Fig. 4. Loss of human NEMP leads to defects in nuclear shape and survival, and EMERIN (EMD) forms a complex with NEMP1.
(A to C) Lentivirus knockdown of hNEMP1 in HT1080 cells causes convoluted nuclei and increased LaminA/C. (A) Quantification of knockdown efficiency and (B) corresponding nuclear shape. (C) Example of nuclei from respective short hairpin RNA (shRNA) transfections. (D) Knockdown of hNEMP1 and hNEMP2 using different combinations of siRNAs (combos A and B) causes a reduction in cell number in various cell types. Cell number remains unchanged in SHP77 cells. (E) Level of hNEMP1 and hNEMP2 knockdown measured by quantitative polymerase chain reaction. (F) Apoptosis of RPE-1 cells assessed by annexin V/PI (propidium iodide) staining at 6 days following combined knockdown of hNEMP1 and hNEMP2. (G) Top 5 hNEMP1 BioID interactors identified by spectral counts. Yellow indicates LEM domain proteins. P value of 2.0 × 10−9 and a Benjamini of 6.2 × 10−8; The database for annotation, visualization and integrated discovery (DAVID) Bioinformatics Resource. (H) Top 5 hNEMP1 AP-MS interactors identified by spectral counts. All proteins listed for BioID and AP-MS are high-confidence <1% false discovery rate. (I) Endogenous hNEMP1 coimmunoprecipitates with EMD-GFP (green fluorescent protein). HEK293 cells transiently transfected with GFP or EMD-GFP were subjected to GFP pull-down. (J) Endogenous EMERIN coimmunoprecipitation with hNEMP1-BirA*-Flag. HEK293 cells stably expressing either hNEMP1-BirA*-Flag or BirA*-Flag were subjected to Flag immunoprecipitation (IP).