Journal: bioRxiv

Article Title: An Atlas of Phosphorylation and Proteolytic Processing Events During Excitotoxic Neuronal Death Reveals New Therapeutic Opportunities

doi: 10.1101/2020.06.15.151456

Figure Lengend Snippet: (A) CRMP2 is cleaved at sites near T509 in its C-terminal tail. Left inset: the abundance (M/L) ratios of the neo-N-terminal peptides at 30 and 240 min after glutamate treatment. Right inset: the abundance ratios of the identified phosphosites in the C-terminal tails of CRMP2 at 30 and 240 min after glutamate treatment. N.D.: not detected. Red scissors: cleavage sites. P in red sphere: phosphorylation. (B) A model depicting the new mechanism of dysregulation of neuronal CRMP2 during excitotoxicity uncovered by our proteomic findings. In control neurons, CRMP2 undergoes hierarchical phosphorylation by Cdk5 and GSK3 at sites in the C-terminal tail. Cdk5 phosphorylates the priming site S522. Upon phosphorylation, pS522 binds GSK3, which catalyses processive phosphorylation of CRMP2 at three other sites in the order of S518, T514 and T509. In excitotoxic neurons, cleavage of CRMP2 generates a long truncated CRMP2 fragment that lacks the priming site S522, abolishing S522 phosphorylation by Cdk5 and in turn suppressing processive phosphorylation of S518, T514 and T509 by GSK3. The truncation and lack of phosphorylation at T509, T514 and S518 may contribute to the accumulation of the immunoreactive CRMP2 signals at the dendritic blebs shown in panel E. (C) Structure of a phosphomimetic mutant of CRMP2 (PDB accession: 5yz5). Dotted line shows the disordered C-terminal tail region. (D) Western blots of lysates from control and glutamate-treated neurons probed with anti-CRMP2, anti-pT509 CRMP2 and tubulin antibodies. Asterisks: potential hyper-phosphorylated forms of intact CRMP2 detected by the anti-CRMP2 and anti-pT509 CRMP2 antibodies. (E) Fluorescence microscopy images showing actin (phalloidin), CRMP2 and nuclei (DAPI) in control and glutamate-treated neurons. White arrows indicate dendritic blebs. The close-up views of the images in the rectangles marked by white dotted lines are shown. Inset: The number of dendritic blebs per mm 2 in control and the glutamate treated neurons in three biological replicates. **: p < 0,01; ***: p <0.001.

Article Snippet: For all experiments, DNA counterstain DAPI (Molecular Probes, Thermo Fisher Scientific) was applied before coverslipping with ProLong gold anti-fade reagent (Invitrogen).

Techniques: Phospho-proteomics, Control, Mutagenesis, Western Blot, Fluorescence, Microscopy

Journal: PLoS Genetics

Article Title: Genome-Wide Analysis of Cell Type-Specific Gene Transcription during Spore Formation in Clostridium difficile

doi: 10.1371/journal.pgen.1003756

Figure Lengend Snippet: Cells of the C. difficile 630Δerm strain, the spoIIID or spoVT mutant and the complemented strains, CDIP262, carrying a multicopy spoIIID gene controlled by its native promoter or CDIP263, carrying a multicopy spoVT gene controlled by its native promoter were collected after 24 h of growth in SM broth, stained with the DNA stain DAPI and the membrane dye FM4-64 and examined by phase contrast and fluorescence microscopy.

Article Snippet: The cells were washed with 1 ml of PBS and ressuspended in 0.1 ml of PBS supplemented with the membrane dye FM4-64 (10 μg.ml −1 ) and the DNA stain DAPI (4′,6-diamidino-2-phenylindole; 50 μg.ml −1 ) (Molecular Probes, Invitrogen).

