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: American Journal of Cancer Research

Article Title: METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m 6 A modifications and splicing factors

doi:

Figure Lengend Snippet: Establishment of stable METTL3-knockdown cell lines using osteosarcoma U2OS cells. A. Using the CRISPR-Cas9 system, METTL3-knockdown cells were established (2-9 and 2-14) along with control cells (NT2 and NT3). METTL3-knockdown efficiency was confirmed by the decreased METTL3 and METTL14 protein expression using western blot. B. Levels of m6A in total RNA isolated from each stable cell line was measured via ELISA.

Article Snippet: U2OS cells were purchased from the Korean Cell Line Bank.

Techniques: Knockdown, CRISPR, Control, Expressing, Western Blot, Isolation, Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m 6 A modifications and splicing factors

doi:

Figure Lengend Snippet: Enriched RBP motif in alternatively spliced genes. A. rMAPS captured a total of 19 RBPs that were enriched in alternatively spliced genes in METTL3-knockdown cells. Each RBP is represented with a different color based on the type of alternative splicing (alternative 3’ splice site: red, alternative 5’ splice site: blue, cassette exon: black, intron retention: green). B. The Pearson’s correlation coefficients between each RBP and METTL3 expression were analyzed in 12,839 TCGA pan-cancer patients. C. The m6A modification of the 3’UTR of SFPQ mRNA. The m6A sites in SFPQ mRNA was analyzed using SRAMP (https://www.cuilab.cn/sramp; left panel) and a red arrow indicates the predicted m6A site in the 3’UTR. MeRIP-qPCR analysis was performed to validate the predicted m6A site in U2OS cells (right panel). D. The mRNA expression levels of SFPQ and IGF2BP3 after knockdown of IGF2BP3. The mRNA expression levels were estimated by real-time PCR after transfecting 25 nM of IGF2BP3 siRNA for 48 h. E. Enrichment of IGF2BP3 in the 3’UTR of SFPQ mRNA. RIP-qPCR analysis was performed to detect binding of IGF2BP3 to the 3’UTR of SFPQ mRNA. F. The number of SFPQ peaks located within a distance of 1000 bp of alternatively spliced genes was counted.

Article Snippet: U2OS cells were purchased from the Korean Cell Line Bank.

Techniques: Knockdown, Alternative Splicing, Expressing, Modification, Real-time Polymerase Chain Reaction, Binding Assay

Journal: American Journal of Cancer Research

Article Title: METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m 6 A modifications and splicing factors

doi:

Figure Lengend Snippet: Alternatively spliced genes in METTL3-knockdown HULEC-5a and A375 cells. A. Alternative splicing was analyzed using JUM in METTL3-knockdown HULEC-5a and A375 cells using shRNA. The number of alternatively spliced genes is represented using a bar graph (p-value < 0.05 being significant). B. Venn diagram showing the number of common alternatively spliced genes between U2OS, HULEC-5a and A375 cells. C. Top 10 enriched GO terms of alternatively spliced genes in HULEC-5a and A375 cells.

Article Snippet: U2OS cells were purchased from the Korean Cell Line Bank.

Techniques: Knockdown, Alternative Splicing, shRNA