Journal: Cell Research
Article Title: Derivation of totipotent-like stem cells with blastocyst-like structure forming potential
doi: 10.1038/s41422-022-00668-0
Figure Lengend Snippet: a Western blot analysis showing the levels of histone H3 and H4 acetylation and H3K79me2 of EPS and TPS cells. Similar results were obtained in at least 2 independent experiments. b qPCR analysis of expression levels of classical RAR downstream target genes in EPS and TPS cells. n = 3 biological replicates. c qPCR analysis of expression levels of representative totipotency marker genes on day 3 upon treatment of different small molecule combinations. In the CPEC condition, EPZ004777, VPA, CD1530 and CHIR 99021 were replaced by small molecules targeting DOT1L, HDAC, RA signaling and GSK3β, respectively. n = 2 technical replicates. Similar results were obtained in at least 2 independent experiments. EPS, EPS cells; Basal, EPS cells cultured in the basal medium of CPEC condition. EPZ rep, VPA rep, CD1530 rep and CHIR rep indicate small molecules that target DOT1L, HDAC, RA and GSK3β, respectively. d qPCR analysis of Hdac1 and Hdac2 expression in EPS cells after shRNA knockdown of Hdac1/2 . sh Hdac , Hdac1/2 shRNA. n = 2 biological replicates. e qPCR analysis of expression levels of representative totipotency marker genes on day 3 after knocking down Hdac1/2 in EPS cells during TPS cell induction. EPS, EPS cells; CPEC-V + sh Hdac , replacement of VPA with Hdac1/2 knockdown. n = 2 biological replicates. f qPCR analysis of Dot1l expression in EPS cells after shRNA knockdown of Dot1l . sh Dot1l , Dot1l shRNA. n = 3 biological replicates. g qPCR analysis of expression levels of representative totipotency marker genes on day 3 after knocking down Dot1l in EPS cells during TPS cell induction. EPS, EPS cells; CPEC-E + sh Dot , replacement of EPZ004777 with Dot1l knockdown. n = 3 biological replicates. h qPCR analysis of the effect of inhibiting RAR signaling on totipotency induction (left panel) and maintenance (right panel) in TPS cells. Expression of representative totipotency marker genes were analyzed. RARγi, LY2955303; RARα/βi, LE135; RXRi, UVI3003. n = 3 biological replicates. i qPCR analysis of the effect of CPEC chemical cocktail on totipotency maintenance in early preimplantation embryos. Small molecules were added from the 2-cell embryo stage for 2 days. DMSO was used as a negative control. 2C, 2-cell embryo. n = 2 biological replicates. j qPCR analysis of the effect of Dux knockdown on maintaining totipotency in TPS cells. Expression of representative totipotency marker genes were analyzed. Dux KD, dux knockdown. n = 2 biological replicates. k qPCR analysis of the effect of Dux knockdown on inducing totipotency from EPS cells. Expression of representative totipotency marker genes were analyzed. Dux KD, dux knockdown. n = 2 biological replicates. l qPCR analysis of the effect of p53 knockdown on inducing totipotency from EPS cells. Expression of representative totipotency marker genes were analyzed. p53 KD, p53 knockdown. n = 2 biological replicates.
Article Snippet: To inhibit RAR signaling during culturing of TPS cells, RARγ inhibitor LY2955303 (1 μm; MCE, HY-107765), RARα/β inhibitor LE135 (2 μm; MCE, HY-107436), and RXR inhibitor UVI3003 (1 μm; MCE, HY-107500) were individually added into the CPEC medium.
Techniques: Western Blot, Expressing, Marker, Cell Culture, shRNA, Knockdown, Negative Control
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