Fig 2: Minimal co-localization of MATR3 with lamin A/C or histones. Mouse C2C12 cells were grown on glass cover slips and transiently transfected with pEF.Bos vectors encoding MATR3:YFP variants noted on the figure. At 24 hours post-transfection, the cells were fixed, immunostained with antibodies to lamin A/C (Sigma-Aldrich #SAB4200236) or histone H3 (Abcam #ab8898) and imaged. The images shown are representative of images seen in 3 independent transfections, viewing 30 to 50 cells on each cover slip. Each image is from a single z-plane captured on a confocal microscope (see Methods). Nikon elements software was used to determine the extent of interaction between MATR3 and lamin A/C or histone H3 (Supplemental Figures S16 and S17). MATR3 shows limited co-localization with either lamin A/C or histone H3.

Fig 3: Sln expression extends the lifespan of Lmna -/- mice. (a) Microarray analysis for the differentially expressed genes between 30-day-old male wild-type (WT; n = 3) and Lmna -/- (n = 3) femoris muscle. The upregulated genes are shown in red, and the down-regulated genes are shown in green. Genes that are associated with endoplasmic reticulum (ER) function are highlighted in blue letters. (b) Relative expression levels of the indicated genes in 30-day-old male Lmna -/- skeletal muscle compared to the WT cohorts by qRT-PCR. (c) Schematic representation of the construction strategy for Sln knockout mice using the CRISPR/Cas9 techniques. The Sln knockout mice used in this study lost the entire coding region of Sln. (d) Western blotting for verification of lamin A/C and Sln protein expression in the soleus muscle of 30-day-old male WT, Lmna -/-, Lmna -/- Sln -/-, and Sln -/- mice. For each genotype, the expression profiles from three mice were shown. Immunoblots of ß-tubulin and Gapdh are shown as loading controls. (e) Growth curve in body weight of WT, Lmna -/-, Lmna -/- Sln -/-, and Sln -/- mice (male and female). (f) Kaplan–Meier survival curve of WT, Lmna -/-, Lmna -/- Sln -/-, and Sln -/- mice (male and female). Comparing Lmna -/- and Lmna -/- Sln -/- mice, p = .0579 using log-rank (Mantel–Cox) test, and p = .0111 using Gehan–Breslow–Wilcoxon test. All values are mean ± SEM

Fig 4: The interaction of nuclear lamins and endoplasmic reticulum (ER) proteins. (a and b) Normal and HGPS skin fibroblasts were transfected with a Sln expression vector tagged with HA (i.e., Sln-HA). Cells were fixed after 96 hr of transfection and immunofluorescent stained to visualize HA (green), cell endogenous SERCA2 or lamin A (red), and lamin B (gray). The nuclei were counterstained using Hoechst 33,342 (blue). Transverse intensity line scans along the white lines of the corresponding cell images are presented on the right. (c) HEK293TN cells were co-transfected with Sln-HA, FLAG-progerin and/or FLAG-lamin A. Cells transfected with (+) or without (-) the indicated expression plasmids are noted at the top of the Western blot profiles. Cell lysates were immunoprecipitated using mouse anti-HA and mouse anti-FLAG agarose, respectively. The cell lysate input and co-IP products were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. The signals of the co-immunoprecipitated Calnexin and SERCA2 were quantified and shown below the immunostaining profiles

Fig 5: The changes in METTL16 subcellular distribution after UVA and UVC irradiation. Quantitative immunofluorescence analysis of METTL16 distribution was performed in selected cell lines. (A) The graph depicts the normalized ratios of METTL16 fluorescence intensity (FI) values in the nucleoplasm to METTL16 FI in the cytoplasm. Panel (B) shows normalized ratios of METTL16 FI in the nucleolus to METTL16 FI in the cytoplasm (from left to right the number of measurements were: n = 25, 20, 23, 30, 30, 31, 34, 20, 20, 34, 20, 20, 48, 32, 20). Error bars represent standard deviations (S.D.). Statistical analysis was performed using Mann–Whitney rank-sum test. Statistically significant differences are shown at * a = 0.05, ** a = 0.001. (C) Immunofluorescence labeling of METTL16 (green) in mESCs and dual labeling of METTL16 (green) with fibrillarin (red) in MEFs and MCF7 cells. (D) Western blot analysis shows the level of the METTL16 protein in MEFs and MCF7 cells exposed to UVA and UVC irradiation (original data see in Supplementary Figure S1). Data were normalized to the total protein levels and to the level of a-tubulin. (E) Co-IP and Western blots of METTL16 with the DDB2 protein or METTL16 with ?H2AX, studied in wt MEFs and LMNA dn cells.