Fig 1: Bach1-KO promotes mesendodermal differentiation by activating Nodal signaling.(A) mRNA levels of Nodal and the Nodal receptors ALK4 and ACVRIIB were evaluated in WT and Bach1-KO hESCs on day 3 of differentiation via qRT-PCR, and the results were normalized to measurements in WT cells (n = 3). **P < 0.01 compared with WT; t test. (B) Protein levels of Nodal, ACVRIIB, phosphorylated Smad2 and Smad3 (p-Smad2 and p-Smad3, respectively), and total Smad2/3 were evaluated in WT and Bach1-KO hESCs on day 3 of differentiation via Western blot. (C) DoxBach1-transfected WT hESCs were transfected with plasmids containing luciferase reporter constructs controlled by WT or mutated versions of the Nodal promoter; then, luciferase activity was measured in cells that had been treated with (Dox+) or without Dox (Dox-) on day 3 of differentiation, and results were normalized to measurements in Dox- cells (n = 3). *P < 0.05 compared with Dox-; t test. (D) p-Smad2, total Smad2/3, and histone 3 protein levels were evaluated in the nucleus and cytoplasm of WT and Bach1-KO hESCs on day 3 of differentiation via Western blot. (E) The binding of promoter sequences for mesendodermal genes and for the signaling molecules Wnt3 and Nodal was evaluated in WT and Bach1-KO hESCs and in DoxBach1-transfected WT hESCs that had (Dox+) or had not (Dox-) been treated with Dox on day 3 of differentiation via ChIP-qPCR; results were normalized to measurements in WT or Dox- cells (n = 3). *P < 0.05; **P < 0.01 compared with WT or Dox-; t test. (F and G) mRNA levels of mesendodermal genes were evaluated in WT and Bach1-KO hESCs and in WT and Bach1-KO hESCs that had been treated with SB-431542 (20 µM), which inhibits TGF-ß receptors (F) or transfected with adenoviruses coding for ß-catenin shRNA and treated with SB-431542 (G), and assessments were performed on day 3 of differentiation via qRT-PCR; results were normalized to measurements in WT cells (n = 3). **P < 0.01 compared with WT, DMSO (F) or WT, DMSO, Ad-shCtrl (G); ##P < 0.01 compared with KO, DMSO (F) or KO, DMSO, Ad-shCtrl (G); one-way analysis of variance. Data were collected from three independent replicates and are shown as means ± SD. (H) Model showing that Bach1 plays an important role in the maintenance of hESC self-renewal and suppression of mesendodermal differentiation. Bach1 interacts with Nanog, Sox2, and Oct4 and facilitates deubiquitylation of these pluripotency factors by recruiting DUB Usp7 and therefore stabilizes pluripotency factors and maintains the stem cell self-renewal. Bach1 also impedes mesendodermal differentiation via recruitment of PRC2, which leads to the deposition of H3K27me3 and gene silencing, as well as inhibiting Wnt/ß-catenin and Nodal/Smad2/3 signaling.
