Journal: Cell Death and Differentiation

Article Title: ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

doi: 10.1038/cdd.2013.186

Figure Lengend Snippet: ROS-mediated activation of JNK contributes to the p53-mediated apoptosis, DDR and transcriptional repression of oncogenes. ( a ) Dose-dependent induction of p-JNK, p-Ser33-p53, p-Ser15-p53, γ H2AX, PARP cleavage and inhibition of Wip1, Mcl1 and MdmX by RITA as assessed by western blotting. ( b ) HCT116 and HCT116 p53−/− cells were treated with 1 μ M RITA; analyzed as in ( a ). ( c ) Pretreatment with NAC for 6 h prevented the induction of p-JNK, γ H2AX, PARP cleavage and inhibition of Wip1, Mcl1 and MdmX by RITA as analyzed by immunoblotting. ( d ) JNK inhibitor SP600125 prevented the induction of p-JNK, p-Ser33-p53, γ H2AX, PARP cleavage and inhibition of Wip1, Mcl1 and MdmX by RITA, as assessed by western blotting. ( e ) SP600125 blocked the induction of p-JNK, p-Ser33-p53, γ H2AX, PARP cleavage and inhibition of MdmX and Wip1 by combination treatment with 0.05 μ M RITA and 1 μ M Aura for 24 h, as assessed by western blotting.( f , g ) Depletion of JNK by siRNA prevented the induction of γ H2AX ( f ), p-Ser33-p53 and inhibition of Wip1 and MdmX ( g ), analyzed by immunoblotting. ( h ) Inhibition of JNK by siRNA prevented apoptosis induction by RITA, as measured by FACS of PI-stained cells. ( i ) SP600125 blocked the repression of MCL1, PPM1D, PIK3CA, PIK3CB, EIF4E and MDM4 (MdmX) mRNA upon RITA treatment as assessed by qPCR (mean±S.E.M., n =3) in HCT116 (12 h treatment) and MCF7 (8 h treatment) cells

Article Snippet: Plasmids encoding FLAG-Wip1 and shRNA for Wip1 were kindly provided by René H. Medema, Utrecht, Netherlands.

Techniques: Activation Assay, Inhibition, Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

doi: 10.1038/cdd.2013.186

Figure Lengend Snippet: Inhibition of Wip1 promotes the induction of γ H2AX upon RITA treatment. ( a ) Wip1 mRNA was repressed after 8 h treatment with 1 μ M RITA, but not 0.1 μ M RITA, as assessed by qPCR (mean±S.E.M., n =3). ( b ) Downregulation of Wip1 protein level correlated with the induction of γ H2AX upon RITA treatment as analyzed by immunoblotting. ( c ) MCF7 and U2OS cells stably transfected with empty vector shRNA or Wip1 shRNA were treated with 0.1 and 1 μ M RITA for indicated periods and γ H2AX was assessed as in ( b ). ( d ) HCT116 and U2OS cells transfected with either empty vector or FLAG-Wip1 were treated with 1 μ M RITA for indicated times. Proteins were detected by western blotting

Article Snippet: Plasmids encoding FLAG-Wip1 and shRNA for Wip1 were kindly provided by René H. Medema, Utrecht, Netherlands.

Techniques: Inhibition, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, shRNA