Techniques: Mutagenesis, Staining, Membrane, Fluorescence, Microscopy

Journal: PLoS Genetics

Article Title: Genome-Wide Analysis of Cell Type-Specific Gene Transcription during Spore Formation in Clostridium difficile

doi: 10.1371/journal.pgen.1003756

Figure Lengend Snippet: Cells of the C. difficile 630Δerm strain, the spoIIR mutant and the complemented strain, CDIP246, carrying a multicopy spoIIR gene controlled by its native promoter were collected after 24 h of growth in SM broth, stained with the DNA stain DAPI and the membrane dye FM4-64 and examined by phase contrast and fluorescence microscopy.

Article Snippet: The cells were washed with 1 ml of PBS and ressuspended in 0.1 ml of PBS supplemented with the membrane dye FM4-64 (10 μg.ml −1 ) and the DNA stain DAPI (4′,6-diamidino-2-phenylindole; 50 μg.ml −1 ) (Molecular Probes, Invitrogen).

Techniques: Mutagenesis, Staining, Membrane, Fluorescence, Microscopy

Journal: PLoS Genetics

Article Title: Genome-Wide Analysis of Cell Type-Specific Gene Transcription during Spore Formation in Clostridium difficile

doi: 10.1371/journal.pgen.1003756

Figure Lengend Snippet: Cells of the C. difficile 630Δerm strain, and of the spo0A , sigF and sigE mutants carrying a P spoIIR -SNAP Cd transcriptional fusion in a multicopy plasmid were collected 24 h of following inocculation in SM broth. Cells were labelled with the fluorescent substrate TMR to allow localization of SNAP Cd production driven by the spoIIR promoter, stained with the DNA marker DAPI and the membrane dye MTG and examined by phase contrast and fluorescence microscopy.

Article Snippet: The cells were washed with 1 ml of PBS and ressuspended in 0.1 ml of PBS supplemented with the membrane dye FM4-64 (10 μg.ml −1 ) and the DNA stain DAPI (4′,6-diamidino-2-phenylindole; 50 μg.ml −1 ) (Molecular Probes, Invitrogen).

Techniques: Plasmid Preparation, Staining, Marker, Membrane, Fluorescence, Microscopy

Journal: Nature communications

Article Title: Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site

doi: 10.1038/ncomms1386

Figure Lengend Snippet: a , WC-21 contains a FITC group and a photoactive azide (N 3 ) moiety, and displays high sigma-2 receptor affinity and selectivity; K i = 8.7 ± 1 nM for sigma-2 receptor and K i > 4,000 nM for sigma-1 receptor. b , Following UV cross-linking, ligand-labeled proteins were enriched and separated through SDS-PAGE; WC-21 cross-linked proteins can be visualized using an anti-FITC antibody; SS is silver staining and WB is western blot. c , Proteomic studies of the FITC positive band identified two proteins which shared the same genetic name: progesterone receptor membrane component 1 (PGRMC1). The protein structure shows the peptides (bold) detected by mass spectrometry as well as the Cytochrome b5 domain (underlined).

Article Snippet: 24 h after plating, the cells were transfected with the pCMV6-XL4 vector only or human PGRMC1 cDNA (OriGene Technologies, Inc., Rockville, MD) using a Fugene 6 transfection reagent (Roche Applied Science, Indianapolis, IN).

Techniques: Labeling, SDS Page, Silver Staining, Western Blot, Mass Spectrometry

Journal: Nature communications

Article Title: Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site

doi: 10.1038/ncomms1386

Figure Lengend Snippet: a, Competitive binding studies of [ 125 I] RHM-4 were carried out with the PGRMC1 ligand AG-205, sigma-2 receptor ligands DTG, WC-26 , SV119 , and siramesine or with the sigma-1 receptor ligand (+)-pentazocine. [ 125 I] RHM-4 binding was blocked by AG-205 and the sigma-2 ligands but not by (+)-pentazocine. n = 2, sample in triplicate. b , Typical western blot confirming PGRMC1 protein expression knockdown in PGRMC1 specific siRNA treated HeLa cells relative to nontargeting siRNA treated controls compared to actin (loading control). c , The bar graph shows reduced sigma-2 receptor binding activity in the PGRMC1 knockdown cells; * p<0.01, Student's t-test, n=3. d , Typical western blot confirming increased PGRMC1 protein expression in PGRMC1 transfected HeLa cells relative to vector transfected controls. e , Bar graph showing increased sigma-2 receptor binding activity in PGRMC1 transfected cells; * p<0.01, Student's t-test, n=3. Error bars in a , c , d represent SEM.

