Skip to main content
. 2019 Mar 21;104(4):749–757. doi: 10.1016/j.ajhg.2019.02.021

Figure 3.

Figure 3

Immunofluorescence Analysis of Mutant LEMD2 in U2OS Cells

(A) Irregular nuclear localization of mutant LEMD2 during transient overexpression with pCMV6-LEMD2-FLAG constructs containing either the LEMD2 WT or mutant (c.1436C>T) sequence in U2OS cells. 48 h after transfection, immunofluorescence staining of FLAG (green) and lamin A and C (red) reveals patchy accumulations of mutant LEMD2 (white arrows), whereas the WT construct shows more even distribution within the nuclear lamina.

(B) Manual comparison of cells transfected with WT and mutant LEMD2 constructs shows a higher percentage of abnormal distribution in cells transfected with mutant LEMD2. Nuclear distribution of LEMD2 was characterized as normal or irregular, and the percentage of nuclei with irregular distribution was calculated. Combined results (mean ± SEM) of three separate experiments are shown. (A total of 225 cells transfected with WT and 209 cells transfected with mutant LEMD2 were analyzed.)

(C) Quantitative colocalization analysis of WT and mutant LEMD2 demonstrates disturbed colocalization of mutant LEMD2 and lamin A and C. Nuclei of cells transfected with both constructs (pCMV6-LEMD2-FLAG WT and mutant) were delimited and analyzed with the ImageJ software (Coloc2 plugin). The Pearson correlation coefficients of LEMD2-FLAG and lamin A and C of each individual nucleus, as well as averages and p values (according to the Wilcoxon rank sum test with continuity correction), are shown.

(D) WT and mutant LEMD2 are both predominantly localized within the nucleus. The Subcellular Protein Fractionation Kit (Thermo Fisher) was used to collect fractionated cell lysates 48 h after transfection with WT and mutant LEMD2 constructs. Immunoblot analysis revealed similar distribution patterns of both WT and mutant LEMD2 within cytoplasm (Cyto), membranes (Mem), and nuclear (Nuc) fractions. Comparison of band intensity normalized for total protein (shown as x-fold change with regard to the normalized band intensity of the cytoplasm fraction) from three separate experiments (mean ± SEM) did not reveal significant differences between WT and mutant constructs.

(E–G) Proteins of the nuclear lamina display different localization patterns relative to LEMD2-FLAG constructs. U2OS cells were transfected through the use of pCMV6-LEMD2-FLAG constructs (WT or mutant), and immunofluorescence stainings were performed 48 h after transfection. Whereas emerin and BANF1 colocalize with irregular nuclear accumulations of mutant LEMD2 (E and F), LAP1B does not (G).