1. Volume 2, Issue 1, Pages 1-9 (July 2012)
Unique Preservation of Neural Cells in Hutchinson- Gilford Progeria Syndrome Is Due to the Expression of the Neural-Specific miR-9 MicroRNA 
Xavier Nissan, Sophie Blondel, Claire Navarro, Yves Maury, Cécile Denis, Mathilde Girard, Cécile Martinat, Annachiara De Sandre- Giovannoli, Nicolas Levy, Marc Peschanski 
Cell Reports 
Volume 2, Issue 1, Pages 1-9 (July 2012)
DOI: /j.celrep
Copyright © 2012 The Authors Terms and Conditions

2. Cell Reports 2012 2, 1-9DOI: (10.1016/j.celrep.2012.05.015)
Copyright © 2012 The Authors Terms and Conditions

3. Figure 1 Molecular Characterization of Control and HGPS iPSC
(A) qRT-PCR analysis of pluripotency and self-renewal markers OCT4, LEFTB, NANOG, CRIPTO, DNMT3B in control and HGPS fibroblasts and iPSC. Data are normalized on teratocarcinoma cells (EP2102). Each histogram represents the means ± SD of three independent experiments.
(B) Flow cytometry analysis of pluripotency and self-renewal markers TRA 1-81 and SSEA4 in fibroblasts in control and HGPS fibroblasts and iPSC. Values indicate means ± SD of three independent experiments.
(C) Immunostaining of lamin A/C (JOL2 and ab8984 antibodies) and lamin B1 and pluripotency and self-renewal markers OCT4, TRA 1-81, and SSEA4 in HGPS fibrobasts, undifferentiated control and HGPS iPSC. ∗Boxes represent higher magnification. Scale bar is 50 μm.
See also Figure S1.
Cell Reports 2012 2, 1-9DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

4. Figure 2
Characterization of Lamins Expression Profile in iPSC Derivatives
(A) Lamin A/C immunostaining (JOL2 antibody) in HGPS fibrobasts, undifferentiated HGPS iPSC and its derivatives into mesenchymal stem cells (MSC-iPSC), melanocytes (Mel-iPSC), keratinocytes (Ker-iPSC), retinal pigment epithelial cells (RPE-iPSC), astrocytes (Astrocytes-iPSC), neural stem cells (NSC-iPSC), neurons (Neurons-iPSC) and motoneurons (Motoneurons-iPSC). ∗Boxes represent higher magnification. Scale bar is 50 μm.
(B) Flow cytometry analysis of lamin A/C (JOL2 antibody) and lamin B1 expression in fibroblasts, iPSC, MSC-iPSC, NSC-iPSC and Neurons-iPSC. Values indicate means ± SD of three independent experiments.
See also Figure S2.
Cell Reports 2012 2, 1-9DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

5. Figure 3 miR-9 Regulates Lamin A and Progerin Expression
(A) Predictive alignment of microRNAs potentially targeting the 3′ UTR sequence of lamin A using three independent algorithms: TargetScan, miRanda, and miRDB.
(B) qRT-PCR analysis of miR-9 in control and HGPS iPSC, fibroblasts, MSC-iPSC, NSC-iPSC, and Neurons-iPSC. Values represent the means ± SD of three independent experiments.
(C) Western blot analysis of lamin A/C (JOL2 antibody) and β-actin expression in control and HGPS MSC-iPSC transfected with a premiR control (Mock) or premiR-9.
(D) and (E) Quantification by western blot analysis of lamin A/C and β-Actin expression in control and HGPS MSC-iPSC transfected with a premiR control (Mock) or premiR-9. ∗p value < 0.05.
(F) Luciferase activity of control MSC-iPSC transfected or not with lamin A-Luc 3′ UTR plasmid in presence of a premiR control (Mock), premiR-203, anti-miR-9, or premiR-9. ∗p value < 0.05.
(G) Sequences alignment of the normal and mutated lamin A 3′ UTR.
(H) Luciferase activity of control MSC-iPSC transfected or not with the normal or mutated lamin A-Luc 3′ UTR plasmids in presence of a premiR control (Mock) or premiR-9. Values represent the means ± SD of three independent experiments. ∗p value < 0.05.
See also Figure S3.
Cell Reports 2012 2, 1-9DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