Journal: Cell Death and Differentiation

Article Title: ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

doi: 10.1038/cdd.2013.186

Figure Lengend Snippet: Depletion of Wip1 confers a sustained transcriptional activation of p53 target genes, but does not facilitate transrepression. ( a and b ) Microarray analysis of MCF7 cells with (indicated in violet) or without (indicated in gray) Wip1 depletion by shRNA, treated with 0.1 μ M RITA or DMSO for indicated time points revealed that p53-mediated transactivation was enhanced by Wip1 silencing. ( c ) Wip1 downregulation led to the increased induction of p53-activated genes (upper panel) but did not augment the repression of pro-survival genes by p53 (lower panel) upon low dose of RITA as analyzed by qPCR (mean±S.E.M., n =3). Insert demonstrates the efficiency of Wip1 depletion, as assessed by immunoblotting. ( d ) MCF7 cells transfected with either empty vector or FLAG-Wip1 were treated with 1 μ M RITA or DMSO for 8 h and mRNA levels of PIK3CA , PIK3CB and IGF-1R were assessed by qPCR (mean±S.E.M., n =3). ( e ) MCF7 cells stably transfected with shWip1 or control shVector were treated with 0.1 and 1 μ M RITA or DMSO for 24 h, and cells were stained with Annexin V followed by FACS analysis (mean±S.E.M., n =3, * P <0.05, ** P <0.01, by two-tailed t -test). ( f ) Model of the synthetic lethality upon activation of p53 and inhibition of TrxR. Inhibition of TrxR lead to the accumulation of ROS and activation of JNK, facilitating p53 function upon its release from Mdm2. In turn, activated p53 induces pro-oxidant genes, which increases the level of ROS, further activating JNK, and thus, p53. Activated JNK converts p53 to an inhibitor of Wip1 and MdmX, therefore amplifying p53 activity. Transcriptional repression of Mcl1, eIF4E and PI3K abolishes survival signaling, contributing to apoptosis induction. Thus, the dual targeting of p53 and TrxR (i.e., by RITA) leads to the robust apoptosis

Article Snippet: Plasmids encoding FLAG-Wip1 and shRNA for Wip1 were kindly provided by René H. Medema, Utrecht, Netherlands.

Techniques: Activation Assay, Microarray, shRNA, Western Blot, Transfection, Plasmid Preparation, Stable Transfection, Control, Staining, Two Tailed Test, Inhibition, Activity Assay

Journal: The Journal of Cell Biology

Article Title: The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis

doi: 10.1083/jcb.201206013

Figure Lengend Snippet: A kinome-wide siRNA screen identifies MARK4 as positive regulator of ciliogenesis. (A) Screen flowchart. (B) Kinases positively regulating ciliogenesis. Functional notes are obtained from the Entrez Gene database. Asterisks indicate kinases presented in the ciliome database. (C) RPE1 cells, treated with nontargeting (control) or MARK4 siRNA, were serum starved for 48 h and stained for IFT88, polyglutamylated tubulin, and DNA (DAPI). The left images show merged images. Regions within the white boxes are shown at higher magnification on the right. (D) Percentages of ciliated cells. polyglu. tub., polyglutamylated tubulin; acetyl., acetylated; detyr., detyrosinated. (E) Total cell extracts of control and MARK4-depleted RPE1 cells were analyzed by immunoblotting. Actin served as a loading control. (F) NIH 3T3 cells treated with control or MARK4 siRNA were serum starved for 24 h and stained for polyglutamylated and γ-tubulin and DNA. Insets show higher magnification of the centrosome region. (G) Percentages of ciliated cells determined using polyglutamylated tubulin. (H) Same as in E, except using extracts of NIH 3T3 cells. The asterisk indicates an unspecific band. Data are means ± SD of three independent experiments.

Article Snippet: After siRNA transfection, GFP-EB1–expressing RPE1 cells were reseeded onto 35-mm μ-dishes (Ibidi GmbH).

Techniques: Functional Assay, Control, Staining, Western Blot

Journal: The Journal of Cell Biology

Article Title: The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis

doi: 10.1083/jcb.201206013

Figure Lengend Snippet: MARK4 localizes to the basal body in ciliated cells. (A) RPE1 cells treated with control or MARK4 siRNA were serum starved for 48 h and stained for MARK4, polyglutamylated tubulin, and DNA. (B and C) RPE1 cells were transiently transfected with GFP-hMARK4L and stained for γ-tubulin (B) or acetylated tubulin (C) and DNA. (D) After control or MARK4 depletion, LAP-hMARK4L or LAP-hMARK4L-kd (kinase-dead mutant) expression was induced by the addition of 100 ng/ml doxycycline (dox), the cells were serum starved for 24 h, and percentages of ciliated cells were determined using acetylated tubulin as a cilia marker. Data are means ± SD of four independent experiments. *, P < 0.05. (E) Immunoblot analysis of the indicated NIH 3T3 cell extracts using anti-GFP and anti-MARK4 antibodies. Actin served as a loading control. Asterisks indicate unspecific bands. exo., exogenous; endo., endogenous. In A–C, the right images show merged images. Insets show higher magnification.

