Journal: Nucleic Acids Research

Article Title: B-type lamins maintain transcriptional homeostasis by spatially controlling chromatin–speckle proximity

doi: 10.1093/nar/gkag283

Figure Lengend Snippet: The absence of B-type lamins changes the global expression pattern of genes especially adjacent to the nuclear speckles. ( a ) Schematic diagram contrasting chromatin activity in a normal state (left) and possible transcription regulation defect in lamin-depleted cells (right). ( b ) Staining of EU incorporation (green) and immunostaining for Lamin B (red; WT and LMNB2 −/− : Lamin B1 Ab, LMNB1 −/− : Lamin B2 Ab). Cells were incubated for 4 h after treatment with 0.2 mM EU. Confocal images display EU-positive signals in the nuclei of three different cell types. P -values are calculated using a two-tailed, unpaired t -test (**** P < 0.0001); scale bar: 10 µm. ( C ) RNA FISH detecting poly(A) mRNA expression (green; poly(dT) probe), with DNA stained using DAPI (blue), and quantification of poly(dT) signals in both the nucleus and the whole cell area, with significance determined by a two-tailed, unpaired t -test (**** P < 0.0001); scale bar: 10 µm. ( d ) WT and LMNB1 −/− or WT and LMNB2 −/− cells were respectively co-cultured. RNA FISH detecting poly(A) mRNA expression (green; poly(dT) probe) and immunostaining for Lamin B (red), along with quantification of poly(dT) and Lamin B intensities in the whole cell area; scale bar: 10 µm. ( e ) Box plot of expression changes (log -fold changes in FPKM ratios) categorized into quartiles based on expression levels in WT cells (Q1 = upper quartile; most active genes), with the box representing the interquartile range (IQR) and whiskers plotted using the Tukey method, ignoring outliers; significance was assessed using a two-tailed Mann–Whitney test (**** P < 0.0001). ( f ) Genome tracks of LADs and SPADs detected by the EDD with a 20 kb bin size in four different cell types (H1, K562, HCT116, and HFFc6), showing constitutive, facultative, and constitutive-inter LADs and SPADs. Up- and downregulated genes in LMNB1 −/− and LMNB2 −/− cells are separately visualized in red and blue, respectively, and plotted onto their genomic loci, with point size representing the P -value and height denoting the level of expression change (log -fold changes in FPKM ratios). ( g ) Bar plot showing the proportion of up- and downregulated genes in each type of LAD and SPAD. See also and , and , , , , and .

Article Snippet: According to the manufacturer’s instructions, the TruSeq mRNA Library Prep Kit (Illumina) generated the RNA-Seq library.

Techniques: Expressing, Activity Assay, Staining, Immunostaining, Incubation, Two Tailed Test, Cell Culture, MANN-WHITNEY