Article Snippet: 24 h after plating, the cells were transfected with the pCMV6-XL4 vector only or human PGRMC1 cDNA (OriGene Technologies, Inc., Rockville, MD) using a Fugene 6 transfection reagent (Roche Applied Science, Indianapolis, IN).

Techniques: Binding Assay, Western Blot, Expressing, Activity Assay, Transfection, Plasmid Preparation

Journal: Nature communications

Article Title: Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site

doi: 10.1038/ncomms1386

Figure Lengend Snippet: a, Chemical structure of WC-26 , a selective sigma-2 receptor ligand. b, Knockdown of PGRMC1 in HeLa cells resulted in decreased caspase-3 activation induced by WC-26. c, WC-26 stimulated PGRMC1 expression in the same manner as AG-205 , a PGRMC1 ligand; * p<0.01, Student's t-test, n=3. Error bars in b represent SEM.

Article Snippet: 24 h after plating, the cells were transfected with the pCMV6-XL4 vector only or human PGRMC1 cDNA (OriGene Technologies, Inc., Rockville, MD) using a Fugene 6 transfection reagent (Roche Applied Science, Indianapolis, IN).

Techniques: Activation Assay, Expressing

Journal: Nature communications

Article Title: Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site

doi: 10.1038/ncomms1386

Figure Lengend Snippet: a , Sigma-2 receptors labeled with SW120 and PGRMC1 partially colocalized with mitochondria markers ( b, c ) and endoplasmic reticulum markers ( d, e ) in HeLa cells. For imaging sigma-2 receptors, live cells were incubated with SW120 and either MitoTracker or ER-Tracker and then imaged by confocal microscopy. For imaging PRGMC1, cells were fixed and incubated with goat anti-PGRMC1 antibody followed by a FITC-conjugated secondary antibody (Mito-marker or ER-marker). Antibody stained HeLa cells were then coverslipped and imaged by confocal microscopy. Scale bar represents 20 μm.

Article Snippet: 24 h after plating, the cells were transfected with the pCMV6-XL4 vector only or human PGRMC1 cDNA (OriGene Technologies, Inc., Rockville, MD) using a Fugene 6 transfection reagent (Roche Applied Science, Indianapolis, IN).

Techniques: Labeling, Imaging, Incubation, Confocal Microscopy, Marker, Staining

Journal: EBioMedicine

Article Title: The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

doi: 10.1016/j.ebiom.2015.10.017

Figure Lengend Snippet: PGRMC1 knockout using a CRISPR/Cas9 approach. A. Schematic of the PGRMC1 sgRNA/Cas9-expressing lentiviral constructs for knocking out PGRMC1. Gray areas show the three candidate sgRNA sequences in the exon-1 of the PGRMC1 gene, of which two were used for the CRISPR/Cas9 constructs. B. A representative DNA gel of control and PGRMC1 knockout (clones 38 and 207) NSC34 cells verifying the Cas9 cleavage of the genomic DNA. Control refers to the NSC34 cells transfected with the control vector expressing Cas9 but not an sgRNA. C. Western blotting detection of PGRMC1 in control and PGRMC1 knockout (clones 38 and 207) NSC34 cells.

Article Snippet: PGRMC1 cDNA was obtained from Origene (Rockville, MD).

