Nucleoporin insufficiency disrupts a pluripotent regulatory circuit in a pro-arrhythmogenic stem cell line

Figures

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  Figure 1. Nucleoporin insufficiency blunts ESC colony mass. (a,b) Representative images of alkaline phosphatase staining in mouse ESC colonies in WT (left panel) and nup155+/− (right panel) conditions showing positive stain in both cell lines. (c) Image analysis revealed significant reduction in size of nup155+/− ESC colonies (52%, p < 0.05) as well as (d) lower percent area of colonies per field compared to WT (21% vs 30% respectively, p < 0.05). (e) No change was observed in overall colony number between conditions (p = 0.99).

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  Figure 2. Remodeling of the non-coding sub-transcriptome in nup155+/− ES cells. (a) Venn diagram intersecting significantly changing genic regions from GSE111596 dataset (8,822; light orange) with all known non-coding RNA (ncRNA) entries from the mouse GENCODE database (18,734; light blue). From this, 96 distinct ncRNAs were unique to the present study (light yellow). (b) Transcriptome deconvolution revealed that the majority of ncRNAs detected in our analysis were long non-coding RNAs, antisense RNAs and microRNAs, with the remainder belonging to other RNA species (see text). Shown is the categorical breakdown for down regulated ncRNAs enriched following analysis (43 entities, fold change >2, p < 0.05). Abbreviations – lncRNA, long non-coding RNA; misc, miscellaneous; miRNA, microRNA; rRNA, ribosomal RNA; snoRNA, small nucleolar RNA; snRNA, small nuclear RNA.

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  Figure 3. Nucleoporin insufficiency decreases ES cell expression of the miR290–295 cluster. (a) RNAseq track data for WT (pink) and nup155+/− (purple) ES cells showing diminished numbers of reads for the miR290–295 cluster. Members of the cluster include 7 miRNAs: miR290, miR291a, miR291b, miR292, miR293, miR294, and miR295. (b) Individual plots of the four statistically significant down regulated miRNAs identified in our dataset. (c) Remaining members of the miR290–295 cluster recapitulate the down regulated trend, but did not reach statistical significance. Shown are changes for miR290, miR292 and miR295. (d) Confirmation of down regulated expression by independent RT-qPCR. Bar graph shows decreased expression of miR291a, miR291b, miR293, and miR294 in nup155+/− cells compared to WT (*p < 0.05, n = 7).

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  Figure 4. Decreased pluripotency factor protein expression in nup155+/− cells. (a) Representation of the miR290–295 cluster maintenance of pluripotency circuit in mouse ESCs, which is conducive to preservation of the self-renewal state of ESCs. (b) Western blots probing for OCT4, SOX2, and NANOG shows decreased protein expression in nup155+/− cells relative to WT, β-tubulin was used as loading control. Shown are 4 biological replicates for WT and nup155+/− ESC samples. Blots are shown as cropped images. Uncropped western blot images are included in Supplementary Files. (c) Densitometry readings for each lane were normalized to β-tubulin and analyzed using a two tailed, homoscedastic T-test. All three proteins in nup155+/− cells exhibit decreased expression, with SOX2 (p = 0.027) and NANOG (p = 0.001) reaching statistical significance.

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  Figure 5. Expression of pluripotency factors is decreased in nup155 deficient ESCs. (a,b) Representative images of OCT4 (red), Lamin B (green), and DAPI (blue) fluorescence signal depicting OCT4 intranuclear localization with overall decrease in signal profile in nup155+/− (right panel) compared to ESC colonies. Bottom panel represents individual cell intensity profiles. (c,d) Maximum (Max) intensity of OCT4 in nup155+/− ESC colonies was significantly decreased compared to WT, along with the OCT4:DAPI signal ratio (*p < 0.001). (e,f) SOX2 (red), Lamin B (green), and DAPI (blue) triple staining of nup155+/− and WT ESC colonies show no overall change in intranuclear signal profile, as well as (g) no change in SOX2 max signal intensities between conditions (p = 0.42). (h) SOX2:DAPI signal ratio is higher in NUP155 deficient cells compared to WT. (i,j) NANOG (red) fluorescent signal profile was diminished in nup155+/− compared to WT. (k,l) NANOG (red) max intensity and NANOG:DAPI ratio were significantly decreased in nup155+/− ESC colonies compared to WT (*p < 0.001).