Article Snippet: After siRNA transfection, GFP-EB1–expressing RPE1 cells were reseeded onto 35-mm μ-dishes (Ibidi GmbH).

Techniques: Control, Staining, Transfection, Mutagenesis, Expressing, Marker, Western Blot

Journal: The Journal of Cell Biology

Article Title: The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis

doi: 10.1083/jcb.201206013

Figure Lengend Snippet: MARK4 depletion does not affect MT dynamics. (A) After control or MARK4 depletion, RPE1 cells were serum starved for 48 h and treated with 8 µM nocodazole at 37°C for 90 min to depolymerize MTs. Cells were stained for α- and γ-tubulin and DNA. (B–E) After control or MARK4 depletion, GFP-EB1–expressing RPE1 cells were serum starved for 24 h and either stained for MARK4 and DNA (B) or analyzed by live-cell imaging ( Videos 1 and 2 ). The left images in B show merged images. Regions within the white boxes are depicted at higher magnification on the right showing GFP-EB1 tracks in the cytoplasm (1) or the centrosome (2). Time-lapse sequences were analyzed using automated tracking software. There is no significant difference in the mean growth speeds (C), lifetimes (D), and track lengths (E) between cells treated with control or MARK4 siRNA (control, n = 15; MARK4, n = 22). Boxes show the top and bottom quartiles (25–75%) with a line at the median, and whiskers extend from the minimum to the maximum of all data.

Article Snippet: After siRNA transfection, GFP-EB1–expressing RPE1 cells were reseeded onto 35-mm μ-dishes (Ibidi GmbH).

Techniques: Control, Staining, Expressing, Live Cell Imaging, Software

Journal: The Journal of Cell Biology

Article Title: The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis

doi: 10.1083/jcb.201206013

Figure Lengend Snippet: ODF2 overexpression rescues cilia loss after MARK4 depletion. (A and B) RPE1 cells were transiently transfected with GFP-hODF2 (A) or GFP (B) and subsequently treated with control or MARK4 siRNA for 24 h before serum withdrawal and incubation for another 24 h. Cells were stained for DNA, γ-tubulin, and acetylated tubulin. The left images show merged images. Regions within the white boxes are shown at a higher magnification on the right. (C) Percentages of ciliated cells based on acetylated tubulin as a cilia marker. UT, untransfected. (D) Efficiency of MARK4 depletion in untransfected and GFP-hODF2–expressing cells were determined by quantitative fluorescence microscopy using MARK4-specific antibodies. Boxes show the top and bottom quartiles (25–75%) with a line at the median, and whiskers extend from the minimum to the maximum of all data. (E and F) HEK293T cells were transiently transfected with the indicated constructs. Immunoprecipitations (IP) were performed using anti-FLAG agarose. (E) Interacting proteins were detected by immunoblotting. (F) Quantification of E. (G) NIH 3T3 cells and NIH 3T3 cells expressing low levels of LAP-MARK4L were serum starved for 24 h, and immunoprecipitations were performed on the lysates using anti-GFP beads and probed for endogenous ODF2 and MARK4. exo., exogenous; endo., endogenous. The asterisk indicates an unspecific band. (H) For in vitro kinase assays, purified 6His-MARK4L, either catalytic active (ca) or kinase dead (kd), was incubated with recombinant ODF2 truncations. GST was used as a negative control. Samples were subjected to SDS-PAGE followed by autoradiography ( 32 P) and Coomassie Brilliant blue staining (CBB). An aliquot of each reaction was analyzed by immunoblotting with anti-MARK4. (I) Relative phosphorylation of ODF2-F1, ODF2-F2, and ODF2-F3 normalized to protein amounts. (J) Schematic representation of ODF2 truncations. A.U., arbitrary unit. Data are means ± SD of three independent experiments. *, P < 0.05; **, P < 0.001.

Article Snippet: After siRNA transfection, GFP-EB1–expressing RPE1 cells were reseeded onto 35-mm μ-dishes (Ibidi GmbH).

