Journal: Stem Cells (Dayton, Ohio)

Article Title: A Regulatory Network Involving β‐ C atenin, e‐ C adherin, PI3k / A kt, and S lug Balances Self‐Renewal and Differentiation of Human Pluripotent Stem Cells In Response to W nt Signaling

doi: 10.1002/stem.1944

Figure Lengend Snippet: Dual function of β‐catenin in canonical Wnt signaling pathway is responsible for hESC self‐renewal and differentiation. (A): βS33Y‐Tet‐On hESCs were treated with doxycycline (DOX, 2 µg/ml) for 24 hours, followed by clonogenic assays (mean ± SD, n = 3, **, p < .01). (B–D): βS33Y‐Tet‐On hESCs were treated with different dosages of DOX for 4–6 days, followed by immunofluorescence staining of β‐catenin and Nanog (B), and quantification of mean pixel intensity of Nanog (C, n >100 cells/group). The nuclear translocation of β‐catenin was observed in 1,000 ng/ml of DOX group (D). Nuclei were counterstained with DAPI (blue). White arrows indicate cytoplasmic β‐catenin. Scale bars = 10 µm (insets in D: 1 µm). (E, F): Clonogenic assays (E, mean ± SD, n = 3, *, p < .05; **, p < .01) and Western blot analysis (F) for the same cells as described in (B)–(D). (G): TCF‐eGFP reporter activity remains invisible in the absence of DOX for 4–6 days but visible in the nuclear region in a DOX dose‐dependent manner, scale bars = 20 µm. (H, I): Flow cytometric analysis of 7xTCF‐eGFP hESCs as described in (G), pregated on single 7‐AAD‐negative live cells. The TCF‐eGFP reporter activity was measured by the percentage of GFP‐positive cells after treatment with different concentrations of DOX for 4–6 days (H). The expression of GFP‐positive cells is normalized to DOX‐untreated group that was arbitrarily set at 1 (I). (J): Flow cytometric analysis of the cells in (H) and (I) for E‐cadherin expression (E‐cad+) on the cell membrane indicates an inverse association with TCF‐eGFP activity as shown in (G)–(I), pregated on single 7‐AAD‐negative live cells. Data in (I) and (J) are means ± SD, n = 3, two independent experiments; **, p < .01 compared to DOX‐untreated group. Abbreviations: GFP, green fluorescent protein; hESC, human embryonic stem cell; TCF, T‐cell factor.

Article Snippet: Four Wnt reporter hESC sublines (7xTCF‐eGFP‐EcadΔβ‐Tet‐On, 7xTCF‐eGFP‐βS33Y‐Tet‐On, 7xTCF‐eGFP‐vector‐Tet‐On, and 7xTCF‐eGFP) were generated by transduction of the established EcadΔβ‐, βS33Y‐, and vector‐Tet‐On or wild‐type hESCs with a 7×TCF‐eGFP//SV40‐PuroR lentiviral vector containing seven Tcf/Lef‐binding sites and a puromycin resistance gene (Addgene #24305, a kind gift from Dr. Roel Nusse ).

Techniques: Immunofluorescence, Staining, Translocation Assay, Western Blot, Activity Assay, Expressing

Journal: Stem Cells (Dayton, Ohio)

Article Title: A Regulatory Network Involving β‐ C atenin, e‐ C adherin, PI3k / A kt, and S lug Balances Self‐Renewal and Differentiation of Human Pluripotent Stem Cells In Response to W nt Signaling

doi: 10.1002/stem.1944

Figure Lengend Snippet: E‐cadherin associates with PI3K/Akt activation in response to short‐term Wnt/β‐catenin signaling‐enhanced human embryonic stem cell (hESC) self‐renewal. (A): Decreased phosphorylated AKT (pAKT S473 ) in hESCs after E‐cadherin knockdown for 48 hours (Western blot). (B): Increased phosphorylated AKT (pAKT S473 ) after E‐cadherin upregulation. E‐cadherin‐Tet‐On hESCs were treated with DOX (2 µg/ml) for 48 hours, followed by Western blot analysis. (C): Inhibition of PI3/Akt with LY294002 abrogates the enhanced clonogenic capacity induced by E‐cadherin upregulation. E‐cadherin‐Tet‐On hESCs were treated with or without DOX (2 µg/ml) in the presence or absence of LY294002 (5 µM) for 24 hours, followed by clonogenic assays ( n = 3). (D): E‐cadherin knockdown abolishes short‐term GSK3 inhibition‐induced upregulation of E‐cadherin and phosphorylated Akt, while specific Akt activator SC79 counteracts the effect of E‐cadherin knockdown. After siRNA knockdown for 44 hours, hESCs (H9 and H1 lines) were treated with either CHIR99021 (6 µM) or vehicle (DMSO) for 6 hours, followed by Akt inhibitor VIII (6 µM), or Akt activator II SC79 (6 µg/ml), or vehicle for an additional 30 minutes. All data in this figure are mean ± SD and were generated from H1 and H9 lines. **, p < .01.

