Expansion of the Nucleoplasmic Reticulum Requires the Coordinated Activity of Lamins and CTP:Phosphocholine Cytidylyltransferase

Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure 2 - Localization of CCT-GFP and CCT-mGFP. (A) Two representative images of NE-localized CCT-GFP in CHO58 cells treated with oleate as described in the legend to Figure 1. Images were taken using a 100x objective (B) Localization of CCT-mGFP in untreated (no addition, NA) and oleate-treated CHO58 cells. Images were taken using a 63x objective.
• Supplemental Figure 3 - Distribution of NR tubules in CHOK1 and CHO58 cells expressing GFP lamins and CCT-DsRed. The distribution of tubules in individual nuclei (0-1, 2-4 and >4) was quantified with AlexaFluor647-ConA in control (no addition, NA) and oleate-treated CHOK1 cells transfected with GFP (A), GFP-lamin A (B) or GFP-lamin B1 (C). Results are from the examination of 40 nuclei each in 3 separate experiments. CHO58 cells were transfected with vectors (pGFP and pDsRed) (D), GFP-lamin A and CCT-DsRed (E) or GFP-lamin B1 and CCT-DsRed (F). Results are from the examination of 40 nuclei each in 3 separate experiments.
• Supplemental Figure 4 - Representative fields of CHOK1 cells with knockdown of lamin expression by RNAi. CHOK1 cells were transfected with control (siNT) or siRNAs specific for lamin A, lamin A/C or lamin B1 for 48 h, and fixed and processed for immunofluorescence detection of lamins or ConA as described in the legend to Figure 5. Confocal microscope settings were optimized for detecting lamin and ConA images in control cells (and were equivalent within an experiment) but lead to weak signals when lamin expression was reduced by 70-90% in the knockdown cells.
• Supplemental Figure 1 - Movie of CCT-GFP translocation in oleate stimulated CHO58 cells. Cells were treated as described in the legend to Figure 1.