Journal: Molecular cancer research : MCR

Article Title: Hyperphosphorylation of CDH1 in glioblastoma cancer stem cells attenuates APC/C CDH1 activity and pharmacological inhibition of APC/C CDH1/CDC20 compromises viability

doi: 10.1158/1541-7786.MCR-18-1361

Figure Lengend Snippet: (a) The APC/C holoenzyme was immunoprecipitated (IP) using anti-CDC27 from G1 synchronized NSTCs and CSCs from GBM 3832 with resulting levels of the indicated APC/C subunits identified by silver staining. Control lane = anti-CDC27 antibody and beads only. Antibody heavy chain is indicated (IgG). (b) The APC/C holoenzyme was immunoprecipitated using anti-CDC27 from G1 synchronized NSTCs and CSCs from GBM 3832 with resulting immunoprecipitates analyzed by LC-MS/MS. Protein abundance for the indicated APC/C holoenzyme subunits and CDH1 were quantified and resulting fold change graphed (normalized to the NSTCs). (c-e) Whole-cell lysates from G1 synchronized NSTCs and CSCs from GBM 3832 (c), 3691 (d), and 08-387 (e) were probed for APC4 and CDH1. βactin served as a loading control. (f-h) The APC/C holoenzyme was immunoprecipitated from G1 synchronized NSTCs and CSCs from GBM 3832 using anti-CDC27 (f) or anti-APC4 (g) and from 3691 using anti-APC4 (h). Resulting immunoprecipitates were probed for CDC27 or APC4 and CDH1 (top two panels). Control lane = anti-CDC27 or anti-APC4 antibody and beads only. Bound CDH1 relative to CDC27 or APC4 as an indication of active APC/C was quantified and resulting fold change graphed (normalized to NSTCs). n = 3 biological replicates, error bars represent standard deviation; *P < 0.05; **P < 0.01; ***P < 0.001. Bottom two panels represent inputs for the CDC27 or APC4 immunoprecipitation. βactin or α-tubulin served as loading controls. For all immunoblots, the molecular weight (MW) of resulting bands is given in kilodaltons (kD).

Article Snippet: Immunoprecipitation (all used at 1 ug per reaction); mouse anti-CDC27, clone AF3.1 (Sigma-Aldrich), mouse anti-APC4, clone CIV1.1 (Novus Biologicals), or mouse anti-CDH1, clone DH01 (Calbiochem/MilliporeSigma).

Techniques: Immunoprecipitation, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Standard Deviation, Western Blot, Molecular Weight

Journal: Molecular cancer research : MCR

Article Title: Hyperphosphorylation of CDH1 in glioblastoma cancer stem cells attenuates APC/C CDH1 activity and pharmacological inhibition of APC/C CDH1/CDC20 compromises viability

doi: 10.1158/1541-7786.MCR-18-1361

Figure Lengend Snippet: (a-c) Whole-cell lysates from G1 synchronized NSTCs and CSCs from GBM 3832 (a), 3691 (b), and 08-387 (c) were probed for EMI1. βactin served as a loading control. (d-e) The APC/C holoenzyme was immunoprecipitated (IP) from G1 synchronized NSTCs and CSCs from GBM 3832 (d) or 3691 (e) using anti-APC4. Resulting immunoprecipitates were probed for APC4 and EMI1 (top two panels). Control lane = anti-APC4 antibody and beads only. The line in (d) indicates that two non-relevant lanes were removed from the image between the control lane and the 3832 immunoprecipitation lanes. Bound EMI1 relative to APC4 was quantified and resulting fold change graphed (normalized to NSTCs). n = 3 biological replicates, error bars represent standard deviation; ns = no significance. Bottom two panels represent input for EMI1 immunoprecipitation. α-tubulin served as a loading control. For all immunoblots, the molecular weight (MW) of resulting bands is given in kilodaltons (kD).

Article Snippet: Immunoprecipitation (all used at 1 ug per reaction); mouse anti-CDC27, clone AF3.1 (Sigma-Aldrich), mouse anti-APC4, clone CIV1.1 (Novus Biologicals), or mouse anti-CDH1, clone DH01 (Calbiochem/MilliporeSigma).

Techniques: Immunoprecipitation, Standard Deviation, Western Blot, Molecular Weight

Journal: PLoS Pathogens

Article Title: Memory like NK cells display stem cell like properties after Zika virus infection

