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. 2019 Mar 28;10:249. doi: 10.3389/fgene.2019.00249

Figure 5.

Figure 5

The FMRpolyG protein reduces cell viability and disrupts the lamin architecture. (A) Effect of FMRpolyG on cell viability. HEK293 cells were transfected with the indicated constructs, and cell death measured in GFP positive cells. Cell transfected with GFP-C1 were used as negative controls. Cells were counted as non-viable based on the incorporation of propidium iodide detected by FACS. For each transfection >50 000 cells were counted per experiment. ***p < 0.001; **p < 0.01; *p < 0.05. The exact p-values, from left to right, are as follows: 0.0009 and 0.0081. (B) Effect of FMRpolyG on lamin architecture. HEK293 cells were transfected with the indicated constructs and stained for Lamin B1 24 h after transfection. Transfection with GFP-C1 served as negative control. Cells were analyzed by confocal imaging, and the fraction of cells with disrupted lamin architecture counted in GFP positive cells (left) and in cells with aggregates (right). The number of GFP positive cells included in the analysis was >440 for GFP-C1 and wtHP-99Gly-GFP. For wtHP-99Gly-GFP > 170 aggregate bearing cells were counted. Due to the low levels of GFP positive cells among those transfected with mutHP-90Gly-GFP, a total of 80 GFP-positive cells and 70 aggregate bearing cells were included from mutHP-90Gly-GFP transfected cell populations. The exact p-values, from left to right, are as follows: 0.0021 (**), 0.0004 (***), 0.0123 (*), and 0.0113 (*). (C) Representative confocal images showing normal lamin rings in cells expressing GFP (GFP-C1), but disrupted lamin architecture in aggregate-containing cells after transfection of wtHP-99Gly-GFP or mutHP-90Gly-GFP. (D) Effect of stably expressed FMRpolyG on lamin architecture. Expression of GFP or FMRpolyG-GFP were induced with doxycycline for 72 h, followed by Lamin B1 staining, confocal imaging, and counting of GFP positive cells with disrupted lamin architecture. Cell expressing GFP (from GFP-C1) were used as negative controls. For GFP-C1 and wtHP-99Gly-GFP, a minimum of 830 GFP positive cells were quantified. Due to low expression levels in cells expressing mutHP-90Gly-GFP, 150 GFP-positive cells were included for this construct. For wtHP-99Gly-GFP and mutHP-90Gly-GFP, a minimum of 80 aggregate bearing cells were analyzed per construct. The exact p-values, from left to right, are as follows: 0.0186 (*), 0.0007 (***), 0.00001 (***), and 0.0004 (***). (E) Representative confocal images showing normal lamin rings in HEK-FlpIn cells expressing GFP and disrupted lamin structures in aggregate-containing cells expressing FMRpolyG-GFP. All graphs in (A,B,D) are based on quantifications of a minimum of three individual experiments and error bars represent SD.