Techniques: Knock-Out, CRISPR, Expressing, Construct, Control, Clone Assay, Transfection, Plasmid Preparation, Western Blot

Journal: EBioMedicine

Article Title: The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

doi: 10.1016/j.ebiom.2015.10.017

Figure Lengend Snippet: [ 125 I]-IAF photolabeling of the S2R is protected by S2R-specific ligands but not affected by PGRMC1 knockout. A. [ 125 I]-IAF photolabeling of the S2R in PC12 membranes is protectable by CM compounds. [ 125 I]-IAF photolabeling of both S1R and S2R was protected by 20 μM DTG (D) while CM compounds (CM 353, CM 398, CM 775, and CM 777, see Fig. S1 for affinities to sigma receptors) selectively blocked the labeling of the S2R but not the S1R. Two concentrations (1 and 10 μM) were used for CM compounds. B. [ 125 I]-IAF photolabeling of the S2R in membranes prepared from control and PGRMC1 knockout (clones 38 and 207) NSC34 cells. 5 μM (+)-pentazocine (P) protected against [ 125 I]-IAF photolabeling of the S1R while 20 μM DTG (D) protected against photolabeling of both the S1R and the S2R. Note that the background photolabeled bands were not protected by these specific sigma ligands.

Article Snippet: PGRMC1 cDNA was obtained from Origene (Rockville, MD).

Techniques: Knock-Out, Labeling, Control, Clone Assay

Journal: EBioMedicine

Article Title: The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

doi: 10.1016/j.ebiom.2015.10.017

Figure Lengend Snippet: Eliminating PGRMC1 protein does not alter [ 3 H]-DTG binding to the S2R in cell membranes. A. A representative of [ 3 H]-DTG saturation binding in membranes prepared from control and PGRMC1 knockout (clones 38 and 207) NSC34 cells, (+)-pentazocine (100 nM) was included to mask [ 3 H]-DTG binding to the S1R such that [ 3 H]-DTG would be bound only to the S2R and measured as specific S2R binding. Nonspecific binding was measured (by adding haloperidol) and subtracted. Control refers to the NSC34 cells transfected with the control vector for the expression of Cas9 but not an sgRNA. B. Statistics. Maximum binding (B max ) and equilibrium dissociation constants (K D ) for [ 3 H]-DTG were calculated using a Prizm software and reported as mean ± SEM of three separate experiments each performed in triplicates. Not significant (n.s.).

Article Snippet: PGRMC1 cDNA was obtained from Origene (Rockville, MD).

Techniques: Binding Assay, Control, Knock-Out, Clone Assay, Transfection, Plasmid Preparation, Expressing, Software

Journal: EBioMedicine

Article Title: The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

doi: 10.1016/j.ebiom.2015.10.017

Figure Lengend Snippet: PGRMC1 overexpression does not change [ 3 H]-DTG binding to the S2R in cell membranes. A. A representative Western blot detecting PGRMC1 in membranes prepared from control and PGRMC1-3 × HA overexpressing NSC34 cells. Control refers to the NSC34 cells transfected with the same expression vector but without the PGRMC1 gene. B. A representative experiment of [ 3 H]-DTG saturation binding (as described in A) in membranes prepared from control and PGRMC1-3 × HA overexpressing NSC34 cells. C. Statistics. Maximum binding (B max ) and equilibrium dissociation constants (K D ) for [ 3 H]-DTG were calculated using a Prizm software and reported as mean ± SEM of three separate experiments each performed in triplicates. Not significant (n.s.).

Article Snippet: PGRMC1 cDNA was obtained from Origene (Rockville, MD).

