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. 2014 Aug 5;111(34):12426–12431. doi: 10.1073/pnas.1413299111

Fig. 6.

Fig. 6.

Loss of function experiments to test roles of KLF4 and LTR7s in reprogramming and the DD phenotype. (A) KLF4 is responsible for HERV-H expression. Shown are relative expressions of HERV-H in DD-iPSCs (D) transduced with KLF4 shRNA (shKLF4), LTR7 shRNA-1 (shLTR7-1), or shRoR, and normal iPSCs (N) compared with those of Mock-transduced DD-iPSCs. Each value was normalized to that of G3PDH. n = 3. Error bars are SDs. n = 3. *P < 0.05 vs. Mock was calculated by Dunnett test. (B) Knockdown of LTR7 expression. Bars show relative expression of ABHD12B, HHLA1, C4ORF51, lincRNA-RoR, and NANOG in normal iPSCs (N) and DD-iPSCs (D) transduced with empty vector (Mock), LTR7 shRNA-encoding vectors (shLTR7-1 and -2), or shRoR compared with Mock analyzed by microarray. Error bars are SDs. N =2. *P < 0.05 vs. Mock was calculated by Dunnett test. (C) Suppression of KLF4/HERV-H LTR7 rescues the DD phenotype. Shown are the relative proportions of residual TRA-1-60 (+) cells on day 14 after neural differentiation of DD-iPSCs (D) carrying empty vector (Mock), LTR7 shRNAs (shLTR7-1), or shRoR, compared with normal iPSCs (N). n = 3. *P < 0.05 vs. Mock was calculated by Dunnett test. (D) Suppression of LTR7 rescues the KLF4-induced DD phenotype. Shown are the percentages of residual TRA-1-60 (+) cells on day 14 after neural differentiation of normal iPSCs carrying dox-inducible KLF4, and empty vector or LTR7 shRNA (shLTR7-1). Differentiation was performed in the presence (+) or absence (−) of Dox. Error bars are SDs. n = 2. (E) LTR7 activity enhances reprogramming efficiency. Shown are the percentages of TRA-1-60 (+) cells on days 7 (black) and 11 (green) posttransduction of OSKM with empty vector (Mock), LTR7 shRNA-encoding vectors (shLTR7-1 and -2), or shRoR vector. n = 3. Error bars are SDs. *P < 0.05 vs. Mock was calculated by Dunnett test. (F) LTR7 activity facilitates iPSC generation. Shown are the relative numbers of iPSC colonies on day 25 posttransduction of OSKM with empty vector (Mock), LTR7 shRNA-encoding vectors (shLTR7-1 and -2), or shRoR vector. Error bars are SDs. n = 4. *P < 0.05 vs. Mock was calculated by Dunnett test.