Stem Cell Reports, Volume 2 Supplemental Information Involvement of ER Stress in Dysmyelination of Pelizaeus-Merzbacher Disease with PLP1 Missense Mutations Shown by ipsc-derived Oligodendrocytes Yuko Numasawa-Kuroiwa, Yohei Okada, Shinsuke Shibata, Noriyuki Kishi, Wado Akamatsu, Masanobu Shoji, Atsushi Nakanishi, Manabu Oyama, Hitoshi Osaka, Ken Inoue, Kazutoshi Takahashi, Shinya Yamanaka, Kenjiro Kosaki, Takao Takahashi, and Hideyuki Okano Inventory of Supplemental Information Supplemental Figures Figure S1. Reproducible novel protocol (Related to Figure2) Figure S2. No compensatory proliferation of OPCs (Related to Figure5) Figure S3. Myelinating neurites in vitro (Related to Figure6) Supplemental Tables Table S1 Primers used for direct sequencing (Related to Figure1) Table S2 Primers used for pyrosequencing (Related to Figure1) Table S3 Primers used for qpcr (Related to Figure1, 2,4 and S1)
FigureS1 Numasawa et al.
FigureS2 Numasawa et al.
FigureS3 Numasawa et al.
FIGURE LEGENDS Figure S1. The expression of cell type specific markers during differentiation (Related to Figure 2) A. Quantitative RT-PCR analysis of the expression of cell type specific markers in each differentiation stage. Expression profiles were similar among the control (201B7, WD39, and TIG121), PMD1 (PMD1-7, 15, and 27), and PMD2 (PMD2-6, 10, and 22) (n=3, mean ± SEM, independent experiments). B. Quantitative RT-PCR analysis of differentiated neurospheres for the markers of neurons ( III tubulin), astrocytes (GFAP), and oligodendrocytes (CNP). The expression profiles were similar among control (201B7, WD39, TIG121), PMD1 (PMD1-7, 15, 27) and PMD2 (PMD2-6, 10, 22) (n=3, mean ± SEM, independent experiments). Figure S2. The expression of proliferative and apoptotic markers in oligodendrocyte cultures (Related to Figure 5) A. Representative image of immunocytochemistry of KI67 and OLIG2. Scale bar, 100 μm. B. Quantitative analysis of the number of KI67 + cells and OLIG2 + cells. The proportion of OLIG2 + cells and KI67 + /OLIG2 + cells were unchanged between the control (201B7, WD39, and TIG121), PMD1 (PMD1-7, 15, and 27), and PMD2 (PMD2-6, 10, and 22; n=3, mean ± SEM, independent experiments, t-test). C. Representative image of immunocytochemistry for cleaved caspase-3 and neuronal marker ( III tubulin). No apoptosis in neurons derived from either control or PMD ipscs was detected. Scale bar, 50 μm. Figure S3. Myelinating neurites in vitro (Related to Figure 6) A. Representative image of immunocytochemistry for neurofilament (NF200) and MBP. Some parts of the neurofilament-positive neurites were wrapped by MBP-positive processes of oligodendrocytes. Scale bar, 5 μm. SUPPLEMENTAL TABLES Table S1. Primers used for direct sequencing (Related to Figure 1) Temp. Product size Sense Antisense 5 UTR 63 436 GCTGCATATCCCACACCAATT GCTGGCTAGTCTGCTTTGTG Exon1 55 349 CAGTGAAAGGCAGAAAGAGA CTGTGTCCTCTTGAATCTTC Exon2 63 389 ATCTCCGAGCCTGTGAGCAC CCACCCCAGTCCCCTGCTAG Exon3 55 470 TGTATACCTCACTTATGTCGGG CGCTTCCTCACTCAAATGCCAG Exon4 60 472 CCACTCCAGGATCTCCCAG GCCAATGTGAATGGCCAAGAA Exon5 55 315 TAGAGATGGAAGAAGGGCTC AGGCACACTTAGCCAACATG Exon6 55 264 AAAGATATCAACACATTCAG TCAAGGATGGAAGCAGTCTA Exon7 55 305 ATCGGACTTCCAGTCAAGTG TGTGGTTAGAGCCTCGCTAT Temp: annealing temperature
Table S2. Primers used for pyrosequencing (Related to Figure 1) Temp. Product size Sense Antisense Sequencing primer PMD1(c.757 T>A) 56 56 ATGACCTTCCACCTGTTTATTGCT TTGCCTTTCAGAATAGCTGTACC CTGCAGCTACACTGGTT PMD2(c.643 C>T) 211 134 GAATCCAACTTTACAGCCAGGATA AACCCAAATAACCCACTCACCTC TTCTCCCATGGAATGCTTTC Temp: annealing temperature Table S3. Primers used for RT-qPCR (Related to Figures 1, 2, 4, and S1) Temp. Product size Sense Antisense SOX2 tg 58 270 ACGGCCATTAACGGCACACTG CCCTTTTTCTGGAGACTAAATAAA OCT4 tg 58 227 TCTGGGCTCTCCCATGCATTCAAAC CCCTTTTTCTGGAGACTAAATAAA KLF4 tg 58 266 CACCTCGCCTTACACATGAAGAG CCCTTTTTCTGGAGACTAAATAAA c-myc tg 58 262 CTTGAACAGCTACGGAACTCTTG CCCTTTTTCTGGAGACTAAATAAA -ACTIN 62 180 GATCAAGATCATTGCTCCTCCT GGGTGTAACGCAACTAAGTCA NANOG 62 218 CCTATGCCTGTGATTTGTGG TGTTTCTTGACTGGGACCTTG SOX1 62 285 GAAATAGCCAATGCCAGGTG CCGTGAATACGATGAGTGTTACC BRACHYURY 62 274 GCCCTCTCCCTCCCCTCCACGCAGAG CGGCGCCGTTGCTCACAGACCACAGG SOX17 62 215 TGTCCCAAAACAGCTTCCTC TCACCCTTTTCGAGGATGAG IIItubulin 62 289 CATTCTGGTGGACCTGGAAC ATACTCCTCACGCACCTTGC GFAP 62 291 ACATCGAGATCGCCACCTAC CGGAGCAACTATCCTGCTTC CNP 62 231 AAGGAGAAGAACCAGTGGCA CAAGTCCATCTTCTCCCTGG BIP 62 73 TGTTCAACCAATTATCAGCAAACTC TTCTGCTGTATCCTCTTCACCAGT CHOP 62 67 AGAACCAGGAAACGGAAACAGA TCTCCTTCATGCGCTGCTTT Spliced XBP1 62 59 CTGAGTCCGCAGCAGGTGCAG ATCCATGGGGAGATGTTCTGG Temp: annealing temperature