Lamin A/C expression silencing leads to transformation.

<p><b>(A)</b> Lamin A/C was monitored by WB after shRNA depletion by SK-N-SH-lamin-A/C-shRNA or SK-N-SH-scramble-shRNA. Actin was used as a loading control. <b>(B)</b> Cumulative cell numbers of SK-N-SH-lamin-A/C-shRNA and SK-N-SH-scramble-shRNA, respectively. Three independent experiments were performed in triplicate (n = 9) using cells at less than eight passages, and error bars represent the s.d. Statistical significance was assessed using Student’s t-test;(p<0.01). Representative images showing differential cell growth on day 8 of the experiment. Scale bar, 100μm. <b>(C)</b> Wounding assay of confluent cell layers of SK-N-SH-lamin-A/C-shRNA or SK-N-SH-scramble-shRNA.Number of cells that migrated into a delimited wound area after 12h is plotted. Cells in three defined area per group per experiment were quantified in three independent experiments with three technical replicates. Error bars represent the s.d. Statistical significance was assessed using Student’s t-test; (*p<0.01). Representative images; scale bar, 100μM. <b>(D)</b> Quantification of the Matrigel chamber migration assay for SK-N-SH-lamin-A/C-shRNA and SK-N-SH-scramble-shRNA. Error bars represent the s.d. (n = 9). Statistical significance was assessed by Student’s t-test; (**p<0.01). Scale bar, 10μM. <b>(E)</b> SK-N-SH-lamin-A/C increased the number of colonies in soft agar compared with the SK-N-SH-scramble-shRNA control. The number of colonies per well was counted and plotted. Three independent experiments (n1 = 3) with 3 replicates per experiment (n2 = 9) were performed. Error bars represent the s.d. (n = 9). Statistical significance was assessed using Student’s t-test (**p<0.01). Scale bar, 100μM.</p

Silencing of Lamin A/C in unmethylated neuroblastoma cells induces changes in different cytoskeletal components.

<p>Immunofluorescence staining showing changes in SK-N-SH-lamin-A/C-shRNA compared with SK-N-SH-scramble-shRNA in <b>(A)</b> β-actin filaments, <b>(B)</b> F-actin filaments. <b>(C)</b> Vimentin filaments, and <b>(D)</b> α-tubulin. Lamin A/C is shown in green; β-actin, F-actin, vimentin, and α-tubulin are shown in red.DNA is stained in blue (DAPI). Scale bar, 10μM.</p

Methylation analysis of the Lamin A/C promoter.

<p><b>RNA and protein analysis (A)</b> Schematic of the Lamin A/C CpG Island around the transcription start site (TSS) (long black arrow). CpG dinucleotides are represented as black bars.The locations of bisulphite genomic sequencing PCR primers and methylation-specific PCR primers are indicated as white and black arrows, respectively. The bisulphite sequencing PCR (BSP)results are shown for 10 individual clones for each cancer cell line studied and primary normal human fetal brain neuronal progenitor cells(PNP) cells and IVD αs negative and positive controls, respectively.Two chromatograms are shown to compare one methylated and one unmethylated cell line. <b>(B)</b> Methylation-specific PCR (MSP) of the Lamin A/C gene in neuroblastoma cell lines. The presence of a PCR band under lane M indicates methylated genes, while the presence of a PCR band under lane U indicates unmethylated genes. <b>(C)</b> RT-PCR analysis of Lamin A/C expression. <b>(D)</b> Treatment with the demethylating agent 5-aza reactivated Lamin A/C gene expression in the methylated neuroblastoma SK-N-DZ cell line at the RNA level. <b>(E-F)</b> Treatment with 5-aza-2’ deoxycytidine (5-aza) reactivated Lamin A/C gene expression in the methylated neuroblastoma SK-N-DZ cell line at the protein level evaluated by WB and IF, respectively. GAPDH and actin were used as loading controls for RNA and protein expression, respectively. Lamin A/C is observed in green and DNA in blue (DAPI). Scale bar, 10μM.</p

Progerin introduction in SK-N-DZ cells induces changes in cytoskeletal components and mechanical properties.

<p><b>(A)</b> Immunofluorescence staining showing Progerin. <b>(B)</b> Changes in β-actin, F-actin, vimentin filaments, and α-tubulin after Progerin introduction. Progerin is shown in green; β-actin, F-actin, vimentin, and α-tubulin are shown in red; and DNA is stained in blue (DAPI). Scale bar, 10μm. <b>(C)</b> Representative AFM images of height measurement in SK-N-DZ-ev cells. In red and blue height profile across lines in different cells from height AFM image are shown Scale bar, 10μm <b>(D)</b>The AFM tip is positioned directly above the lamellar region. Scale bar 10μm <b>(E)</b> Normalized histogram of the data obtained from both groups for the lamellar region. Each histogram was fitted to a Gaussian curve to obtain the mean value and the standard deviation of Young’s Modulus. Three independent experiments (n1 = 3) with 3 replicates per experiment (n2 = 9) were performed. A Student’s t-test confirmed that the difference was significant (**p<0.01).</p

Lamin A/C reintroduction induces reorganizational changes in the different cytoskeletal components.