Techniques: Over Expression, Binding Assay, Western Blot, Control, Transfection, Expressing, Plasmid Preparation, Software

Journal: EBioMedicine

Article Title: The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

doi: 10.1016/j.ebiom.2015.10.017

Figure Lengend Snippet: Assessment of DTG and haloperidol binding affinities for PGRMC1. Experiments were performed with rat liver membranes. Shown on the left is a representative inhibition curve of [ 3 H]-progesterone binding (at 30 nM) to PGRMC1 in the presence of increasing concentrations of non-radioactive DTG or haloperidol. Nonspecific binding was measured by the addition of 10 μM of nonradioactive progesterone and subtracted, as previously described by . Inhibition constants (K I ) are reported as mean ± SEM from three separate experiments each performed in triplicates for DTG and two separate experiments each performed in triplicates for haloperidol.

Article Snippet: PGRMC1 cDNA was obtained from Origene (Rockville, MD).

Techniques: Binding Assay, Inhibition

Journal: Cancer Research

Article Title: DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor–Positive Breast Cancer Following Endocrine Therapy

doi: 10.1158/0008-5472.can-19-1110

Figure Lengend Snippet: Figure 1. E2-mediated ER activation is required for DAXX protein expression. A, MCF7, BT474, and T47D cells were treated with ethanol (0 nmol/L E2), 5 nmol/L E2, or E2 þ fulvestrant (Fulv; 100 nmol/L) for 3 days. ERa, NOTCH4, and DAXX protein levels were detected by Western blotting. B, MDA-MB-231 (231) and MDA-MB-468 (468) cells were treated with ethanol (0 nmol/L E2), 5 nmol/L E2 for 3 days, followed by Western blotting for DAXX. C, PDX BCM-5097 tumors were harvested for detection of ERa, NOTCH4, and DAXX protein levels. D, MCF7 and T47D cells were transfected with nonspecific (SCBi) or DAXX-specific (DAXXi) siRNA for 2 days and then grown for 3 days with 0 nmol/L E2, 5 nmol/L E2, or 5 nmol/L E2 þ 100 nmol/L fulvestrant, followed by Western blotting to detect NOTCH4 and DAXX proteins. After 7 days, mammospheres were harvested by pooling together three wells and NOTCH4 and DAXX proteins were detected by Western blotting.

Article Snippet: A mammalian expression vector, pCMV6-entry, containing a human DAXX cDNA was purchased from Origene and used to transiently overexpress DAXX in cell lines.

Techniques: Activation Assay, Expressing, Western Blot, Transfection

Journal: Cancer Research

Article Title: DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor–Positive Breast Cancer Following Endocrine Therapy

doi: 10.1158/0008-5472.can-19-1110

Figure Lengend Snippet: Figure 2. DAXX protein stabilization is mediated by E2-mediated ER activation. A, Real-time PCR was performed to measure relative transcript levels of DAXX and PS2 in MCF7, BT474, and T47D cells after 3 days of treatment with 0 or 5 nmol/L E2. Bar graphs show mean SD values normalized to HPRT and compared with 0 nmol/L E2 based on three independent experiments using the 2DDCt calculation. A one-way ANOVA was performed on DCt values after normalization to HPRT. B, The same cells were treated with 0 or 5 nmol/L E2 for 3 days; 12 hours prior to harvesting, cells were treated with vehicle (DMSO) or 10 mmol/L MG132. Western blotting was performed to detect DAXX protein levels. C, MCF7 cells were grown in 0 nmol/L E2, 5 nmol/L E2, or E2 þ 100 nmol/L fulvestrant for 24 hours, following which, cells were treated with 10 mmol/L cycloheximide (CHX) for the indicated times. Western blotting was performed to detect DAXX protein levels. MCF7 cells treated with 0 nmol/L E2 or 5 nmol/L E2 þ 100 nmol/L fulvestrant were treated with 10 mmol/L cycloheximide for a shorter time course. Western blotting was performed to detect DAXX protein levels. Blots are representative of three independent experiments. D, ImageJ was used to measure densitometry of DAXX protein levels as a ratio to b-actin. The estimated half-life (t1/2) of the DAXX protein for each independent experiment was calculated by linear regression analysis. Means of DAXX protein half-life SD were statistically compared by a one-way ANOVA. #, P < 0.05; $, P < 0.01; @, P < 0.001.

