Fig. S1. eif6 expression in HEK293 transfected with shrna against eif6 or pcmv-eif6 vector.

Transcription

1 Fig. S1. eif6 expression in HEK293 transfected with shrna against eif6 or pcmv-eif6 vector. (a) Western blotting analysis and (b) qpcr analysis of eif6 expression in HEK293 T cells transfected with either the control plasmid (Scramble) or shrna against eif6. GAPDH was used as an internal control in Western blotting, while 18s rrna was used as an internal control in qpcr. All the data are presented as the means±sem. *P<0.05.

2 Fig. S2. eif6 expression in HEK293 cells transfected with pcmv-eif6 vector. qpcr analysis of eif6 mrna expression in HEK293 cells transfected with either the control vector (pcmv) or the eif6-overexpression vector (pcmv-eif6). GAPDH was used as an internal control. All the data are presented as the means±sem. *P<0.05.

3 Fig.S3. The association of eif6 and Histone proteins in vitro (a) Western blotting analysis of Histone H2B protein expression in WT (eif6 +/+ ) and eif6 +/- fibroblasts (n=3). Representative images are shown. GAPDH was used as an internal control. (b) qpcr and (c) Western blotting analyses of eif6 and H2A.Z expression in cultured passage 2 fibroblasts isolated from the skin of wild-type and eif6 +/- mice (n=3). β-actin was used as an internal control. All the data are presented as the means±sem. *P<0.05.

4 Fig. S4. Decreased level and altered subcellular localization of eif6 in hypertrophic scars (HS). (a) Western blotting analysis of eif6 expression in paired tissue samples of normal skin (NS) and hypertrophic scars (HS) obtained from patients (n=10). GAPDH was used as an internal control. (b) Immunohistochemical staining of eif6 in the sfb of hypertrophic scar (upper line) and in the normal skin-derived fibroblasts (FB, lower line). Some of the eif6+ cells were identified by arrows. Left panels: scale bar=100µm; right panels, scale bar=50µm. (c) Immunofluorescence of eif6 in the sfb of hypertrophic scar (A-F) and in the normal skin-derived fibroblasts (FB, G-L). The eif6+ cells were identified by white arrows. A-C and G-I: scale bar=100µm; D-F and J-L, scale bar=50µm.

5 Table S1. The primer sequences for amplification of constructs of TGF-β1 promoter Deletion constructs of TGF-β1 promoter ptgf(-1132/+11) ptgf(-731/+11) ptgf(-453/+11) ptgf(-175/+11) ptgf(+11/+812) ptgf(+11/+271) Sequence of Primers F:5- GATTCGACGCGTGGCTGGCCCCGGCTCC -3 R:5-TAGACCAGATCTTGTCTGGCTGCTCCGC-3 F:5-GATTCGACGCGTCTAGAGACTGTCAGAG-3 R:5- TAGACCAGATCTTGTCTGGCTGCTCCGC-3 F:5- GATTCGACGCGTGACAGACCCTCCTTCT-3 R:5- TAGACCAGATCTTGTCTGGCTGCTCCGC-3 F:5-GATTCGACGCGTCGCCCACGCGAGATGA-3 R:5- TAGACCAGATCTTGTCTGGCTGCTCCGC-3 F:5-GATTCGACGCGTGCGGAGCAGCCAGACA-3 R:5- TAGACCAGATCTGAGCGCGAACAGGGC-3 F:5-GATTCGACGCGTGCGGAGCAGCCAGACA-3 R:5-TAGACCAGATCTCAACGGAAAAGTCTCA-3

6 Table S2. Sequences of each primer pair for qpcr used in this study Primers used for quantitative real-time PCR analysis Gene Forward primer Reverse primer Mouse Tgfb1 5'-CCGCAACAACGCCATCTATG-3' 5'-CTCTGCACGGGACAGCAAT-3' Mouse Gapdh 5'-CGTGCCGCCTGGAGAAAC-3' 5'-AGTGGGAGTTGCTGTTGAAGTC-3' Mouse Acta2 5'-CGTACAACTGGTATTGTGCTGGAC-3' 5'-TGATGTCACGGACAATCTCACGCT-3' Mouse Col1a1 5'-TTCTCCTGGCAAAGACGGACTCAA-3' 5'-AGGAAGCTGAAGTCATAACCGCCA-3' Mouse Col1a2 5'-AGGCGTGAAAGGACACAGTGGTAT-3' 5'-TCCTGCTTGACCTGGAGTTCCATT-3' Mouse Tgfbr2 5'-GACTGTCCACTTGCGACAAC-3' 5'-GGCAAACCGTCTCCAGAGTAA-3' Human TGFB1 5'-TGGAAACCCACAACGAAATCTATGA-3' 5'-TGGAAACCCACAACGAAATCTATGA-3' Human COL1A1 5'-TCCCACCAATCACCTGCGTACA-3' 5'-CGCCGGTGGTTTCTTGGTCG-3' Human ACTA2 5'-CGGCTTTGCTGGGGACGAT-3' 5'-CAGGGGCAACACGAAGCTCAT-3' Human GAPDH 5'-GGGGAAGGTGAAGGTCGGAGTC-3' 5'-TCGCTCCTGGAAGATGGTGATG-3'

7 Table S3. The bisulfite sequencing PCR primer sequences for amplification of TGF-β1 promoter Primer Names Sequence of Primers Product (bp) Ta( ) TGFB1-1-F TGFB1-1-R TGFB1-2-F TGFB1-2-R TGFB1-3-F TGFB1-3-R TTTATTGAATTAYGGGGTAGAAA TCCAAAAAAAATCCCTTCA GTAGGGTTGAAGGGATTTT AATACCAAAAAATCCCCAC GTTTTGAGATTTTTTYGTTGTTAT TCTCAATATACCACCAAAATAAAAA

8 Table S4. The PCR primer sequences of TGF-β1 promoter for H2A.Z and Sp1 binding assays Sequence Definition Sense Primer Anti-sense Primer PCR product region Product Length SP1-1-P1 GCCCACGCTAAGATGAAG CTGGCTGTCTGGAGGATC -120~ SP1-2-P1 CGGGGCGGCTTCAAAACC CTGGCTGTCTGGAGGATC -37~ SP1-3-P1 CCCGCCCACGCTAAG CCCACCCAGGAAGCG -155~ H2AZ-1-P1 CCACGCTAAGATGAAGACAG CTCCTCCCACTCCTCCTC -118~ H2AZ-2-P1 GCCCACGCTAAGATGAAG CTGGCTGTCTGGAGGATC -120~ H2AZ-3-P1 CGGGGCGGCTTCAAAACC CCTCGGCTGCTCCTTTGC -37~