Figure 3.

Requirement of MBD3 in Other Reprogramming Systems

(A) Experimental designs used to analyze the effect of Mbd3 KD and KO on EpiSC reprogramming efficiency. For the KD experiments, wild-type EpiSCs (carrying an Oct4-GFP cassette), stably transfected with pPB-CAG-Klf2.2A.Nanog (K2N) or pPB-CAG-Klf4, were transfected with either siMbd3 or siControl (siCtrl) and, after 24 hr, were plated in 2i/LIF for 12 days. For the KO experiments, Mbd3^−/− or Mbd3^fl− EpiSCs carrying an Oct4-GFP reporter (EOS-GiP), stably transfected with K2N (or empty vector control, EV), were plated in 2i/LIF for 12 days.

(B and C) The efficiency of EpiSC reprogramming after Mbd3 removal, either by KD (B) or KO (C), was assessed by counting Oct4-GFP⁺ colonies. Representative AP stained plates are also indicated. 1.0 × 10⁴ EpiSCs were plated in (B). 1.5 × 10⁴ EpiSCs were plated in (C).

(D) Experimental designs used to analyze the effect of Mbd3 KD and Mbd3 exon 1 KO. For the KD experiments, Nanog-GFP MEFs transfected with doxycycline-inducible MKOS piggyBac transposon (iMKOS) were cultured in S+LIF + DOX + vitamin C (vitC) + Alki 24 hr before lentiviral infections of shMbd3 or shControls. For the KO experiments, Mbd3^ex1fl/ex1fl MEFs transfected with iMKOS were infected with pMX-Cre-ERt2. Reprogramming was carried out in S+LIF + DOX + vitC + Alki, and 4-OHT was added either at the time of DOX administration (0h) or 48 hr later (48h).

(E) Number of Nanog-GFP⁺ iPSC colonies at day 13 of reprogramming upon infection of indicated shRNAs.

(F) Number of Nanog⁺ colony numbers determined by immunofluorescence after 13 days of reprogramming of Mbd3^ex1fl/ex1fl:Cre-ERt2 MEFs. The error bars indicate STDEV. Typical iMKOS positive cell number at day 2 of reprogramming is 1.0–3.0 × 10⁴ cells per well, providing 1%–2% reprogramming efficiency.