Fig 2: MiR-2467/3200/Let7e inhibit self-renewal and attenuate GAS5-mediated NODAL signalling in hESCs.(a) Colony formation assay with different miRNA transfection in hESCs. Quantification of colonies are shown in the right panel. NS, not significant; NC, microRNA mimic control overexpression group. **P<0.01, t-test, n=3. (b) Cell cycle analysis of different miRNA overexpression in hESCs. Comparison of cell ratios in G2 phase are shown in the right panel. NS, not significant; NC, microRNA mimic control overexpression group. **P<0.01, t-test, n=3. (c–e) The mRNA level of pluripotency genes in miRNA-transfected cells combined with rhNODAL incorporation (c) or GAS5 overexpression (d). The relative western blot results are shown in the right panel (e). NC, microRNA mimic control overexpression group. **P<0.01, t-test, n=3. (f) Analysis of mature microRNA and pre-miRNA expressions between different differentiation methods in H1 hESCs. **P<0.01, t-test t, n=3. (g) The analysis of expression changes of pluripotency genes and NODAL signalling-related genes during different differentiation of H1 hESCs. *P<0.01, **P<0.01, t-test, n=3. Here, −bFGF represents bFGF-deprived hESC medium; MEF represents mouse embryonic fibroblasts culture medium (which contains fetal bovine serum). (h) Teratoma formation assay using lentiviral stably transfected hESCs injected into the back cutaneous of NOD/SCID mice. Six weeks after the injection, tumours were dissected, photographed (left panel) and weighed (right panel). The ruler scales are 0.5 mm per minor mark. **P<0.01, t-test, n=3. (i) Levels of GAS5, NODAL, miR-2467, miR-3200 and Let7e in the tumours were evaluated using qPCR. **P<0.01, t-test, n=3. Error bars represent s.d. of the indicated experiment replicates. RNA level of β-actin served as internal reference for qPCR. See also Supplementary Fig. 7.
Fig 3: NODAL is post-transcriptionally regulated by GAS5.(a) A scheme of vectors expressing different GAS5 truncated transcript. The inserted length of each transcript is indicated and the blue square indicates the position of validated GR-binding sequence. 1-651mut represents the GR-binding sequence-mutated construct. The right panel shows the mRNA level of NANOG and NODAL in different treatment groups. **P<0.01, t-test, n=3. (b) The left panel shows the scheme of constructing NODAL promoter reporter. The right panel shows the luciferase activities of NODAL promoter reporter in hESCs with indicated treatment. NC represents NODAL promoter reporter transfected hESCs treated with empty vector, t-test, n=3. (c) The nuclear-plasma separation assay of GAS5 overexpressed or knockdown hESCs are shown in the middle panel using qPCR. Respective FISH images are shown in the left panel, and the GAS5 expression of analysed using total RNAs are shown in the right panel (n=3). NUCL represents nuclear hESC extracts, CYTO represents cytoplasm hESC extracts. Scale bar, 20 μm. All groups were compared with NC or NC-NUCL group respectively. **P<0.01, t-test, n=3. (d) A scheme of MS2-mediated pulldown of GAS5 and its binding protein. (e) The silver staining of pull-down products (n=2), the red triangle indicates the antibody band, whereas black triangle indicates the bands and position that is cutoff for mass spectrometry analysis shown in the right panel. (f) Western blot shows the AGO2 expression in different pulldown groups as indicated by the black triangle (n=2). The red triangle indicates the antibody heavy chain bands, which were used in pull-down experiment and react with the secondary antibody. (g) Confocal microscopy of AGO2 and GAS5 transcript. Scale bar, 100 μm. (h) RNA immune-precipitation analysis of the RNA levels of precipitated GAS5, linc-ROR, H19 using indicated protein antibodies by qPCR. **P<0.01, t-test, n=3. (i) AGO2 competing assay using AGO2 antibody-mediated RNA-IP. RNA levels of precipitated NODAL, linc-ROR, H19 are tested using qPCR. **P<0.01, t-test, n=2. Error bars represent s.d. of the indicated experiment replicates. RNA level of β-actin served as internal reference for qPCR. See also Supplementary Fig. 5.
