Journal: JCI Insight

Article Title: Nuclear membrane ruptures underlie the vascular pathology in a mouse model of Hutchinson-Gilford progeria syndrome

doi: 10.1172/jci.insight.151515

Figure Lengend Snippet: ( A ) Microscopy images of a live SMC (arrow) expressing Nuc-GFP (green) and increased amounts of lamin B1 (see ). Scale bar: 20 μm. ( B ) Microscopy images of Hoechst-stained nuclei in suspended cells expressing lamin B1 (B1), nf-lamin B1 (nf-B1), or lamin B2 (B2) before (left) and after (right) compressing the cells with a glass coverslip. Scale bar: 10 μm. Nuclear lamin expression was induced with Dox (see ). ( C ) Inducing lamin B1 expression (+Dox) increases nuclear area (mean ± SD, numbers of cells examined are reported above each bar; Student’s t test, * P < 0.001). ( D ) Lamin B1 (B1) decreases nuclear stiffness (Young’s modulus) in SMCs, as measured by AFM (mean ± SEM, n = 5 experiments). Measurements were compared with noninduced SMCs by ANOVA (* P < 0.002, ** P < 0.0001). ( E ) Lamin B1 increases nuclear size (spreading) in PreA-SMCs and Progerin-SMCs. Nuclei size was measured as in C (mean ± SD; Student’s t test, * P < 0.001). ( F ) Progerin (Prog) but not nf-Prog increases nuclear stiffness in SMCs (mean ± SEM, n = 5 experiments). Expression data are shown in . All measurements were compared with control SMCs (Con) by ANOVA (* P < 0.0001). ( G ) Western blot showing increased expression of lamin B1 (B1) and nf-B1 in SMCs examined in H . ( H ) Increasing lamin B1 expression but not nf-B1 reduces nuclear stiffness in Prog-SMCs (mean ± SEM, n = 3 experiments). Measurements were compared with SMCs expressing progerin by ANOVA (* P < 0.0001). ( I ) Disrupting the LINC complex with KASH2 (blue bars) reduces nuclear stiffness in Prog-SMCs (mean ± SEM; n = 3 experiments). Nuclear stiffness was compared with cells expressing the inactive mutant KASH2ext (white bars) by the Student’s t test (* P < 0.01).

Article Snippet: The human lamin B2 cDNA was amplified from pCMV6- LMNB2 (Origene, SC106163) with forward primer 5′-ATGCGTGGACCTGGAGAAA-3′ and reverse primer 5′-GTAGCCCCTTGAATTTCACATCACGTAGCAGCCTCTTGA-3′.

Techniques: Microscopy, Expressing, Staining, Control, Western Blot, Mutagenesis

Journal: Cellular signalling

Article Title: Nuclear Lamins and Progerin Are Dispensable for Antioxidant Nrf2 Response to Arsenic and Cadmium

doi: 10.1016/j.cellsig.2017.02.012

Figure Lengend Snippet: A) 30 μg of nuclear extracts from SW480 cells treated with 50 μM sodium arsenite (As) or 50 μM cadmium chloride (Cd) for 2.5 hr, 5 hr, or 8 hr were loaded on 10% SDS-PAGE and expression of Nrf2 and lamins were analyzed by Western blotting. Cleaved lamins A, B1, and B2 are indicated by arrows. Expression of Histone H1 is shown as a loading control of each sample. B) N-terminal or C-terminal epitopes of lamin antibodies used in Fig. 1A are indicated with thick arrows. The positions of conserved Aspartic acid in human lamins known to be cleaved by Caspase 6 are shown. The total amino acid numbers of human mature lamin A, C, B1, and B2 are also shown at their C-terminus. C) 30 μg of nuclear extracts from SW480 cells treated with 50 μM sodium arsenite, 50 μM cadmium chloride, 10 μM and 25 μM cisplatin, 25 μM etoposide, and 100 μM H2O2 for 5 hr or 8 hr were loaded on 10% SDS-PAGE and expression of Nrf2 and lamin A/C were analyzed by Western blotting. The arrow indicates the cleaved lamin A. D) 10 μg of the cytoplasmic and nuclear fractions of human HGPS fibroblasts (AG11498) treated with 25 μM tBHQ or 10 μM sodium arsenite (As) for 22 hr, 25 μg of cadmium chloride-treated SW480 nuclear fractions (used in Fig. 1C), and 10 μg of the nuclear fraction of another HGPS fibroblasts (AG06917) were loaded on 10% SDS-PAGE and the cleaved lamin A (arrow) and progerin were detected by Western blotting.