<p>Immunofluorescence staining showing changes in SK-N-DZ-lamin-A/C compared with SK-N-DZ-ev cells in <b>(A)</b> β-actin filaments, <b>(B)</b> F-actin filaments, <b>(C)</b> Vimentin filaments, and <b>(D)</b> α<b>-</b>tubulin. Lamin A/C is shown in green; β -actin, F-actin, vimentin, and α-tubulin are shown in red.DNA is stained in blue (DAPI). Scale bar, 10μM.</p

Methylation analysis of the Lamin A/C gene in neuroblastoma patients.

<p>Degree of methylation in neuroblastoma tumours at Lamin A/C locus is visualized as colour scale from black(very low methylation) to yellow (fully methylated) in a heatmap, where rows represent CpGs represented on the array (with array probe ID) and columns represent patients. As indicated by coloured bars on the right (green/red), the heatmap illustrates the genomic locations of the Lamin A/C gene from top to bottom, while array locations marked in green represent positions upstream of the TSS, and red marked locations represent the coding sequence In addition, the promoter region -37 to +333 is highlighted in blue. The column tree vizualizes the hierachical clustering on patients. On top of the heatmap clinical parameters: risk (white = low risk, yellow = intermediate risk, blue = high risk) and international neuroblastoma staging system (Inns) stage are given (see colour legend for 6 stages).</p

Tumour suppressor like properties after Lamin A/C reintroduction and senescence induction by Progerin introduction in SK-N-DZ cells.

<p><b>(A)</b> Lamin A/C expression was monitored by WB for the SK-N-DZ infected cell line either with Lamin A/C (SK-N-DZ-lamin-A/C) or ev (SK-N-DZ-ev). Unmethylated SK-N-SH cells were used as a positive control. Progerin expression was also monitored by WB for SK-N-DZ infected with Progerin (SK-N-DZ-progerin), SK-N-DZ-lamin-A/C and SK-N-DZ-ev The HGPS cell line was used as a positive control, and LAN-1 was used as a negative control. Actin was used as a loading control. <b>(B)</b> Cumulative cell numbers of SK-N-DZ-lamin-A/C, SK-N-DZ-progerin, and the SK-N-DZ-ev control. Three independent experiments were performed in triplicate (n = 9) using cells at less than eight passages (n = 9). Statistical significance was assessed by One-way ANOVA test; (**p<0.01). The images above show differential cell growth on day 8 of the experiment and show the different morphologies of these cells. Scale bar, 10μM. <b>(C)</b> Wounding assay of confluent cell layers of SK-N-DZ-lamin-A/C, SK-N-DZ-progerin and the SK-N-DZ-ev control. The number of cells that migrated into a delimited wound area after 12h is plotted. Cells in three defined areas per group per experiment were quantified in three independent experiments with three technical replicates. Statistical significance was assessed by One-way ANOVA test; (**p<0.01). Scale bar, 100μM. <b>(D)</b> Quantification of the Matrigel chamber migration assay of SK-N-DZ-lamin-A/C, SK-N-DZ-progerin and the S–KN-DZ-ev control. Three independent experiments (n1 = 3) with 3 replicates per experiment (n2 = 9) were performed. Error bars represent the s.d. (n = 9). Statistical significance was assessed by One-way ANOVA test; (**p <0.01). Scale bar, 10μm <b>(E)</b> Lamin A/C and Progerin expression have a detrimental effect on anchorage-independent transformation. SK-N-DZ-lamin-A/C formed fewer colonies in soft agar compared to the SK-N-DZ-ev control, whilst SK-N-DZ-progerin formed almost no colonies. The number of colonies per well were counted and plotted. Three independent experiments (n1 = 3) with 3 replicates per experiment (n2 = 9) were performed. Error bars represent the s.d. (n = 9); Statistical significance was assessed by One-way ANOVA test; (**p<0.01). Scale bar, 100μm. <b>(F)</b> Quantification of senescence using β-galactosidase staining. Percentage of senescent cells in SK-N-DZ-progerin compared with SK-N-DZ-lamin-A/C and SK-N-DZ-ev. Representative images of β-galactosidase are stained (blue). Three independent experiments with three technical replicates were performed. Error bars represent the s.d. (n = 9) Statistical significance was assessed by One-way ANOVA test; (**p<0.01). Scale bar, 10μM.</p