6. Figure 4 miR-9 Mediated Improvement of HGPS Nuclear Abnormalities
(A) Lamin A/C immunostaining (JOL2 antibody) in HGPS MSC-iPSC transfected with a premiR control (Mock) or premiR-9. ∗Boxes represent higher magnification. Scale bar is 50 μm.
(B) Quantification of nuclear abnormalities (lamin A/C immunostaining with JOL2 antibody) in HGPS MSC-iPSC transfected with a premiR control (Mock) or premiR-9. Values represent the means ± SD of two hundreds nuclei in three independent experiments. ∗p value < 0.05.
(C) Quantification of nuclear abnormalities HGPS MSC-iPSC transfected with increasing concentrations of premiR-9. Values represent the means ± SD of 200 nuclei in three independent experiments. ∗p value < 0.05.
See also Figure S4.
Cell Reports 2012 2, 1-9DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

7. Figure S1
Molecular Characterization of Mesenchymal Stem Cells Derived from HGPS iPSC, Related to Figure 1
(A) Morphological observations of control and HGPS MSC. Scale bar is 10 μm.
(B) Flow cytometry analysis of pluripotency and self-renewal markers TRA 1-81 and SSEA4 and mesenchymal stem cells markers ALCAM, integrin β1, hyaluronate receptor and SH3/NT5E in MSC derived from HGPS iPSC (HGPS MSC). Values indicate means of three independent experiments.
(C) Immunostaining of lamin A/C (JOL2 antibody) in control and HGPS MSC. Values represent the percentage of abnormal nuclei. Scale bar is 50 μm.
(D) Measure of β-galactosidase activity in control and HGPS MSC. Values represent the means ± SD of three independent experiments.
(E) Measure of Ki67 positives cells in control and HGPS MSC. Values represent the means ± SD of three independent experiments.
Cell Reports 2012 2, 1-9DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

8. Figure S2
Neural Induction of Control and HGPS iPSC, Related to Figure 2
(A) Schematic representation and phenotypical characterization (PAX3/PAX6, NESTIN/PAX6, and TUJ1/lamin B1 immunostaining) of iPSC neural induction process using the dual SMAD inhibition method (Noggin and SB431542). Scale bar is 50 μm.
(B) qRT-PCR analysis of pluripotency and self-renewal (OCT4, LEFTB, NANOG) and neural progenitor markers (SOX2, NESTIN, PAX6, NEUROD1) in control and HGPS undifferentiated iPSC and neural stem cells (NSC-iPSC). Values represent the mean ± SD of three independent experiments.
(C) qRT-PCR analysis of pluripotency and self-renewal (OCT4, NANOG) and neuronal markers (PSD95, Synaptophysin, MAP2, SNAP25) in HGPS undifferentiated iPSC, neural stem cells (NSC-iPSC) and neuronal induced NSC-iPSC after 7, 14 and 21 days. Human fetal brain was used as a positive control of neuronal markers. Values represent the mean ± SD of three independent experiments.
(D) Immunostaining of lamin A, lamin A/C (Abcam antibody) and lamin B1 in control and HGPS NSC-iPSC and in control NSC derived from hESC (H9 and SA01). Scale bar is 50 μm.
Cell Reports 2012 2, 1-9DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

9. Figure S3
Genomic Sequences of Lamin A and C 3′ UTR, Related to Figure 3
(A) Genomic sequences of lamin A 3′ UTR. Red and blue highlights represent the two sites potentially targeted by miR-9.
(B) Genomic sequences of lamin C 3′ UTR.
Cell Reports 2012 2, 1-9DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions

10. Figure S4
Dose-Dependent Regulation of miR-9 on Nuclear Abnormalities, Related to Figure 4
(A) Immunostaining of lamin A/C (JOL2 antibody) representing nuclei considered as normal or affected in this study.
(B) miR-9 expression in untransfected or transfected HGPS MSC with 10 nM, 30 nM, 100 nM or 300 nM of premiR-9. Values are normalized on RNU48 and expressed as a % of untransfected cells.
(C) Immunostaining of lamin A/C (JOL2 antibody) in untransfected or transfected HGPS MSC with 10 nM, 30 nM, 100 nM or 300 nM of premiR-9.
Cell Reports 2012 2, 1-9DOI: ( /j.celrep )
Copyright © 2012 The Authors Terms and Conditions