Article Snippet: 100–300 pmol of non-targeting siRNA or 100 pmol each of siRNA (all from Qiagen) for lamin A/C, lamin B1, and/or lamin B2 were electroporated into 100 μl suspension of SW480, HaCaT, or K562 cells in siRNA transfection medium (Santa Cruz Biotechnology) using Gene Pulser X-Cell (BioRad) and incubated at room temperature for 10 min.

Techniques: SDS Page, Expressing, Western Blot, Control

Journal: Cellular signalling

Article Title: Nuclear Lamins and Progerin Are Dispensable for Antioxidant Nrf2 Response to Arsenic and Cadmium

doi: 10.1016/j.cellsig.2017.02.012

Figure Lengend Snippet: A) HaCaT cells were transfected with non-targeting control siRNA or 3 different lamin A/C siRNAs (#9, 10, and 13: Qiagen), treated with 50 μM sodium arsenite for 3 hr, and 15 μg of whole cell extracts were loaded on 10% SDS-PAGE to measure expression levels of Nrf2, HMOX1, and NQO1 by Western blotting. Lamin A/C Western blot for the assessment of knockdown is also shown. B) HaCaT cells were transfected with non-targeting control siRNA or a mixture of lamin B1 and B2 siRNAs, treated with 50 μM sodium arsenite or 50 μM cadmium chloride for 4 hr, and 10 μg of cytoplasmic and nuclear extracts were loaded on 10% SDS-PAGE to measure expression levels of Nrf2 and HMOX1 by Western blotting. Western blots for lamins are shown for the assessment of lamin B1 and B2 knockdown. Quantification of Nrf2 and HMOX1 bands was analyzed with Image J software. Relative density after normalization to corresponding β-actin band is shown in parentheses. SiControl/arsenite(As) signal band was defined as 1.0. Representative Western blots from four experiments are shown.

Article Snippet: 100–300 pmol of non-targeting siRNA or 100 pmol each of siRNA (all from Qiagen) for lamin A/C, lamin B1, and/or lamin B2 were electroporated into 100 μl suspension of SW480, HaCaT, or K562 cells in siRNA transfection medium (Santa Cruz Biotechnology) using Gene Pulser X-Cell (BioRad) and incubated at room temperature for 10 min.

Techniques: Transfection, Control, SDS Page, Expressing, Western Blot, Knockdown, Software

Journal: Cellular signalling

Article Title: Nuclear Lamins and Progerin Are Dispensable for Antioxidant Nrf2 Response to Arsenic and Cadmium

doi: 10.1016/j.cellsig.2017.02.012

Figure Lengend Snippet: A) HaCaT or B) SW480 cells were transfected with non-targeting control siRNA or a mixture of lamin A/C, B1 and B2 siRNAs, and treated with 50 μM sodium arsenite or 50 μM cadmium chloride for 4 hr (A) or 4hr and 8 hr (B). 15 μg of cytoplasmic and nuclear extracts in A) or only nuclear extracts in B) were loaded on 10% SDS-PAGE to measure expression levels of Nrf2 and HMOX1 by Western blotting. Western blots for lamins are shown for the assessment of each lamin knockdown. Quantification of Nrf2 and HMOX1 bands was analyzed with Image J software. Relative density after normalization to corresponding β-actin band is shown in parentheses. SiControl/arsenite(As) signal band was defined as 1.0. Representative Western blots from four experiments are shown.

Article Snippet: 100–300 pmol of non-targeting siRNA or 100 pmol each of siRNA (all from Qiagen) for lamin A/C, lamin B1, and/or lamin B2 were electroporated into 100 μl suspension of SW480, HaCaT, or K562 cells in siRNA transfection medium (Santa Cruz Biotechnology) using Gene Pulser X-Cell (BioRad) and incubated at room temperature for 10 min.

Techniques: Transfection, Control, SDS Page, Expressing, Western Blot, Knockdown, Software