doi: 10.1371/journal.ppat.1009132

Figure Lengend Snippet: ( A ) Hallmark pathway gene sets analysis in FACS purified CD27 + memory like and non-memory CD27 - NK cells a month post ZIKV infection. RNA-seq data are from 3 biological replicates for each group. ( B ) ATAC-seq tracks for selected loci of Wnt/β-catenin pathway genes in CD27 + memory like (green) and non-memory CD27 - NK cells (blue). ( C-D ) GSEA enrichment plots ( C ) and heat maps ( D ) for Wnt/β-catenin pathway ( C left ) and canonical Wnt targets ( C right ) among CD27 + memory like and non-memory CD27 - NK gene sets. (E - G ) Immunofluorescence images of FACS purified CD27 + memory like and non-memory CD27 - NK cells, isolated a month post ZIKV infection, and stained for TCF-1 (yellow) and β-catenin (red) show co-localization of both molecules in CD27 + cells. Scale bar corresponds to 10 μm. Top-CD27 + NK cells, Bottom-CD27 - NK cells. Data are representative of 2 independent experiments. ( G ) Relative fluorescent intensity of TCF-1 in CD27 + memory like and non-memory CD27 - NK. Data are representative of 2 independent experiments (n = 4 per experiment). ( H ) Transcription factor motif enrichment analysis of ATAC-seq from CD27 + memory like NK cells. ( I ) % of target sequences with TCF-1 motifs in genomic regions of CD27 + memory like and non-memory CD27 - NK cells. Chromatin opening by ATAC-seq ( J ) and gene expression by RNA-seq ( K ) of selected TCF-1 target genes in CD27 + memory like NK cells. Fold change values for peaks are plotted in ( J ). Mean ± s.d. two-sided Student’s t-test, *** P ≤ 0.001.

Article Snippet: This was followed by antibody staining step and cells were incubated with TCF-1-PE (4:100) (S33-966, BD Biosciences, #564217) and β-catenin-APC (4:100) (REA480, Miltenyi Biotech, #130124453) antibodies in PBS with 0.5% BSA for 45 min. Then, cells were washed with PBS containing 0.5% BSA, pelleted and subjected to cytospin for 2 minutes and taken on the slides.

Techniques: Purification, Infection, RNA Sequencing, Immunofluorescence, Isolation, Staining, Gene Expression

Journal: The Journal of Cell Biology

Article Title: The APC/C recruits cyclin B1–Cdk1–Cks in prometaphase before D box recognition to control mitotic exit

doi: 10.1083/jcb.200912084

Figure Lengend Snippet: Binding of phosphorylated APC/C Cdc20 to cyclin B1 starts in mitosis. (A) Cyclin B1 IPs from synchronized G2 phase and nocodazole-arrested mitotic cells (mitosis) are shown. See Materials and methods for synchronization details. IPs were analyzed for coprecipitating proteins by Western blotting probed for anti-APC3 (top; showing only phosphorylated APC3) or anti–cyclin B1 (middle). (bottom) The presence of unphosphorylated (lane 1) and phosphorylated (lane 2) APC3 in whole cell extracts (WCE) used for the IPs (10% of input) is shown. (B) APC/C IPs using anti-APC6 antibodies on extracts of G2 phase cells and mitotic cells. (C) Cyclin B1 IPs on extracts from G2 or mitotic cells were used in in vitro ubiquitination assays. Mitotic cells arrested in nocodazole were released but kept in mitosis by the proteasome inhibitor MG132 (NR, MG132). Control, GFP IPs. Purified E1 enzyme, the APC/C-specific E2 enzyme UbcH10, ATP, and HA-ubiquitin (HA-Ub) were added to start the reactions. HA-ubiquitin–conjugated reaction products were probed with anti-HA antibody (for further controls see Fig. S3 ). Asterisks indicate an aspecific background band in the control GFP IPs. (D) Comparison of mitotic cells arrested in the spindle checkpoint (Noco), released from the spindle checkpoint arrest but kept in mitosis by MG132, or allowed to enter mitosis in the presence of MG132; the latter contain high cyclin A levels (top). Cyclin B1 IPs are shown; negative control, securin (Sec) IP. Western blots were analyzed for coprecipitating proteins with the indicated antibodies. (top) Phosphorylated forms of APC3 in cyclin B1 IPs are shown. (E) Cells were transfected and selected for expression of the indicated cyclin–GFP fusion proteins. Mitotic cells that had entered mitosis in the presence of the proteasome inhibitor MG132 were collected by shake off. GFP IPs were probed for coprecipitation of Cdc20. (F) Cells were synchronized in G2 and mitosis as indicated. Anti–cyclin A IPs, anti–cyclin B1 IPs, and mitotic extracts were blotted for APC3, Cdc20, and Mad2. Comparable polyclonal antibodies were used detecting the N-terminal 430 amino acids of the respective cyclins. Markers are given in kilodaltons.

Article Snippet: For detecting proteins, the following antibodies were used: rabbit anti-APC2 (provided by H. Yu, University of Texas Southwestern Medical Center, Dallas, TX), mouse anti-APC3/Cdc27 (Transduction Laboratories), goat anti-APC4 (C18S; Santa Cruz Biotechnology, Inc.), rabbit anti-GFP (26), mouse anti-HA (HA-7; Sigma-Aldrich), mouse anti-Mad2 (MBL International), mouse anti–cyclin A (Thermo Fisher Scientific), mouse anti–cyclin B1 (GNS1), mouse anti-securin (Abcam), rabbit anti-Plk1 (Invitrogen), mouse anti-Cdc20 (E7; Santa Cruz Biotechnology, Inc.), mouse anti-Cdh1 (Thermo Fisher Scientific), mouse anti-Cdk1 (Transduction Laboratories), and rabbit anti-Cdk2 (M2; Santa Cruz Biotechnology, Inc.).

Techniques: Binding Assay, Western Blot, In Vitro, Purification, Negative Control, Transfection, Expressing