Article Snippet: A mammalian expression vector, pCMV6-entry, containing a human DAXX cDNA was purchased from Origene and used to transiently overexpress DAXX in cell lines.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cancer Research

Article Title: DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor–Positive Breast Cancer Following Endocrine Therapy

doi: 10.1158/0008-5472.can-19-1110

Figure Lengend Snippet: Figure 3. DAXX is required to inhibit NOTCH4 and TIC-survival. A, MCF7 and T47D cells were transfected with a nonspecific (SCBi) or DAXX-specific (DAXXi) siRNA for 2 days and then treated with 0 or 5 nmol/L E2 for 3 days. DAXX and NOTCH4 proteins were detected by Western blotting. B, A total of 50,000 cells were plated into an ultralow attachment plate containing methylcellulose mammosphere-forming medium. After 7 days, mammospheres were imaged at 20 magnification, harvested, and %MFE calculated. Representative images of mammospheres taken by light microscopy are shown. Scale bars, 100 mm. Bar graphs show %MFE SD from three independent experiments. Statistical significance was calculated using a two-way ANOVA with a Tukey post hoc test for multiple comparisons. Symbols denote statistical significance between 5 nmol/L and 0 nmol/L E2, SCBi and DAXXi, and E2 and E2þ fulvestrant (FUL). C and D, Real-time PCR was used to detect transcript levels of PS2 and DAXX (Fig. 1C) and NOTCH targets DELTEX, HES1, and HEY1 (Fig. 1D). Bar graphs show mean values SD of relative transcript expression normalized to HPRT and compared with SCBi þ 0 nmol/L E2 conditions from three independent experiments using the 2DDCt calculation. A two-way ANOVA was performed on DCt values after initial normalization to HPRT. Symbols denote statistical significance between SCBi and DAXXi and 0 and 5 nmol/L E2. #, P < 0.05; $, P < 0.01; @, P < 0.001.

Article Snippet: A mammalian expression vector, pCMV6-entry, containing a human DAXX cDNA was purchased from Origene and used to transiently overexpress DAXX in cell lines.

Techniques: Transfection, Western Blot, Light Microscopy, Real-time Polymerase Chain Reaction, Expressing

Journal: Cancer Research

Article Title: DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor–Positive Breast Cancer Following Endocrine Therapy

doi: 10.1158/0008-5472.can-19-1110

Figure Lengend Snippet: Figure 4. DAXX is necessary to restrict TIC frequency in vivo. MCF7 cells were transfected with a nonspecific (SCBi) or DAXX-specific (DAXXi) siRNA for 2 days. 10,000 (1 104), 100,000 (1 105), or 1,000,000 (1 106) cells were resuspended into Matrigel solution (1:1 Matrigel:PBS) and injected into the mammary fat pads of 5 female, athymic, nude mice/dilution. Silastic capsules containing E2 were also implanted for up to 8 weeks. A, DAXX protein levels were detected by Western blotting. B, Tumor area was measured weekly using Vernier calipers. Total number of tumors per group were counted. Tumor images were taken after 8 weeks. C, Kaplan–Meier curve represents the rate of tumor incidence across all cell dilutions. Statistical significance was calculated by the log-rank (Mantel–Cox) test. D, Estimate of TIC frequency for each cell dilution group was calculated using the ELDA software. SCBi and DAXXi TIC-frequency was compared by the x2 test to determine statistical significance. E, Using Kmplotter.com, RFS for women with ERþ breast cancer was compared in DAXX high expressing tumors versus DAXX low expressing tumors by "best cutoff" as determined by the kmplotter software. The data represent the mean of two probes using the multigene analyzer. Two cohorts of patients were analyzed: systemically untreated (left) and endocrine therapy only (right; excluded for chemotherapy). All parameters used to generate these curves are summarized in Supplementary Table S1C. F, Coexpression analysis of the METABRIC cohort comparing DAXX RNA expression versus ALDH1A1 in patients with ERþ breast cancer. DAXX versus ALDH1A1 RNA expression was evaluated by correlation coefficient (CC) and Q value.

