosomal associations. (A) DNA FISH analysis of the association between αSat 17 and rDNA in HeLa cells expressing the indicated V5-tagged transgenes (luciferase, p150N [aa 1–310], or p150C [aa 245–938]). The percentage of αSat 17 alleles colocalized with the rDNA is indicated, with mean and SDs from three experiments. For V5-luciferase cells, total alleles assayed = 471 for sh-Luc and 480 for sh-p150; for V5-p150N cells, 525 for sh-Luc and 489 for sh-p150; and or V5-p150C cells, 450 for sh-Luc and 468 for sh-p150. p values comparing the sh-Luc and sh-p150 samples are indicated, demonstrating statistically significant rescue by the p150N but not the p150C transgene. (B–D) Data from the experiment in A replotted to display the number of αSat 17 alleles per cell associated with the rDNA. Note that these HeLa-derived lines have three copies of chromosome 17. For V5-luciferase cells, total cells examined = 157 for sh-Luc and 160 for sh-p150; for V5-p150N cells, 175 for sh-Luc and 163 for sh-p150; and for V5-p150C cells, 150 for sh-Luc and 156 for sh-p150. Comparisons for which p ≤ 0.05 are indicated by asterisks, and a number sign indicates p ≤ 0.01. (E) Fluorescence microscopy images of representative cells from an experiment from A. rDNA is colored red, the αSat 17 is green, and DAPI is blue. Scale bar, 5 μm.

Article Snippet: In addition, rabbit polyclonal anti-p150 antibodies (sc-10772, Santa Cruz Biotechnology, Santa Cruz, CA; and sera specifically generated for this study) were used for immunoblotting and ChIP.

Techniques: Expressing, Luciferase, Derivative Assay, Fluorescence, Microscopy

Journal: Molecular Biology of the Cell

Article Title: A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

doi: 10.1091/mbc.e14-05-1029

Figure Lengend Snippet: FIGURE 8: The p150 SIM and sumoylation proteins are required for nucleolar interchromosomal associations. (A) Immuno-FISH analysis of the association between αSat 17 and fibrillarin in HeLa cells expressing the indicated V5-tagged transgene derivatives of full-length p150. The percentage of αSat 17 alleles colocalized with fibrillarin is indicated, with mean and SDs from three experiments. For ΔPIP cells, total alleles assayed = 510 for sh-Luc and 477 for sh-p150; for ΔHP1 cells, 522 for sh-Luc and 513 for sh-p150; and for I99A cells, 486 for sh-Luc and 480 for sh-p150. p values comparing the sh-Luc and sh-p150 samples are indicated, demonstrating statistically significant rescue by the ΔPIP and ΔHP1 but not the I99A transgenes. (B–D) Data from the experiment in A replotted to display the number of αSat 17 alleles per cell associated with the rDNA. Note that these HeLa-derived lines have three copies of chromosome 17. For ΔPIP cells, total cells examined = 170 for sh-Luc and 159 for sh-p150; for ΔHP1 cells, 174 for sh-Luc and 171 for sh-p150; and for I99A cells, 162 for sh-Luc and 160 for sh-p150. Comparisons for which p ≤ 0.05 are indicated by asterisks. (E) Immuno-FISH analysis of MCF10A cells treated with the indicated siRNAs. The percentage of αSat 17 alleles colocalized with fibrillarin is indicated, with mean and SDs from three experiments. The total alleles assayed = 328 for si-luciferase–treated cells, 346 for si-p150–treated cells, 324 for si-UBC9–treated cells, and 342 for si-SUMO2-treated cells. p values comparing the sh-Luc and other samples are indicated, demonstrating statistically significant reductions in association by each of the three test depletions. (F) Data from the experiment in E replotted to display the number of αSat 17 alleles per cell associated with fibrillarin. Note that these MCF10A-derived lines have two copies of chromosome 17. (G) Quantitative PCR analyses of esiRNA treatments demonstrating reduced steady-state mRNA levels in the targeted cells. (H) Immunoblot of representative whole-cell extracts from an experiment in E, with fibrillarin as loading control. (I) Fluorescence microscopy images of representative cells from an experiment from E and F. Fibrillarin is colored green, the αSat 17 is red, and DAPI is blue. Scale bar, 5 μm.

Article Snippet: In addition, rabbit polyclonal anti-p150 antibodies (sc-10772, Santa Cruz Biotechnology, Santa Cruz, CA; and sera specifically generated for this study) were used for immunoblotting and ChIP.

Techniques: Expressing, Derivative Assay, Luciferase, Real-time Polymerase Chain Reaction, esiRNA, Western Blot, Control, Fluorescence, Microscopy