Journal: iScience

Article Title: Daughter cell fate choice instructed preemptively by mother cells facing nutrient limitation

doi: 10.1016/j.isci.2023.107198

Figure Lengend Snippet:

Article Snippet: pMK232 , Addgene , Cat #72834.

Techniques: Recombinant, Negative Control, Software, Time-lapse Microscopy, Imaging

Journal: bioRxiv

Article Title: Super-resolution Imaging Reveals 3D Structure and Organizing Mechanism of Accessible Chromatin

doi: 10.1101/678649

Figure Lengend Snippet: ( A ) Acute depletion of CTCF and Cohesin in mESCs by the auxin-induced protein depletion system. The mini auxin-inducible degron (mAID)-HaloTag (halo) was bi-allelically knocked into N-terminus of CTCF or C-terminus of Rad21 (Cohesin subunit) by CRISPR/Cas9 genome editing technology (upper panel) in a mouse ES line stably expressing the plant derived E3 ligase adaptor protein osTir1. Adding plant derived hormone analogue (auxin) triggers rapid degradation of target protein RAD21 or CTCF (See also Figure S5A-B) as revealed by single cell immunofluorescence (lower panel). OCT4 immunostaining was used as a control. Scale bar, 5 μm. ( B and C ) Single-cell 3D illustration of ATAC-PALM localizations upon CTCF ( B ) and RAD21 ( C ) depletion. The color bar indicates localization density calculated by using a canopy radius of 250 nm. See the 3D rotatory presentation in Movie S5 . Scale bar, 2 μm. ( D ) CTCF and RAD21 depletion promotes global accessible chromatin clustering. The error bar represents standard error (SE) of the mean and Mann-Whitney U test was applied for comparing data points at g(0) . ( E ) The dose-dependent effect of Cohesin depletion on global accessible chromatin clustering revealed by the inverse relationship between clustering amplitude ( A ) and residual RAD21 levels measured by RAD21-GFP-mAID fluorescence intensities (arbitrary fluorescent units). Specifically, two different auxin concentrations (5 μM and 500 μM) were used to generate a gradient of RAD21 level in single cells. ( F ) Acute CTCF or RAD21 depletion affects ACD formation through distinct mechanisms. The upper panel shows the violin plot of localization density of top 100 rank ordered ACDs among 4 individual cells for each condition. The lower panel shows the violin plot of normalized radius of top 100 ranked ACDs among 4 individual cells for each condition. The black bar indicates the median value for each data set. For statistical test, data from 4 individual cells for each condition were pooled together and Mann-Whitney U test was applied. ( G ) 3D iso -surface reconstruction of ACDs (green) identified by using the DBSCAN algorithm for WT (left panel), CTCF depletion (middle) and RAD21 depletion (right panel) conditions. The iso-surface in grey outlines the nuclear envelope. The lower panels show 4× magnification of local regions under each condition. Scale bar, 1 μm.

Article Snippet: The pLenti-EF1-osTir1-9Myc-P2A-Bsd was constructed by PCR amplifying the Oryza Sativa Tir1 (osTir1) cDNA from the pBabe Puro osTIR1-9Myc (Addgene #80074) and inserted into the AgeI/BamHI site of the lentiCas9-Blast construct (Addgene #52962).

Techniques: CRISPR, Stable Transfection, Expressing, Derivative Assay, Immunofluorescence, Immunostaining, MANN-WHITNEY, Fluorescence