Article Snippet: A mammalian expression vector, pCMV6-entry, containing a human DAXX cDNA was purchased from Origene and used to transiently overexpress DAXX in cell lines.

Techniques: In Vivo, Transfection, Injection, Capsules, Western Blot, Software, Expressing, RNA Expression

Journal: Cancer Research

Article Title: DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor–Positive Breast Cancer Following Endocrine Therapy

doi: 10.1158/0008-5472.can-19-1110

Figure Lengend Snippet: Figure 5. Nuclear DAXX is enriched on TIC gene promoters and is necessary to repress TIC genes. A, MCF7 (left) and T47D (right) cells were treated with 0 or 5 nmol/L E2 for 3 days. DAXX protein was detected by Western blot analysis in total lysates or cellular fractions. Actin was used as a cytosolic control and PARP1 as a nuclear control. B, MCF7 and T47D cells were treated with 0 or 5 nmol/L E2 for 1 day. Cells were fixed, chromatin was isolated and probed using either a nonspecific IgG or DAXX-specific antibody (2 mg of antibody for each condition), and DNA fragments were isolated and purified. Fold DAXX enrichment as compared with IgG at TIC-associated gene promoters was quantified by real-time PCR. Fold-enrichment was determined by 2DCt calculation. Bar graphs show mean SD values of fold enrichment for each gene promoter measured. DCt values for each gene promoter from cells treated with 0 nmol/L E2 versus 5 nmol/L E2 were compared and statistical significance was calculated using a nonpaired, two-sided Student t test. Symbols denote statistical significance between 0 nmol/L and 5 nmol/L E2. C, MCF7 and T47D cells were transfected with a nonspecific (SBCi) or DAXX-specific siRNA for 2 days. Cells were treated with 0 or 5 nmol/L E2 for 1 day. Transcript levels of SOX2, OCT4, NOTCH4, NANOG, and ALDH1A1 (ALDH) were detected by real-time PCR from MCF7 and T47D cells. Bar graphs show mean SD values of relative transcript expression normalized to HPRT and compared with SCBi 0 nmol/L þE2 conditions from three independent experiments using the 2DDCt calculation. A two-way ANOVA was performed using DCt values after initial normalization to HPRT. Symbols denote statistical significance between 0 nmol/L and 5 nmol/L E2 and SCBi and DAXXi groups. #, P < 0.05; $, P < 0.01; @, P < 0.001.

Article Snippet: A mammalian expression vector, pCMV6-entry, containing a human DAXX cDNA was purchased from Origene and used to transiently overexpress DAXX in cell lines.

Techniques: Western Blot, Control, Isolation, Real-time Polymerase Chain Reaction, Transfection, Expressing

Journal: Cancer Research

Article Title: DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor–Positive Breast Cancer Following Endocrine Therapy

