Journal: Nature Methods

Article Title: Optimal precision and accuracy in 4Pi-STORM using dynamic spline PSF models

doi: 10.1038/s41592-022-01465-8

Figure Lengend Snippet: Mic60 is a component of the MICOS complex, and is involved in the formation and maintenance of crista junctions that connect the crista membrane with the inner boundary membrane. a , Mic60 in a U-2 OS cell, labeled with primary and secondary antibodies. The Mic60 signals appear as structured, punctate clusters. The localizations are color coded according to their z coordinate (identical color scales in a – d ). Scale bar, 200 nm. b , Magnified view of the boxed region in a . Scale bar, 50 nm. c , Mic60 in a COS-7 cell, in which the crista junctions exhibit a linear organization over segments of the inner boundary membrane. Scale bar, 200 nm. d , Magnified view of the boxed region in c . Scale bar, 50 nm. e , f , Unwrapped views of the Mic60 localization density around the surface of the mitochondria, showing the nanoscale distribution of Mic60. In U-2 OS cells, Mic60 appears predominantly punctate, with pairs or clusters of signal density separated by 20–40 nm (Extended Data Fig. and Supplementary Fig. ). In COS-7 cells, Mic60 appears to have a zigzag or double-line arrangement, with a typical width of approximately 25 nm (Extended Data Fig. and Supplementary Fig. ). Dashed lines indicate the extent of the data in f . g , Two-color image of Mic60 (blue) and mitochondrial nucleoids (yellow) in a COS-7 cell, stained with antibodies labeled with Alexa Fluor 647 and Cy5.5, respectively. Scale bar, 1 µm. h , Detailed view of the boxed region in g . Lower density of Mic60 close to the DNA signal, suggesting fewer crista junctions in these regions. i , Cross-section ( x – z ) through the region indicated by the dashed lines in h , showing Mic60 at the inner boundary membrane, and a DNA cluster in the center of the mitochondrion. j , A 3D perspective view of the mitochondrion shown in h and i , where the Mic60 and DNA signals have been rendered as isosurfaces. Scale bars, 250 nm ( h – j ).

Article Snippet: Experiments were performed using either standard COS-7 cells or U-2 OS cells obtained from American Type Culture Collection (ATCC), or gene-edited U-2 OS cells expressing a SNAP-tagged version of the nucleoporin Nup107 (CLS Cell Lines Service, U-2OS-ZFN-SNAP-Nup107 clone 294) or Nup96 (CLS Cell Lines Service, U-2OS-CRISPR-NUP96-SNAP clone 33) .

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: Deep learning enables fast and dense single-molecule localization with high accuracy

doi: 10.1101/2020.10.26.355164

Figure Lengend Snippet: a) DECODE can reduce acquisition times by one order of magnitude. The same sample of microtubules, labeled with anti- α tubulin primary and AF647 secondary antibodies, imaged with different UV activation intensities to result in different emitter densities between 0.08 and 0.86 emitters per frame per μm 2 and acquisition times between 93 and 1120 s, while keeping the total number of localizations the same. For high-density activation, we show a comparison with CSpline. b) Fourier Ring Correlation curves for DECODE and CSpline for different emitter densities. c) Resolution estimates obtained using the Fourier Ring Correlation and 0.143 criterion across densities for both methods. d) Fast live-cell SMLM on the nuclear pore complex protein Nup96-mMaple acquired in 3 seconds. e) DECODE enables ultra-high labeling densities. Microtubules labeled with a high concentration of anti- α and anti- β tubulin primary and AF647 secondary antibodies. e1, e2) Magnified regions as indicated in a. Data acquired with high-density labeling shows continuous structures. As a comparison, the same sample was acquired after pre-bleaching of the fluorophores to reach the single-molecule blinking regime. Here, single labels are resolved in the superresolution reconstruction and lead to a sparse decoration of the microtubules. e3, e4) Side view reconstructions of regions as indicated in e1, e2 resolving the hollow, cylinder-like structure of immunolabeled microtubules. f) Representative raw camera frames for the high-density and single-emitter acquisitions, respectively. Scale bars: 10 μm (d inset, f), 1 μm (a, d, e, e1, e2), 100 nm (e3,e4).

Article Snippet: For imaging of live cells, coverslips containing Nup96-mMaple cells (catalog no. 300461, CLS Cell Line Service, Eppelheim, Germany) were rinsed twice with warm PBS before they were mounted onto a custom manufactured sample holder in 1 mL growth medium containing 20 mM HEPES buffer and imaged directly.

Techniques: Labeling, Activation Assay, Concentration Assay, Immunolabeling

Journal:

Article Title: General Transcriptional Coactivator PC4 Activates p53 Function

doi: 10.1128/MCB.24.5.2052-2062.2004

Figure Lengend Snippet: PC4 directly interacts with p53 in vitro and in vivo. (A) Induction of p53 expression in the U2OS cell line. The levels of p53 and PC4 present in adriamycin (2 μg/ml)-treated U2OS cells were assessed by Western blotting using anti-p53 (upper panel) and anti-PC4 (lower panel) antibodies. (B and C) In vivo interaction of PC4 with p53. (B) Lane 1, an adriamycin (2 μg/ml)-induced U2OS cell extract was immunoblotted with polyclonal PC4 antibody N17. Lane 2, immunoprecipitation of endogenous PC4 from an induced U2OS cell lysate was performed using anti-p53 monoclonal antibody DO1 followed by immunoblotting with anti-PC4 polyclonal antibody. Lane 3, immunoprecipitation using mouse preimmune serum used as a control. (C) Lane 1, immunoprecipitation of endogenous p53 from an induced U2OS cell extract, using anti-PC4 polyclonal antibody N17 followed by immunoblotting with anti-p53 monoclonal antibody DO1. Lane 2, adriamycin-induced U2OS cell extract immunoblotted with monoclonal p53 antibody DO1. Lane 3, immunoprecipitation reaction with goat preimmune serum used as a control. (D, E, and F) Interaction of PC4 with p53 in an in vitro GST pulldown assay. (D) Schematic representation of GST and GST-p53 fusion proteins. ++, strong interaction of PC4 with respective GST-p53 fusion protein; +, weaker interaction; −, no interaction. (E) SDS-PAGE (10%) and Coomassie blue R250 staining of immobilized GST-p53 fusion proteins. Lane 1, GST-p53 (full length); lane 2, GST-p53(1-73), lane 3, GST-p53(120-290); lane 4, GST-p53(284-330), lane 5, GST-p53(328-368); lane 6, GST-p53(364-393). GST fusion proteins predominantly contain intact proteins (indicated with asterisks) with minimum low-molecular-weight breakdown products. (F) One microgram of GST (lane 2) or GST-p53 fusion proteins (lanes 3 to 8) was incubated with bacterial extract containing 200 ng of PC4 and analyzed by immunoblotting with anti-PC4 N17 antibody. Lane 1, 5% input of bacterial cell lysate. IP, immunoprecipitation; IB, immunoblot; WCE, whole-cell extract; AD, activation domain; DBD, DNA binding domain; OD, oligomerization domain.

Article Snippet: Immunoprecipitation was performed by incubating the drug-treated U2OS cell lysate with protein A-agarose beads conjugated to either anti-p53 mouse monoclonal antibody DO1 (Oncogene) or anti-PC4 goat polyclonal antibody N17 (Santa Cruz).

Techniques: In Vitro, In Vivo, Expressing, Western Blot, Immunoprecipitation, GST Pulldown Assay, SDS Page, Staining, Molecular Weight, Incubation, Activation Assay, Binding Assay