Techniques: Over Expression, Transfection, Control, Incubation, Staining, Marker, Expressing, Fluorescence, Microscopy, Construct, Western Blot, In Vitro, Purification, Recombinant, Negative Control, SDS Page, Autoradiography, Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis

doi: 10.1083/jcb.201206013

Figure Lengend Snippet: Ultrastructural analysis of MARK4 depletion phenotypes. (A) A schematic model depicting the different stages of ciliogenesis, according to , ). The mother centriole is characterized by the presence of distal (DA) and subdistal appendages (SDA; 0). The initial step of ciliogenesis is the docking of ciliary vesicles (CV) to the mother centriole (1). The CVs increase in size by fusion with nearby vesicles (V; 2) and become invaginated by the ciliary bud (CB), an accumulation of electron-dense material at the distal end of the mother centriole (3). MT doublets start to elongate from the basal body giving rise to the axonemal shaft (AS) and ciliary pocket (CP; 4). The elongated ciliary vesicle fuses with the plasma membrane (PM), and the cilium emerges in the extracellular environment (5). (B and C) Electron micrographs showing serial sections of unstarved RPE1 cells. Cells were seeded 24 h before fixation and cultivated in medium with 10% serum. (D and E) Electron micrographs showing serial sections of RPE1 cells treated with control siRNA. Cells were starved for 24 h to enrich early stages of ciliogenesis. (F–H) Electron micrographs showing serial sections of RPE1 cells treated with control (F) or MARK4 siRNA (G and H). Cells were serum starved for 48 h before fixation for TEM analysis. (I) After control or ODF2 depletion, RPE1 cells were serum starved for 48 h and analyzed by immunofluorescence. Percentages of ciliated cells were determined using polyglutamylated tubulin. Data are means ± SD of three independent experiments. (J) Immunostaining with anti-ODF2 revealed residual low levels of ODF2 at the centrosome in cells treated with ODF2 siRNA. Box and whisker plots show the relative ODF2 intensity at the centrosome after control and ODF2 depletion. One representative experiment out of three is shown. Boxes show the top and bottom quartiles (25–75%) with a line at the median, and whiskers extend from the minimum to the maximum of all data. A.U., arbitrary unit; polyglu. tub., polyglutamylated tubulin. (K and L) Electron micrographs showing serial sections of RPE1 cells treated with ODF2 siRNA. Cells were serum starved for 48 h before fixation for TEM analysis. (M) Quantification of ultrastructural phenotypes in control, MARK4-, and ODF2-depleted RPE1 cells after the indicated time of serum starvation. For unstarved control cells (0 h), only cells in G1 with unduplicated centrioles were considered. The asterisk indicates that the ODF2-depleted RPE1 cells showed an additional defect in the subdistal appendage formation; subdistal appendages were either completely absent (77%) or grossly impaired (23%).

Article Snippet: After siRNA transfection, GFP-EB1–expressing RPE1 cells were reseeded onto 35-mm μ-dishes (Ibidi GmbH).

Techniques: Clinical Proteomics, Membrane, Control, Immunofluorescence, Immunostaining, Whisker Assay

Journal: The Journal of Cell Biology

Article Title: The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis

doi: 10.1083/jcb.201206013

Figure Lengend Snippet: CP110 persists at the mother centriole after MARK4 depletion. (A) RPE1 cells, treated with the indicated siRNAs, were serum starved for 48 h and stained for IFT88, polyglutamylated tubulin (polyglu. tub.), and DNA. (B–E) Same as in A; however, cells were stained for CP110 (B) or Cep97 (D), polyglutamylated tubulin, and DNA. (C and E) Quantification of B and D, respectively. The number of ciliated cells and CP110 (C) or Cep97 dots (E) per centrosome are indicated. (F and G) Codepletion of CP110 rescues the loss of cilia in MARK4- or ODF2-depleted RPE1 cells. (F) After treatment with control, MARK4, or ODF2 siRNA for 24 h, RPE1 cells were subjected to control or CP110 depletion for 24 h and subsequent serum starvation for 24 h. Percentages of ciliated cells based on polyglutamylated tubulin as a cilia marker. *, P < 0.05. (G) RPE1 cells were treated as in F and stained for IFT88, polyglutamylated tubulin, and DNA. In A, B, D, and G, merged images are shown. Regions within the white boxes are shown at higher magnification at the left (A, B, and D) or in the bottom images (G). Data are means ± SD of three independent experiments.

Article Snippet: After siRNA transfection, GFP-EB1–expressing RPE1 cells were reseeded onto 35-mm μ-dishes (Ibidi GmbH).