doi: 10.1158/0008-5472.can-19-1110

Figure Lengend Snippet: Figure 6. DAXX requires DNMT1 to restrict TIC survival and transcript expression. A, MCF7 and T47D cells were treated with 0 or 5 nmol/L E2 for 3 days. DNMT1 and DAXX protein levels were detected by Western blot analysis using actin as a loading control. B and C, MCF7 and T47D cells were transfected with a mock vector (EV) or the human DAXX cDNA (DAXX) for 2 days and then retransfected with the nonspecific (SCBi) or DNMT1-specific (DNMT1i) siRNA for an additional day. B, DAXX and DNMT1 proteins were detected by Western blotting. C, After 7 days, mammospheres were imaged, isolated, measured, and %MFE was calculated. Bar graphs show mean SD. %MFE from three independent experiments. Statistical significance was calculated using two-way ANOVA with a Tukey post hoc test for multiple comparisons. Symbols denote statistical significance between empty vector and DAXX, SCBi and DNMT1 under DAXX conditions, and 0 nmol/L and 5 nmol/L E2. D, MCF7 cells were transfected with a nonspecific (SCBi), DAXX-specific (DAXXi), or DNMT1-specific (DNMT1i) siRNA for 2 days. Cells treated with 0 or 5 nmol/L E2 for 1 day. Total DNA was isolated and subjected to bisulfite treatment. Bisulfite-converted DNA was amplified using SOX2 promoter–specific primers or NOTCH4-CpG island–specific primers that anneal to bisulfite-treated DNA. The PCR product was purified and sent for DNA sequencing. CpG sites that were read as "T" were considered unmethylated and sites read as "C" were considered methylated. Bar graphs show mean (total number of methylated CpG sites)/(total CpG sites read 100) SD from five independent experiments. Statistical significance was calculated using two-way ANOVA with a Tukey post hoc test for multiple comparisons. Symbol denotes statistical significance between 0 nmol/L and 5 nmol/L E2 and SCBi and DAXXi/DNMT1i groups. #, P < 0.05; @, P < 0.001.

Article Snippet: A mammalian expression vector, pCMV6-entry, containing a human DAXX cDNA was purchased from Origene and used to transiently overexpress DAXX in cell lines.

Techniques: Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Isolation, DNA Sequencing, Methylation

Journal: Cancer Research

Article Title: DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor–Positive Breast Cancer Following Endocrine Therapy

doi: 10.1158/0008-5472.can-19-1110

Figure Lengend Snippet: Figure 7. DAXX is sufficient to restrict NOTCH4 and TIC-survival in response to endocrine therapy (ET). A, MCF7 and T47D cells were transfected with the empty vector (EV) or DAXX-containing vector using polyethylenimine for 2 days and then treated with 0 or 5 nmol/L E2 for 3 additional days. DAXX and NOTCH4 protein levels were detected by Western blot analysis using b-actin as a loading control. B and C, A total of 50,000 MCF7 and T47D cells were plated in ultralow attachment plates containing mammosphere-forming medium for 7 days. Mammospheres were imaged, isolated, counted, and %MFE calculated from three independent experiments. Bar graphs show mean %MFE SD from three independent experiments. Statistical significance was calculated using two-way ANOVA with a Tukey post hoc test for multiple comparisons. Symbols denote statistical significance between empty vector and DAXX and 0 nmol/L and 5 nmol/L E2 groups. MCF7 (C) and T47D (D) cells were transfected with the empty vector or DAXX-containing vector for 2 days and then treated with 0 or 5 nmol/L E2 for 1 day. SOX2, OCT4, NOTCH4, NANOG, and ALDH1A1 transcripts were detected by real-time PCR. Bar graphs show mean values SD of relative transcript expression normalized to HPRT and compared with SCBi 0 nmol/L þE2 from three independent experiments using the 2DDCt calculation. Symbols denote statistical significance between empty vector and DAXX and 0 nmol/L and 5 nmol/L E2. A two-way ANOVA was performed on DCt values after initial normalization to HPRT. E, Working model that endocrine therapy selects for TICs by depleting DAXX protein levels. DAXX depletion results in derepression of TIC genes promoting TIC survival. TICs contribute to heterogeneous and resistant recurrent tumors. Therapies that stabilize DAXX may prevent TIC survival and prevent ERþ breast cancer recurrence. #, P < 0.05; $, P < 0.01; @, P < 0.001. N.S., nonsignificant.

Article Snippet: A mammalian expression vector, pCMV6-entry, containing a human DAXX cDNA was purchased from Origene and used to transiently overexpress DAXX in cell lines.

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Isolation, Real-time Polymerase Chain Reaction, Expressing