Fig 4: Bach1-KO promotes mesendodermal differentiation by activating Wnt/ß-catenin signaling.(A) mRNA levels of components of the Wnt signaling pathway were measured in WT and Bach1-KO hESCs on day 3 of differentiation into EBs via qRT-PCR and normalized to measurements in WT cells (n = 3). *P < 0.05; **P < 0.01 compared with WT; t test. (B) Protein levels of Wnt3, Fzd1, active ß-catenin, and total ß-catenin were evaluated in WT and Bach1-KO hESCs on day 3 of differentiation via Western blot. (C) Total ß-catenin and histone 3 protein levels were evaluated in the nucleus and cytoplasm of WT and Bach1-KO hESCs on day 3 of differentiation via Western blot. (D) WT and Bach1-KO hESCs were transfected with the TOPflash luciferase reporter, which contains eight copies of the binding site for Tcf/lef, a downstream target of Wnt signaling; then, the cells were treated with or without the Wnt inhibitor IWP2 (10 µM), and luciferase activity was evaluated on day 3 of differentiation and normalized to measurements in WT cells (n = 3). **P < 0.01 compared with WT (IWP2-); ##P < 0.01 compared with Bach1-KO (IWP2-); one-way analysis of variance. (E) mRNA levels of mesendodermal genes were evaluated in WT and Bach1-KO hESCs that had been treated with or without IWP2 (10 µM) on day 3 of differentiation (n = 3). **P < 0.01 compared with WT, DMSO; #P < 0.05; ##P < 0.01 compared with Bach1-KO, DMSO; one-way analysis of variance. (F) The binding of promoter sequences for mesendodermal genes and for the signaling molecules Wnt3 and Nodal to activated ß-catenin was evaluated in WT and Bach1-KO hESCs (left) and in DoxBach1-transfected WT hESCs that had (Dox+) or had not (Dox-) been treated with Dox (right) on day 3 of differentiation via ChIP-qPCR; results were normalized to WT (left) or Dox- (right) cells (n = 3). *P < 0.05; **P < 0.01 compared with WT or Dox-; t test. (G) mRNA levels of mesendodermal genes and protein level of ß-catenin were evaluated in WT and Bach1-KO hESCs that had been transfected with adenoviruses coding for ß-catenin shRNA (Ad-shß-catenin) or a control shRNA sequence (Ad-shCtrl) on day 3 of differentiation (n = 3). **P < 0.01 compared with WT, Ad-shCtrl; ##P < 0.01 compared with KO, Ad-shCtrl; one-way analysis of variance. (H) mRNA levels of Wnt3, Nodal, and Bach1 were evaluated in DoxBach1-transfected WT hESCs that had (Dox+) or had not (Dox-) been treated with Dox on day 3 of differentiation via qPCR (n = 3). *P < 0.05 compared with Dox-; t test. (I) DoxBach1-transfected WT hESCs were transfected with plasmids containing luciferase reporter constructs controlled by WT or mutated versions of the Wnt3 promoter; then, luciferase activity was measured in cells that had been treated with (Dox+) or without Dox (Dox-) on day 3 of differentiation, and results were normalized to measurements in Dox- cells (n = 3). *P < 0.05 compared with Dox-; t test. Data were collected from three independent replicates and are shown as means ± SD.
Fig 5: Specific interactome is essential for GAS5 to mediate NODAL signalling.(a,b) The analysis of the expression level of NODAL, GAS5 (b) and their interacting miRNAs (a) in different cell types. **P<0.01, t-test, n=3. (c) The relative mRNA level of pluripotency genes and NODAL in the GAS5 overexpressed groups were compared with different cell types using qPCR. The expression levels of pluripotency genes and NODAL in each cell type is compared with their relative empty vector expressed group, which were shown as a dashed line. **P<0.01, t-test, n=3. (d,e) ELISA analysis of the secreted NODAL in the cell culture medium of different cell types (d) and GAS5 overexpressed or knockdown cells (e). All cells for supernatant collecting were cultured using serum-free medium. **P<0.01, t-test, n=3. (f–h) Standardized quantitative PCR detecting the expression change of indicated miRNA and NODAL transcript in hESCs (f), uMSCs (g) and HEK-293T cells (h) under different concentration of synthetic full-length GAS5 RNA transfection. n=3. (i) The graphical summary of the function and mechanism of GAS5. Error bars represent s.d. of the indicated experiment replicates. RNA level of ß-actin served as internal reference for qPCR. See also Supplementary Fig. 8.
Supplier Page from Abcam for Anti-Nodal antibody [EPR2057]