Techniques: Staining, Control, Marker

Journal: Oncotarget

Article Title: The roles of RRP15 in nucleolar formation, ribosome biogenesis and checkpoint control in human cells

doi: 10.18632/oncotarget.14658

Figure Lengend Snippet: A . Inhibition of cell proliferation by RRP15 depletion in various human cells. RPE1 (up), HeLa (middle) and MCF7 cells (low) were transfected with or without RRP15 siRNA and cell proliferation was determined by MTT assay at indicated times. Results represent means ± standard deviations of five independent experiments. B . RPE1 (up), HeLa (middle) and MCF7 cells (low) were transfected with or without RRP15 siRNA for 48 h and cell cycle profiles were monitored by flow cytometry. Results are representative of three independent experiments. C . RPE1, HeLa and MCF7 cells were transfected with or without RRP15 siRNA for 48 h. The cell lysates were immunoblotted with α-RRP15, α-Caspase 3 and anti-α-Tubulin antibody. D . The effects of RRP15 depletion on BrdU (5-bromo-2′-deoxyuridine) incorporation in various human cells. Cells transfected with or without RRP15 siRNA for 48 h were labeled with 10 mM BrdU (1 h). The cells were fixed and immunostained with mouse α-BrdU. DNA was visualized by DAPI staining.

Article Snippet: The validation of the identity of HeLa, MCF7 and RPE1 cells was determined by Tianjin Weikai Bioeng LTD (China, Tianjin).

Techniques: Inhibition, Transfection, MTT Assay, Flow Cytometry, Labeling, Staining

Journal: Oncotarget

Article Title: The roles of RRP15 in nucleolar formation, ribosome biogenesis and checkpoint control in human cells

doi: 10.18632/oncotarget.14658

Figure Lengend Snippet: A . RRP15 depletion in RPE1 cells enhanced Mdm2 interaction with RPL11. RPE1 cells were transfected with or without RRP15 siRNA for 48 h. Whole-cell lysates were subjected to immunoprecipitation and immunoblotting analyses. B and C . The effects of RRP15 depletion on expression of cell cycle related proteins in various human cells. RPE1, HeLa or MCF7 cells were transfected with or without RRP15 siRNA for 48 h. Whole cell lysates were immunoblotted with indicated antibodies. D . The effects of RRP15 depletion on the expression of DNA damage related proteins in various human cells. HeLa or MCF7 cells were transfected with or without RRP15 siRNA for 48 h. Whole cell lysates were immunoblotted with indicated antibodies. E and F . RRP15 depletion induced increases of γH2AX level and γH2AX foci formation in HeLa and MCF7 cells. RPE1, HeLa or MCF7 cells transfected with or without RRP15 siRNA for 48 h were immunoblotted or immunostained with indicated antibodies. Scale bars, 5 μm. G . Histograms represented average numbers of γH2AX foci detected in 20 cells in (F). ***P<0.001. H . The model of RRP15 involved in regulating ribosome biogenesis and cell proliferation (for detail, see text).

Article Snippet: The validation of the identity of HeLa, MCF7 and RPE1 cells was determined by Tianjin Weikai Bioeng LTD (China, Tianjin).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing

Journal: bioRxiv

Article Title: ExoSloNano: Multi-Modal Nanogold Tags for identification of Macromolecules in Live Cells & Cryo-Electron Tomograms

doi: 10.1101/2024.10.12.617288

Figure Lengend Snippet: (a) Protein domain schematic of relevant histone variants (b) Top panel: RPE1 Halo-mH2A cells labeled with membrane permeant Janelia Fluor which freely diffuses into cells and recognizes the HaloTag. Bottom panel: RPE1 Halo-mH2A cells labeled with 1.4-nm-HAN-594 delivered via SLO (c) Cryo-FIB-ET workflow: RPE1 Halo-mH2A cells were grown on an EM grid (scale bar 100 um), cryo-fluorescence image before FIB-milling (scale bar 30 microns), during milling (scale bar 10 microns), and post-milling (scale bar 5 microns). (d) Denoised and deconvolved in situ tomogram of chromatin, scale bar 10 nm. (e) Subtomogram analysis of all nucleosomes.