Article Snippet: Four Wnt reporter hESC sublines (7xTCF‐eGFP‐EcadΔβ‐Tet‐On, 7xTCF‐eGFP‐βS33Y‐Tet‐On, 7xTCF‐eGFP‐vector‐Tet‐On, and 7xTCF‐eGFP) were generated by transduction of the established EcadΔβ‐, βS33Y‐, and vector‐Tet‐On or wild‐type hESCs with a 7×TCF‐eGFP//SV40‐PuroR lentiviral vector containing seven Tcf/Lef‐binding sites and a puromycin resistance gene (Addgene #24305, a kind gift from Dr. Roel Nusse ).

Techniques: Activation Assay, Western Blot, Inhibition, Generated

Journal: Stem Cells (Dayton, Ohio)

Article Title: A Regulatory Network Involving β‐ C atenin, e‐ C adherin, PI3k / A kt, and S lug Balances Self‐Renewal and Differentiation of Human Pluripotent Stem Cells In Response to W nt Signaling

doi: 10.1002/stem.1944

Figure Lengend Snippet: E‐cadherin sequesters β‐catenin to suppress Wnt‐induced hESC differentiation. (A): Q‐PCR analysis of hESCs cotransfected with βS33Y plasmids and either wild‐type E‐cadherin (WT‐Ecad) or EcadΔβ for 24 hours. pcDNA: control vector. (B): In comparison to control groups, co‐overexpression of EcadΔβ and βS33Y for 24 hours significantly increases Brachyury expression (Q‐PCR). (C): EcadΔβ‐Tet‐On hESCs were treated with or without doxycycline (DOX+, 2 µg/ml) or vehicle in the presence or absence of Wnt3a protein (100 ng/ml) for 4–6 days and analyzed by double‐immunostaining. Nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. (D): Subcellular localization analysis of the expression of active β‐catenin and Brachyury as shown in (C). Plots of the intensity profile of active β‐catenin (red), Brachyury (green), and DAPI (blue) over a linear section of a whole cell (white lines in C) are representative of >100 cells/group analyzed. Data are expressed as AU versus length in microns. The arrowheads indicate the cell‐cell contact areas; the yellow area in the middle of each plot marks the nuclear region; mean ± SD in red on each plot represent the mean pixel intensity of the nuclear active β‐catenin from >100 cells/group. (E): Quantification of the ratio of active β‐catenin nuclear staining/DAPI intensity for the indicated groups in (C). Data are mean ± SD, four replicates from H9 line, **, p < .01 compared to DOX − Wnt3a − group. (F, G): Doxycycline‐induced EcadΔβ over‐expression promotes hESC differentiation in response to Wnt3a treatment as revealed by significant upregulation of Brachyury and Mixl1 (F, Q‐PCR; G, Western blot). (H): Alkaline phosphatase (AP) staining and quantitative analyses. At day 4 of treatments as indicated, cells were dissociated and reseeded. AP‐positive colonies were counted when >50% of the cells within the colony were positive for AP staining. The percentages of AP+ colonies were quantitatively assessed and are shown at the bottom. The dashed line indicates the boundary of the colony. Scale bar = 100 µm. The results represent three replicates ± SD from H1 and H9 lines. *, p <.05; **, p <.01. Abbreviations: AU, arbitrary unit; hESC, human embryonic stem cell.

Article Snippet: Four Wnt reporter hESC sublines (7xTCF‐eGFP‐EcadΔβ‐Tet‐On, 7xTCF‐eGFP‐βS33Y‐Tet‐On, 7xTCF‐eGFP‐vector‐Tet‐On, and 7xTCF‐eGFP) were generated by transduction of the established EcadΔβ‐, βS33Y‐, and vector‐Tet‐On or wild‐type hESCs with a 7×TCF‐eGFP//SV40‐PuroR lentiviral vector containing seven Tcf/Lef‐binding sites and a puromycin resistance gene (Addgene #24305, a kind gift from Dr. Roel Nusse ).

