Inventory of Supplemental Information presented in relation to each of the main figures in the manuscript 1. Figure S1. Related to Figure 1. 2. Figure S2. Related to Figure 2. 3. Figure S3. Related to Figure 3. 4. Figure S4. Related to Figure 4. 5. Figure S5. Related to Figure 5. 6. Figure S6. Related to Figure 6. 7. Figure S7. Related to Figure 7. 8. Table S1. Related to Figures 1 and S1. 9. Table S2. Related to Figures 3, 4, 6 and 7. 10. Table S3. Related to Figure 3. Supplemental Experimental Procedures 1. Patients and samples 2. Cell culture 3. Long term culture-initiating cells (LTC-IC) assay 4. Differentiation of CD34+ cord blood cells 5. Bi-phasic erythroid differentiation assay 6. Clonal assay 7. Mutation and sequencing analysis 8. Bisulphite sequencing and quantitative pyrosequencing 9. Human SNP genotyping 10. Strand-specific cDNA synthesis and PCR 11. Chromatin Immuno-precipitation (ChIP) 12. Immunoprecipitation and western blot 13. Colony genotyping 14. shRNA generation and viral infection 15. Retrovirus generation and transduction 16. Study approval 17. Statistical Methods 18. Primers list Supplementary References (19) 1 Additional SGK2 families 2 3 4 ♂ ♀ ♂ ♀ ♂ ♀ C/G C/C C/C C/G C/C C/G ♀ ♀ ♂ ♀ C/G C/G C/G C/G T T C T G C T A G A T T C T C C T A G A T T C T C C T A G A T T C T C C T A G A T-Cells T-Cells T-Cells T-Cells Additional GDAP1L1 familiy 2 ♂ ♀ G/G A/A ♀ G/A A C A C G G T G Erythroblasts Figure S1 - . )4126 ;-,,-</ L%)K$L( O"I' "=#+(L( L%)K$L # # # # " " ! ! !! # " $"" # ! ! ! ! $ # ! $ " ! !# $ ! # # ! " " # $ " " " "6@4:7EG=H# "6@4:7EG=H# ?6-JGE=H# K3-7@GE=H# K)GE=H# *4,NGO-EGE=H# +K)H!GE=H# / )-E-F16 L%)K$L( O"I' PBQPR )( PBQPR )( # # # # ! $ " "" ! ! $ " "" ! #!!! " $ " $ # !!! " $ " $ ! ! 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"UT !I&JCKL3@ !I&JCKL3@ /++P 0,JCKL3@$ !$JCKL3@$ W VV RQP VV VV VV W Q+P $QP ;SGR/;"T@ +P !I&JCKL3@ !I&JCKL3@ W 0,JCKL3@$ !$JCKL3@$ -+P VV DQP VV W ,+P VV VV /QP SGD/;SG-/ +P D E /$ D E /$ 285);FG6'CH , M1@ M11 - *=NO 01@;4? ; > 7)& ; >=<%;:)98 765&43 D D 01@;4?D ;)A8 ?67)& ;B48CC)&=<% , , , $ $ $ / / / + + + !I&JCKL3@0,JCKL3@/!$JCKL3@/ !I&JCKL3@0,JCKL3@/!$JCKL3@/ !I&JCKL3@0,JCKL3@/!$JCKL3@/ !"#$%&'() A Phase&I&Culture + + + GFP GFP&CFP ScrAshRNA ScrAshRNA ScrAshRNA Day&0 Day&14 L3AshRNA1 S2AshRNA1 DiAshRNA 100% + 75% 50% CD71 Phase&I 25% CD71 0% GPA Phase&II&Culture Day&2 Day&14 100% + 75% 50% GPA 25% Phase&II 0% CD71 2 6 10 14 2 6 10 14 2 6 10 14 GPA Time&(Days) Phase&I + + + + + B GFP&CD71 GFP&CFP&CD71 40% * ** ScrAshRNA L3AshRNA1 S2AshRNA1 DiAshRNA 35% * * 13.8 27.3 20.5 32.1 20% 1.37 4.01 1.81 6.31 10% SAPhase&cells 84.1 67.9 77.1 61.1 BrdU 0% 7AAD L3+S2 rAshRNA AshRNA1 c 3 2AshRNA1 AshRNA Phase&I + + + + + S L S C GFP&CD71 GFP&CFP&CD71 16% ScrAshRNA L3AshRNA1 S2AshRNA1 DiAshRNA + 88.1 6.7 88.4 2.5 76.5 12 85.8 9.3 12% 8% + 4% 4.5 0.2 9.0 0.1 11.1 0.4 4.9 0 7AAD&Anx&V& 0% Annexin&V 7AAD L3+S2 rAshRNA AshRNA1 c 3 2AshRNA1 shRNA S L S 0.0M&5AAC D E F G 0.10 0.8 L3MBTL1 EV L3MBTL1 0.3 HBA 0.2M&5AAC 0.08 0.5M&5AAC 0.6 ABL 0.2 0.06 0.4 0.04 0.1 0.2 0.02 Exp.