Journal: Molecular Biology of the Cell

Article Title: Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes

doi: 10.1091/mbc.E14-06-1109

Figure Lengend Snippet: Mitotic chromosomal association of PRC1 fusion proteins. (A–H) Confocal fluorescence images of PRC1 fusion proteins tagged with either YFP or Cerulean expressed in PGK12.1 ES cells in various phases of mitosis. The mCherry-H2A fusion protein was used to mark mitotic chromosomes. Prometaphase (top), metaphase (middle), and anaphase/telophase (bottom). Representative images from Z -scan stacks are presented in the figures. Scale bar, 5 μm. (I) Quantitative analysis of mitotic binding fraction of PRC1 fusion proteins at mitotic chromosomes. The mitotic binding fraction is the average of individual slices of Z -scan stacks. The data represent at least 10 cells analyzed. Error bars, SD of the mean.

Article Snippet: The sequences coding Cerulean (Addgene, Cambridge, MA), YFP ( Ren et al. , 2008 ), and mCherry (Addgene) fluorescence proteins were amplified by PCR and inserted into the pTRIPZ(M) to produce vectors pTRIPZ(M)-Cerulean, pTRIPZ(M)-YFP, and pTRIPZ(M)-mCherry.

Techniques: Fluorescence, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes

doi: 10.1091/mbc.E14-06-1109

Figure Lengend Snippet: Directly recruiting PRC1 fusion proteins to mitotic chromosomes by YFP-Cbx2 but not by YFP-Cbx2 1-498 . (A) Confocal fluorescence images of Cerulean-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and mCherry-H2A fusion proteins in Cbx2 −/− ES cells in metaphase. Scale bar, 5 μm. (B) Confocal fluorescence images of Cerulean-Ring1b fusion protein coexpressed with YFP-Cbx2 1-498 and mCherry-H2A fusion protein in Cbx2 −/− ES cells in metaphase. Scale bar, 5 μm. (C) Quantification of mitotic chromosomal association of YFP-Cbx2 and YFP-Cbx2 1-498 fusion proteins in Cbx2 −/− ES cells. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (D) Quantitative analysis of mitotic chromosomal association of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and of Cerulea-Ring1b coexpressed with YFP-Cbx2 1-498 in Cbx2 −/− ES cells in metaphase. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (E) Photobleaching FRET images of Cerulean-Ring1b interaction with YFP-Cbx2 and YFP-Cbx2 1-498 at mitotic chromosomes. The YFP-Cbx2, YFP-Cbx2 1-498 , and Cerulean-Ring1b fusion proteins as indicated above images expressed in Cbx2 −/− ES cells. Half-area of fluorescence of YFP-Cbx2 or YFP-Cbx2 1-498 fusion proteins at mitotic chromosomes was photobleached. Z -scan imaging of live cells by confocal laser microscope was performed before (top) and after (bottom) photobleaching. The arrowheads indicate the bleaching areas.

Article Snippet: The sequences coding Cerulean (Addgene, Cambridge, MA), YFP ( Ren et al. , 2008 ), and mCherry (Addgene) fluorescence proteins were amplified by PCR and inserted into the pTRIPZ(M) to produce vectors pTRIPZ(M)-Cerulean, pTRIPZ(M)-YFP, and pTRIPZ(M)-mCherry.

Techniques: Fluorescence, Imaging, Microscopy

Journal: Molecular Biology of the Cell

Article Title: Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes

doi: 10.1091/mbc.E14-06-1109

Figure Lengend Snippet: Analysis of structural elements of YFP-Cbx2 fusion protein required for its targeting and immobilizing. (A) Diagram of structural domains of Cbx2. The dashed rectangles indicate the region required for targeting Cbx2 to mitotic chromosomes (left) and the region required for immobilizing Cbx2 at mitotic chromosomes (right). ATH, AT-hook domain; Cbox, chromobox domain; CHD, chromodomain domain. The numbers in parentheses indicate the starting and ending of amino acid sequence. (B) Confocal images of YFP-Cbx2 and its variant fusion proteins in metaphase of HeLa cells. YFP-Cbx2 mutant and mCherry-H2A fusion proteins were stably expressed in HeLa cells. The mCherry-H2A was used to mark mitotic chromosomes. Scale bar, 5 μm. (C) FRAP curves of interphases and metaphases of YFP-Cbx2 and its variant fusion proteins expressed in HeLa cells. FRAP analysis is described in . (D) Residence time and immobile fraction of YFP-Cbx2 variant fusion proteins at interphasic and mitotic chromatins. The residence time and immobile fraction were calculated from FRAP curves by fitting a first-order kinetic model.

Article Snippet: The sequences coding Cerulean (Addgene, Cambridge, MA), YFP ( Ren et al. , 2008 ), and mCherry (Addgene) fluorescence proteins were amplified by PCR and inserted into the pTRIPZ(M) to produce vectors pTRIPZ(M)-Cerulean, pTRIPZ(M)-YFP, and pTRIPZ(M)-mCherry.