Article Snippet: Approximately 25,000 RPE1 Halo-mH2A cells labeled with 1.4 nm HAN-594 were plated onto four micropatterned EM grids in a 35 mm Mattek dish (P35G-1.5-20-C).

Techniques: Labeling, Membrane, Fluorescence, In Situ

Journal: bioRxiv

Article Title: ExoSloNano: Multi-Modal Nanogold Tags for identification of Macromolecules in Live Cells & Cryo-Electron Tomograms

doi: 10.1101/2024.10.12.617288

Figure Lengend Snippet: (a) Tomographic slices of control or 1.4 nm HAN-594 treated RPE1 mH2A cells strained using ChromEMT (left). Right, shows a chromatin density map where chromatin packing is color coded based on the scale bar. Scale bar is 100 nm. (b) Histogram of chromatin density of the cells shown in A. N= 3 tomograms. (c) Histogram of chromatin diameter of the cells shown in A. N= 3 tomograms.

Article Snippet: Approximately 25,000 RPE1 Halo-mH2A cells labeled with 1.4 nm HAN-594 were plated onto four micropatterned EM grids in a 35 mm Mattek dish (P35G-1.5-20-C).

Techniques: Control

Journal: bioRxiv

Article Title: ExoSloNano: Multi-Modal Nanogold Tags for identification of Macromolecules in Live Cells & Cryo-Electron Tomograms

doi: 10.1101/2024.10.12.617288

Figure Lengend Snippet: (a) Diagram of the knockin and PCR strategy. A V5 epitope and Halo tag are knocked into the N terminus of the human H2AFY (coding the histone variant MacroH2A) locus in retinal pigment epithelial (RPE1) cells. Top shows wild type (WT), bottom shows Halo-knockin. Red arrows highlight PCR primers used for genotyping with the expected fragment size depicted as a black bar below. (b) DNA gel of the PCR described in A. (c) Sanger sequencing of WT and V5-Halo-H2AFY RPE1 cells. (d) Alignment to the indicated genomic sequences.

Article Snippet: Approximately 25,000 RPE1 Halo-mH2A cells labeled with 1.4 nm HAN-594 were plated onto four micropatterned EM grids in a 35 mm Mattek dish (P35G-1.5-20-C).

Techniques: Knock-In, Variant Assay, Sequencing, Genomic Sequencing

Journal: bioRxiv

Article Title: ExoSloNano: Multi-Modal Nanogold Tags for identification of Macromolecules in Live Cells & Cryo-Electron Tomograms

doi: 10.1101/2024.10.12.617288

Figure Lengend Snippet: (a) Absolute copy number of macroH2A molecules per RPE1 Halo-mH2A cell. N=4 experiments. (b) Quantification of 1.4 nm-HAN-594 delivered to RPE1 Halo-mH2A cells, N=3 experiments.

Article Snippet: Approximately 25,000 RPE1 Halo-mH2A cells labeled with 1.4 nm HAN-594 were plated onto four micropatterned EM grids in a 35 mm Mattek dish (P35G-1.5-20-C).

Techniques:

Journal: bioRxiv

Article Title: ExoSloNano: Multi-Modal Nanogold Tags for identification of Macromolecules in Live Cells & Cryo-Electron Tomograms

doi: 10.1101/2024.10.12.617288

Figure Lengend Snippet: (a) Diagram of the possible macroH2A incorporations per nucleosome and the number of ExoSloNano probes (b) Single nucleosome from of RPE1 mH2A-Halo cell treated with 1.4-nm-HAN-594 of the central slice at 8 A/pixel from Denoised, Weighted Back Project (WBP), a normalized subtomogram, and from subtomogram analysis (STA). Scale bar is 10 nm. Yellow arrows point to a density consistent with a 1.4 nm nanogold moiety. Subtomogram analysis on nanogold-labeled nucleosomes. (c) Sub-tomograms mapped back to the original tomogram containing labeled nucleosomes (yellow) and unlabeled nucleosomes (orange).

Article Snippet: Approximately 25,000 RPE1 Halo-mH2A cells labeled with 1.4 nm HAN-594 were plated onto four micropatterned EM grids in a 35 mm Mattek dish (P35G-1.5-20-C).

Techniques: Labeling