&rel.&to& Exp.&rel.&to&ABL Exp.&rel.&to&ABL 0 0.0 0.00 L3MBTL1 SGK2 H 0.20 SGK2 SGK2 L3MBTL1&& 0.5 HBB 150 Number&of&cells SGK2 0.15 120 0.3 90 0.10 60 0.05 0.1 GPA&(MFI) 30 Exp.&rel.&to&ABL Exp.&rel.&to&ABL 0.00 0.0 0 EV M M M K562HEL GPA 0 SGK2 SGK2 0.2 0.5 L3MBTL1 Uninduced& Uninduced A UninducedL3MBTL1 Uninduced GFP ! Empty&vector 50M&Hemin + 50M& L3MBTL1&+&SGK2 GFP L3MBTL1&&&SGK2 &&+&5AAC + + Hemin 50M&Hemin GFP&CFP induction &induction 50M&Hemin &induction &&induction Figure(S6 ((Page(1 I CFU5GM 80 5 BFU5E 40 CFU5GEMM 4 60 30 3 40 20 2 20 1 10 0 0 0 Colonies/1000/cells L3+S2 L3+S2 L3+S2 r5shRNA 5shRNA1 r5shRNA 5shRNA1 r5shRNA 5shRNA1 c 25shRNA1 5shRNA c 25shRNA1 shRNA c 25shRNA1 shRNA S L3 S S L3 S S L3 S J K 15 CFU5S 150 L3mbtl1 Sgk2 120 12 90 9 60 6 30 3 0 Colonies/spleen 0 relative/to/GapdH Normalised/expression 5shRNA 5shRNA5shRNA r5shRNA 25shRNA cr 25shRNA Uninfectedc L3 S S L3 S mL3+mS2 S m m m m Scr25shRNA shRNA L M 150 L3mbtl1 H LSK d 2.0 2.5 CMP p 120 a G 90 2.0 / 1.5 t o t s / 1.5 o e 60 v 1.0 i H t + 1.0 a r l 30 e o r t 0.5 / i t 0.5 n 0 e o i p s 0.0 0.0 s 150 Sgk2 m e o r p 120 C 2.0 GMP MEP / x r e / o d 90 n 1.5 e o s i D l 60 a 1.0 m r 30 o N 0 0.5 0.0 5shRNA5shRNA5shRNA5shRNA cr 3 2 2 25shRNA L S cr S S m + m S 3 L 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA m r 3 2 2 25shRNA r 3 2 2 25shRNA c L S r S c L S r S S c + S c + m m S 3 m m S 3 L L N m m + + + 2.0 BM/B220 BM/CD71 BM/Mac51 1.5 t s o 1.0 H + r o t 0.5 i t e p 0.0 m o Spleen/B220+ + + C 1.5 Spleen/Mac51 Thymus/CD3 / r o n o 1.0 D 0.5 0.0 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA 5shRNA r 3 2 2 25shRNA r 3 2 2 25shRNA r 3 2 2 25shRNA c L S r S c L S r S c L S r S S c + S c + S c + m m S 3 m m S 3 m m S 3 L L L m m m Figure*S6 **Page*2 A 12 MYC 12 ABL 8 8 4 4 Exp.@rel.@ 0 0 L3+S2 L3+S2 )shRNA )shRNA Scr)shRNA Scr)shRNA L3)shRNA2 S2)shRNA3 L3)shRNA1S2)shRNA2 B ph)S2)PolII 3000 6000 2000 4000 1000 2000 0 0 Fold@enrch.@rel.@to@IgG L3+S2 L3+S2 )shRNA Scr)shRNA )shRNA Scr)shRNA L3)shRNA1S2)shRNA1 L3)shRNA1S2)shRNA2 C H3K4me3 2500 3000 2000 2400 1500 1800 1000 1200 500 600 0 0 Fold@enrch.@rel.@to@IgG L3+S2 L3+S2 Scr)shRNA )shRNA Scr)shRNA )shRNA L3)shRNA1S2)shRNA1 L3)shRNA1S2)shRNA1 D L3MBTL1 250 200 200 160 150 120 100 80 50 40 0 0 Fold@enrch.@rel.@to@IgG L3+S2 L3+S2 )shRNA )shRNA Scr)shRNAL3)shRNA1 S2)shRNA1 Scr)shRNAL3)shRNA1 S2)shRNA1 E BRG1 1000 2500 800 2000 600 1500 400 1000 200 500 0 0 Fold@enrch.@rel.@to@IgG L3+S2 L3+S2 )shRNA )shRNA Scr)shRNAL3)shRNA1S2)shRNA1 Scr)shRNAL3)shRNA1S2)shRNA1 F BRG1 900 750 600 500 300 250 0 0 Fold@enrch.@rel.@to@IgG SGK2 SGK2 Empty@vector Empty@vector Figure'S7 Figure S1.