Techniques: Sequencing, Variant Assay, Mutagenesis, Stable Transfection

Journal: bioRxiv

Article Title: Proteasomal Inhibition Triggers Viral Oncoprotein Degradation via Autophagy-Lysosomal Pathway

doi: 10.1101/780171

Figure Lengend Snippet: (A) ∼10 x 10 6 HEK293 cells transfected with either empty vector (pEGFP-C1) or GFP-tagged EBNA3C expression plasmid, either left untreated (DMSO control), or treated with 20 µM MG132 alone or MG132 plus 50 µM chloroquine (CQ). (B) HEK293 cells stably transfected with pTripz-mCherry-Sh-Beclin1 construct expressing sh-Beclin 1 under doxycycline (Dox) inducible promoter were further transfected either empty vector (pA3M) or myc-tagged EBNA3C expressing construct. 36 h post-transfection cells were either left untreated or treated with 20 µM MG132. (A-B) 4 h post-treatment cells were harvested, washed with 1 x PBS, lysed in RIPA buffer and subjected for western blot analyses for the indicated antibodies. (C) ∼5 x 10 4 HEK293 cells stably expressing sh-Beclin 1 with or without doxycycline treatment were transfected with GFP-tagged EBNA3C expressing plasmid using Lipofectamine 3000. 24 h post transfection cells were either left untreated (DMSO control) or treated with 20 µM MG132 for another 4h and subjected to live cell confocal analysis after staining the nucleus with Hochest 33342. Doxycycline treatment induces both mCherry and sh-RNA expression. Scale bars, 5 µm. (D) A colony formation assay was conducted as described in the “Materials and Methods” section using a similar experimental setup in doxycycline inducible sh-Beclin1 stably expressing HEK293 cells transiently transfected with GFP-EBNA3C construct.

Article Snippet: Ltd., India). pEGFP-C1 and pDsRED-Monomer-N1 plasmids were obtained from Clontech Laboratories, Inc. mCherry-tagged p62 expressing plasmid was a kind gift from Edward M. Campbell (Loyola University Chicago, IL, USA). pBabePuro-GFP-LC3 construct was a kind gift from Jayanta Debnath (Addgene plasmid# 22405). pTripz-mCherry-Sh-Beclin1 construct (Clone ID: V2THS_23692) was obtained from GE Healthcare Dharmacon Inc. (Lafayette, CO, USA). pGipz-Sh-Control and pGipz-Sh-EBNA3C were previously described.

Techniques: Transfection, Plasmid Preparation, Expressing, Stable Transfection, Construct, Western Blot, Staining, RNA Expression, Colony Assay

Journal: bioRxiv

Article Title: Proteasomal Inhibition Triggers Viral Oncoprotein Degradation via Autophagy-Lysosomal Pathway

doi: 10.1101/780171

Figure Lengend Snippet: Due to uncontrolled cell proliferation, cancer cells often encounter excess mis-/un-folded protein aggregates, which are subsequently labeled with either K48-linked or K63-linked polyubiquitination for proteolytic degradation. While K48-linked ubiquitin chains are targeted for the proteasomal pathway, K63-linked directs autophagy mediated protein degradation. Upon proteasomal inhibition, EBV oncoprotein EBNA3C is predominantly tagged with K63-linked polyubiquitin chains, translocated to cytoplasm by an unknown mechanism and degraded through autophagy-lysosomal pathway. The N-terminal domain plays a central role in EBNA3C’s degradation through autophagy mechanism by participating within the p62-LC3B complex. Suppression of autophagy pathway (Beclin1 knockdown or chloroquine, CQ treatment) reverses EBNA3C degradation in response to proteasomal inhibition. Additionally, proteasomal inhibitors (such as MG132) induce both autophagy and viral gene transcription that eventually activate viral lytic replication. The results provide foundation to exploit proteasome inhibitors as potential therapeutic approach for EBV associated B-cell lymphomas, where EBNA3C is expressed, typically diagnosed in immunocompromised individuals.

Article Snippet: Ltd., India). pEGFP-C1 and pDsRED-Monomer-N1 plasmids were obtained from Clontech Laboratories, Inc. mCherry-tagged p62 expressing plasmid was a kind gift from Edward M. Campbell (Loyola University Chicago, IL, USA). pBabePuro-GFP-LC3 construct was a kind gift from Jayanta Debnath (Addgene plasmid# 22405). pTripz-mCherry-Sh-Beclin1 construct (Clone ID: V2THS_23692) was obtained from GE Healthcare Dharmacon Inc. (Lafayette, CO, USA). pGipz-Sh-Control and pGipz-Sh-EBNA3C were previously described.

Techniques: Labeling, Inhibition