Fig. 2: Expression of progeria and SMC markers in HGPSΔ2-iPSC SMCs.
From: Vulnerability of progeroid smooth muscle cells to biomechanical forces is mediated by MMP13
a gRNA directs Cas9 nuclease against mutated exon 11 of LMNA gene, upstream the HGPS mutation, disrupting progerin, without altering lamin A and lamin C. Sanger sequencing for LMNA (NM_170707.4 transcript) exon 11 was performed for: N-iPSCs, HGPS-iPSCs and HGPSΔ2-iPSCs, confirming the deletion of two-base pairs in the HGPSΔ2-iPSCs. b Expression of lamin A, progerin, and SMC proteins monitored by immunofluorescence. Scale bar is 100 μm. n = 6 independent experiments. c Expression of progerin (LMNA G608G gene) in HGPS and HGPSΔ2 cell lines. Results are mean ± SEM (n = 4 technical replicates from a pool of three independent experiments). Statistical analyses were performed by a two-tailed unpaired Student’s t test. d Quantification of lamin A, progerin, dysmorphic nuclei, and nuclei blebbing. Results are mean ± SEM (n = 6 independent experiments). **** denotes statistical significance (p < 0.0001). e Percentage of cells that have been differentiated from HGPSΔ2-iPSCs that express SMC markers at protein level. Results are mean ± SEM (n = 5–6 independent experiments). f Number of cells per surface (mm2) as quantified by high-content microscopy (at least three images (×10) have been quantified per time). The number of cells was evaluated after 6 days under arterial flow and was normalized by the number of cells present at day 0. n > 3 images examined over three independent experiments. g MMP13 mRNA transcripts quantified by qRT-PCR analyses in HGPS-iPSC SMCs or HGPSΔ2-iPSC SMCs cultured under flow conditions. MMP13 mRNA transcripts were normalized by GAPDH. n = 4 technical replicates from a pool of three independent experiments. **, *** denote statistical significance (p < 0.01, p < 0.001). Statistical analyses were performed by a two-tailed unpaired Student’s t test.
