Journal: Cell Proliferation

Article Title: Genome‐scale screening in a rat haploid system identifies Thop1 as a modulator of pluripotency exit

doi: 10.1111/cpr.13209

Figure Lengend Snippet: Thop1 ‐KO inhibits phosphorylation of ERK1/2 in the absence of PD0325901. (A) Respective global transcriptome profiles of Thop1 ‐KO GFP (#1 GFP , #2 GFP and #3 GFP ) and DA‐5‐3 GFP were hierarchically clustered. (B) The heatmap of representative pluripotent genes among DEGs of Thop1 ‐KO GFP and DA‐5‐3 GFP . (C) The heatmap of representative apoptosis genes among DEGs of Thop1 ‐KO GFP and DA‐5‐3 GFP . (D) Volcano plot depicting the differential expression analysis between Thop1 ‐KO GFP and DA‐5‐3 GFP and their relationships with the MAPK pathway, the GSK3 pathway and JAK–STAT pathway. Highlight points indicated well‐known genes of the three pathways, considering a fold change of 2.0 and p < 0.05. Only the MAPK pathway well‐known genes located to the DEGs. (E) Phase and FITC merged images of DA‐5‐3 GFP and Thop1‐ KO GFP when cultured withdrawing different factors (CHIR&LIF, PD&LIF, CHIR&PD, 2i&LIF) on Day 5. The red arrows indicated the differentiated cells. Scale bar, 100 μm. (F) FACS analysis of GFP‐positive cells in DA‐5‐3 GFP and Thop1‐ KO GFP when cultured withdrawing different factors (CHIR&LIF, PD&LIF, CHIR&PD, 2i&LIF) on Day 5. (G) Western blotting analysis of p‐ERK1/2, ERK1/2 and α‐TUBULIN in DA‐5‐3 GFP and Thop1‐ KO GFP when cultured in 2i/LIF medium or 2i/LIF medium without PD0325901

Article Snippet: The final concentrations of 2i (PD0325901 [MCE, HY‐10254], CHIR99021 [MCE, HY‐10182]) and human LIF (SinoBiological, 14890‐HNAH) were as follows: PD0325901, 1 μM; CHIR99021, 3 μM and LIF, 1000 U/ml.

Techniques: Expressing, Cell Culture, Western Blot

Journal: Cell Proliferation

Article Title: Genome‐scale screening in a rat haploid system identifies Thop1 as a modulator of pluripotency exit

doi: 10.1111/cpr.13209

Figure Lengend Snippet: Thop1 ‐OE promotes phosphorylation of ERK1/2. (A) Expression levels of Thop1 in DA‐5‐3 GFP , DA‐5‐3 GFP ‐vector and Thop1‐ OE GFP cells. t test, *** p < 0.001. The data were presented as the mean ± SEM. (B) Western blotting analysis of THOP1 and α‐TUBULIN in DA‐5‐3 GFP , DA‐5‐3 GFP ‐vector and Thop1 ‐OE GFP when cultured in ‘2i/LIF’ medium. (C) Phase and FITC merged images of DA‐5‐3 GFP , DA‐5‐3 GFP ‐vector and Thop1 ‐OE GFP cells cultured in 2i/LIF medium on Day 3. The red box was an enlarged image of the differentiated cells. Scale bar, 100 μm. (D) FACS analysis of GFP‐positive cells of DA‐5‐3 GFP , DA‐5‐3 GFP ‐vector and Thop1‐ OE GFP cells cultured in 2i/LIF medium for 3 days. (E) Expression levels of pluripotent genes ( Klf2 , Rex1 , Nanog , Oct4 and Sox2 ) in DA‐5‐3 GFP , DA‐5‐3 GFP ‐vector and Thop1‐ OE GFP cells cultured 2i/LIF medium for 3 days. t test, ** p < 0.01. The data were presented as the mean ± SEM. (F) FACS analysis of GFP‐positive cells in the cell cultures of DA‐5‐3 GFP , DA‐5‐3 GFP ‐vector and Thop1‐ OE GFP differentiated cells on Day 6. (G) The cell viabilities of DA‐5‐3 GFP , DA‐5‐3 GFP ‐vector cells and Thop1‐ OE GFP differentiated cells on Day 6. t test, ** p < 0.01. The data were represented as the mean ± SEM. (H) The western blotting analysis of p‐ERK1/2, ERK1/2 and α‐TUBULIN in DA‐5‐3 GFP , DA‐5‐3 GFP ‐vector and Thop1‐ OE GFP cells cultured in 2i/LIF medium. (I) Thop1 modulated p‐ERK to affect the differentiation of rat ESCs in vitro

Article Snippet: The final concentrations of 2i (PD0325901 [MCE, HY‐10254], CHIR99021 [MCE, HY‐10182]) and human LIF (SinoBiological, 14890‐HNAH) were as follows: PD0325901, 1 μM; CHIR99021, 3 μM and LIF, 1000 U/ml.

Techniques: Expressing, Plasmid Preparation, Western Blot, Cell Culture, In Vitro