Fig 2: Gating strategy for quantification of PGCLC population.Representative scatter plots indicating gating strategy to quantify PGCLC population marked by co-expression of SSEA1 and CD61. Gating for live cells was performed using DAPI. Samples without any antibody staining and samples stained with a single antibody were used to gate SSEA1+CD61+ population as shown.

Fig 3: Alkbh5 depletion induces G2/M cell cycle arrest and impairs the MET process. (A) Cell proliferation of reprogrammed MEFs was assessed by FACS measured by BrdU incorporation on day 3 of reprogramming using either scrambled shRNA or two different shRNAs targeting Alkbh5. (B) Quantification of the mean percentage of each of the populations G1, S, and G2/M from FACS data shown in A. (C) Cell proliferation was assessed by counting Alkbh5f/f cells with or without the addition of 1 µM 4-OH Tam for Alkbh5 depletion. (D) Expression of mitotic checkpoint complex (MCC) factors as assessed by qPCR on day 3 of reprogramming using either scrambled shRNA (SCR) or two different shRNAs targeting Alkbh5 (KD1 and KD2). The data were normalized to the level of the housekeeping gene Gapdh. (E) Immunoblot analysis of the protein levels of several cell cycle regulators on day 3 of reprogramming using either scrambled shRNA or two different shRNAs targeting Alkbh5. a-tubulin (A-TUB) was used as a loading control. (F) Expression of mesenchymal and epithelial genes as assessed by qPCR on day 6 of reprogramming after infection either with scrambled shRNA or two different shRNAs targeting Alkbh5. The data were normalized to the housekeeping gene Gapdh. (G) Estimation of E-cadherin (E-cad) and Thy-1-positive populations by FACS in WT and Alkbh5 KO cells on day 6 of reprogramming. (H) Estimation of E-cad- and PDGFRß-positive populations by FACS in WT and KO Alkbh5 MEFs on day 6 of reprogramming. (I) Estimation of E-cad- and SSEA1-positive populations by FACS in WT and Alkbh5 KO MEFs or MEFs infected with either empty vector (EV) or ALKBH5–HA on day 7 of reprogramming. Images in A, E, G–I are representative of three repeats. Quantitative data are shown as the mean±s.d.; n=3. *P<0.05, **P<0.01, ***P<0.001 (paired Student's t-test).

Fig 4: ALKBH5 overexpression enhances reprogramming efficiency. (A) Immunoblot analysis of ALKBH5 protein levels after lentiviral infection of MEFs with empty vector, ALKBH5 or ALKBH5 tagged with HA (ALKBH5–HA). a-tubulin (A-TUB) was used as a loading control. (B) Fraction of ?-PE-Oct4-GFP-positive cells at day 14 of reprogramming of MEFs, MEFs with empty vector (EV) and MEFs overexpressing ALKBH5–HA determined by FACS analysis. (C) Number of ?-PE-Oct4-GFP-positive colonies on day 14 of reprogramming. (D) Fraction of SSEA1-positive cells at day 14 of reprogramming of MEFs, MEFs with empty vector (EV) and MEFs overexpressing ALKBH5 or ALKBH5–HA as determined by FACS. (E) Reprogramming efficiency in MEFs overexpressing ALKBH5 or ALKBH5–HA was assessed by counting the number of ALP-positive colonies on day 14 of reprogramming. (F) Fraction of SSEA1-positive cells determined by FACS analysis on day 14 of reprogramming. Temporal overexpression of ALKBH5–HA by 1 µg/ml doxycycline (Dox) was carried out from day 1 to day 7, day 7 to day 14 or day 1 to day 14. MEFs were used as a negative control. Images in A, B, D and F are representative of three repeats. Quantitative data are shown as the mean±s.d.; n=3. ***P<0.001 (paired Student's t-test).

Fig 5: Surf4 Promotes iPSCs Generation. (A) Kinetics of Oct4-GFP+ colony formation with or without exogenous Surf4 during the reprogramming process (n = 3). (B) The number of Oct4-GFP+ colonies and the percentage of Oct4-GFP+ cells 15 days after induction induced with or without exogenous Surf4 (n = 3, *p < 0.05, by Student's t test for comparison). (C) Cell proliferation curve with or without exogenous Surf4 during reprogramming. (D) Morphology of primary iPS colonies. Scale bars, 1000 µm. Magnification: ×40. (E) Alkaline phosphatase (AP) staining of the primary iPS colonies. (F) Morphology of an established OSKM + Surf4-iPSC cell line. Scale bars, 1000 µm. Magnification: ×40. (G) Quantitative PCR (qPCR) analysis of pluripotent genes in OSKM + Surf4-iPSCs. The data are presented as the means ± SEM (n = 3). (H) Immunostaining of pluripotent gene products OCT4, NANOG and SSEA1 in OSKM + Surf4-iPSCs. The nuclei were stained with DAPI. Scale bars, 50 µm. (I) Haematoxylin and eosin (H&E) staining of teratomas generated from OSKM + Surf4-iPSCs. Scale bars, 100 µm. See also Figure S1 and Table S1