Journal: bioRxiv

Article Title: Mitotic progression following DNA damage enables pattern recognition within micronuclei

doi: 10.1101/156414

Figure Lengend Snippet: a, RT-qPCR of ISG54 at 6 days following 20Gy in parental MCF10a cells treated +/- CDK1i (RO-3306) or derivatives harboring deletion of cGAS or STING. Error bars represent SEM of three biological replicates. b, Representative western blots of STAT1 activation in CRISPR-CAS9 knockout MCF10A cells for cGAS or STING. c, Immunofluorescence of endogenous cGAS in MCF10A cells. Arrowhead indicates a representative cGAS-positive micronucleus. Scale bar is 10µm d, Quantification of micronuclei in ( c ). Error bars represent SEM of three biological replicates. e, Extracted frames from live cell microscopy of irradiated MCF10A cells stably expressing GFP-H2B to mark chromatin and mCherry-cGAS. Inset depicts time from obvious mitotic induction in hh:mm. f, Representative RNA FISH images (Scale bar is 10µm) and quantification of ISG54 status as described in Methods. Arrowhead indicates an example of a micronucleus. g, Representative western blot of STAT1 activation of naïve cells treated with indicated conditioned media for 24h.

Article Snippet: After a brief rinse in RNA Wash buffer (10% formamide/2X SSC) cells were inverted into hybridization buffer containing Custom Stellaris® FISH Probes designed against ISG54 (NM_001547) by utilizing the Stellaris® RNA FISH Probe Designer (Biosearch Technologies, Inc., Petaluma, CA) available online at www.biosearchtech.com/stellarisdesigner .

Techniques: Quantitative RT-PCR, Western Blot, Activation Assay, CRISPR, Knock-Out, Immunofluorescence, Microscopy, Irradiation, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Parallel evolution between genomic segments of seasonal human influenza viruses reveals RNA-RNA relationships

doi: 10.1101/2021.01.14.426718

Figure Lengend Snippet: A549 cells were infected with A/Perth/16/2009 (H3N2) at an MOI of 2 or mock infected. Cells were fixed at 8 hours post-infection and combination fluorescence in situ hybridization/immunofluoresence (FISH and IF, respectively) was performed. FISH probes targeting the NS, NA and PB2 vRNA segments were labeled with Alexa Fluor 488, Quasar 570 and Quasar 670, respectively. Antibodies targeting nucleoprotein (NP) were used with an anti-mouse Alexa Fluor 594 secondary antibody. Nuclei were labeled with DAPI. Coverslips were mounted and volumetric imaging was performed to obtain Nyquist sampling. A , A maximum projection image of a representative cell is shown after cell segmentation. Scale bar corresponds to 5 pm. B , A3D rendering of the cell after analysis. C , Colocalization of vRNA segments was assessed in 15 individual infected cells. D , Quantification of each pair of vRNA segments within 300 nm of the nuclear border. Each point represents an individual cell (n = 15). Aggregate data from three independently performed experiments are shown. * denotes p < 0.05 (Mann-Whitney U test)

Article Snippet: Custom Stellaris RNA FISH oligonucleotide probes specific for the H3N2 virus NS, NA and PB2 vRNA segments were purchased from BioSearch Technologies (refer to for FISH probe sequences).

Techniques: Infection, Fluorescence, In Situ Hybridization, Labeling, Imaging, Sampling, MANN-WHITNEY

Journal: Molecular cell

Article Title: LncRNA-induced spread of Polycomb controlled by genome architecture, RNA abundance, and CpG island DNA

doi: 10.1016/j.molcel.2019.05.028

Figure Lengend Snippet: (A) Molecules per cell (MPC) of Xist, Airn, Kcnq1ot1 by RNA-Seq. (B) Stability of Xist, Airn, Kcnq1ot1 in TSCs after 5μg/ml of Actinomycin D. Mean ±SD half-lives in parentheses. (C) Boxplots of H3K27me3 density in 40kb sliding bins across Airn domain, and (D) parent-of-origin expression for 61 considered genes (Figure 1) in Airn-overexpression (OE), -wild-type (WT), knockdown (KD), and truncation (KO) TSCs. ***, p<0.001; **, p<0.01, Tukey’s HSD test. Y-axis as in Figure 1E, ​,F.F. (E) H3K27me3 density in 40kb sliding bins across Airn domain in OE, WT, KD, and KO TSCs. Airn MPC is above the density plot. Blue ticks + grey arrows, CGIs in Figure 5A. Green bar, Airn locus. “WT”, TSCs expressing dCas9-VP160 and a non-targeting sgRNA.

Article Snippet: Stellaris FISH probes, Custom Mouse Xist , Quasar 670 , Biosearch Technologies , Stellaris RNA FISH Probe Designer.

Techniques: RNA Sequencing, Expressing, Over Expression, Knockdown

Journal: Molecular cell

Article Title: LncRNA-induced spread of Polycomb controlled by genome architecture, RNA abundance, and CpG island DNA

doi: 10.1016/j.molcel.2019.05.028

Figure Lengend Snippet: (A) Wiggle tracks of RNA-IP data for input, IgG, and HNRNPK in SM33 ESCs + TSCs and CTCF in TSCs across Xist, Airn, and Kcnq1ot1. Blocks above HNRNPK tracks, MACS peaks. Right-justified numbers, signal over IgG. Xist repeats are below Xist diagram. (B) H3K27me3 tiling density and boxplots in Xist, Airn, and Kcnq1ot1 domains in WT and HNRNPK knockdown TSCs. Green bars, lncRNA loci. ***, p<0.001, two-tailed t-test. (C) Boxplots of H3K27me3 density +/− 2kb from CGI centers in Xist, Airn, and Kcnq1ot1 domains. Difference in means between WT and knockdown, upper right corner. ***, p<0.001; **, p<0.01; *, p<0.05, two-tailed t-test. “Nucleation sites” in Airn/Kcnq1ot1 plots, CGIs from Figures 5A/S6A. (D) Model: Super-stoichiometric interactions between proteins such as HNRNPK (pink circles) that bind lncRNAs (squiggles) and PRC1/2 (blue/green ovals) concentrate PRCs in lncRNA foci. These same interactions tether lncRNA foci to CGIs (grey ovals) pre-bound by PRCs, nucleating PRC spread in contacted regions.

Article Snippet: Stellaris FISH probes, Custom Mouse Xist , Quasar 670 , Biosearch Technologies , Stellaris RNA FISH Probe Designer.

Techniques: Knockdown, Two Tailed Test

Journal: Molecular cell

Article Title: LncRNA-induced spread of Polycomb controlled by genome architecture, RNA abundance, and CpG island DNA

doi: 10.1016/j.molcel.2019.05.028

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Stellaris FISH probes, Custom Mouse Xist , Quasar 670 , Biosearch Technologies , Stellaris RNA FISH Probe Designer.

Techniques: Control, Virus, Recombinant, Cell Culture, SYBR Green Assay, DNA Labeling, Reverse Transcription, Sequencing, Variant Assay, Microscopy, Western Blot, Plasmid Preparation, Software, Transfection