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. 2016 Jul 11;5(3):31. doi: 10.3390/cells5030031

Figure 2.

Figure 2

Figure 2

Morpholino treatment downregulates aberrant Prelamin A transcripts production without causing aberrant splicing in off-target genes. (A) Upper panels: Semi-quantitative RT-PCR analyses showing Lamin A, Progerin (Prelamin A Δ50), Prelamin A Δ35, β-actin and GAPDH mRNA levels in Hutchinson-Gilford Progeria Syndrome (HGPS) and HGPS-like fibroblasts treated with morpholino antisense oligonucleotides (AON, 20 µM each) relative to Endo-porter (EP, which is the AON vehicle used as Control), scrambled oligonucleotides (Scr) treated cells and untreated cells (NT). Fragments covering all exons of β-actin (3 fragments, 5 exons) and GAPDH (2 fragments, 9 exons) cDNAs were amplified for each couple of AON used. Given that in patients HGPS-L2 and -L3, the same AON couple as in patient HGPS-L1 was used, the splicing study was only partially replicated. Lower panels: Relative levels of Lamin A, Progerin, Prelamin A Δ35 to GAPDH ex7–ex9 and the indicated fragments of β-actin and GAPDH transcripts ratios using Image J software and compared to EP treated cells. (mean ± SEM, n = 4, Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001, AON-treated vs. EP-control). (B) Quantitative RT-PCR analyses of Lamin A, Progerin (Prelamin A Δ50), Prelamin A Δ35, Prelamin A Δ90 (when detected at baseline, in EP assays), Lamin C and GAPDH mRNA levels in HGPS, HGPS-like and Mandibuloacral Dysplasia type B (MAD-B) fibroblasts treated with AON, 20 µM each relative to Endoporter (EP: vehicle used as Control). The fold change of each transcript was determined by normalizing its value to that of GAPDH for each condition. (mean ± SEM, n = 4, Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001, experimental vs. control).