Techniques: Plasmid Preparation, Over Expression, Expressing, Double Immunostaining, Staining, Western Blot

Journal: Stem Cells (Dayton, Ohio)

Article Title: A Regulatory Network Involving β‐ C atenin, e‐ C adherin, PI3k / A kt, and S lug Balances Self‐Renewal and Differentiation of Human Pluripotent Stem Cells In Response to W nt Signaling

doi: 10.1002/stem.1944

Figure Lengend Snippet: E‐cadherin sequesters β‐catenin and suppresses the functional interaction of β‐catenin and TCF in hESCs. Analysis of 7xTCF‐eGFP‐EcadΔβ‐Tet‐On hESCs after treatment with doxycycline (DOX+, 2 µg/ml) or vehicle in the presence or absence of Wnt3a protein (100 ng/ml) for the indicated time points. The cells were cultured on Matrigel‐coated plates. Media, DOX, and Wnt3a were changed daily. (A): TCF‐eGFP (green) expression pattern of hESCs in the absence or presence of DOX and/or Wnt3a for 6 hours or 4 days. Scale bar = 50 µm. (B–D): Representative flow cytometric plots (B), fold changes of TCF‐eGFP‐positive cells over DOX − Wnt3a − control (C) and MFI (D) in the absence or presence DOX and/or Wnt3a for 4 days, pregated on single 7‐AAD‐negative live cells. Mean ± SD from three independent experiments. *, p < .05; **, p < .01. Abbreviations: GFP, green fluorescent protein; hESC, human embryonic stem cell; MFI, median fluorescence intensity; TCF, T‐cell factor.

Article Snippet: Four Wnt reporter hESC sublines (7xTCF‐eGFP‐EcadΔβ‐Tet‐On, 7xTCF‐eGFP‐βS33Y‐Tet‐On, 7xTCF‐eGFP‐vector‐Tet‐On, and 7xTCF‐eGFP) were generated by transduction of the established EcadΔβ‐, βS33Y‐, and vector‐Tet‐On or wild‐type hESCs with a 7×TCF‐eGFP//SV40‐PuroR lentiviral vector containing seven Tcf/Lef‐binding sites and a puromycin resistance gene (Addgene #24305, a kind gift from Dr. Roel Nusse ).

Techniques: Functional Assay, Cell Culture, Expressing, Fluorescence

Journal: Stem Cells (Dayton, Ohio)

Article Title: A Regulatory Network Involving β‐ C atenin, e‐ C adherin, PI3k / A kt, and S lug Balances Self‐Renewal and Differentiation of Human Pluripotent Stem Cells In Response to W nt Signaling

doi: 10.1002/stem.1944

Figure Lengend Snippet: β‐Catenin‐induced Slug upregulation reinforces human embryonic stem cell (hESC) differentiation by downregulating E‐cadherin and upregulating Brachyury. (A, B): Long‐term but not short‐term BIO treatment enhances Slug expression in hESCs (A, Q‐PCR; B, Western blot). (C): hESCs cotransfected with EcadΔβ and βS33Y, but not with vectors (pcDNA) or with wild‐type E‐cadherin (WT‐Ecad) and βS33Y, enhances Slug expression (Q‐PCR). (D): Over‐expression of EcadΔβ together with short‐term Wnt3a treatment (100 ng/ml) promotes Slug expression in hESCs (Q‐PCR). (E): hESCs infected with pLKO‐siSlug plasmid (siSlug) for 2 days reduce Slug expression (Western blot). Scrambled: pLKO‐puro vector with a scrambled sequence that does not target any mRNA. (F–H): Slug knockdown (pre‐siSlug) followed by BIO treatment for 4 days results in a decrease of differentiation marker Brachyury (F, Q‐PCR; G, Western blot) and clonogenic capacity (H, clonogenic assays, n = 3) in hESCs. All data are mean ± SD, *, p <.05; **, p < .01. (I): The proposed working model (detailed in Supporting Information Fig. S7).

Article Snippet: Four Wnt reporter hESC sublines (7xTCF‐eGFP‐EcadΔβ‐Tet‐On, 7xTCF‐eGFP‐βS33Y‐Tet‐On, 7xTCF‐eGFP‐vector‐Tet‐On, and 7xTCF‐eGFP) were generated by transduction of the established EcadΔβ‐, βS33Y‐, and vector‐Tet‐On or wild‐type hESCs with a 7×TCF‐eGFP//SV40‐PuroR lentiviral vector containing seven Tcf/Lef‐binding sites and a puromycin resistance gene (Addgene #24305, a kind gift from Dr. Roel Nusse ).

Techniques: Expressing, Western Blot, Over Expression, Infection, Plasmid Preparation, Sequencing, Marker

Journal: International Journal of Biological Sciences

Article Title: CircCDK14 Promotes Tumor Progression and Resists Ferroptosis in Glioma by Regulating PDGFRA

doi: 10.7150/ijbs.66114

Figure Lengend Snippet: circCDK14 promotes the proliferation, migration and invasion of glioma. (A and B) Levels of circCDK14 and host gene CDK14 mRNA in U87 cells and U251 cells after transfection of circCDK14 overexpression plasmid or circCDK14 interference sequences, were detected by qRT-PCR. (C) Overexpressing circCDK14 promotes U87 cell proliferation, as determined by CCK-8. (D) CircCDK14 knockdown inhibits U251 cell proliferation, as determined by CCK-8. (E and F) Wound healing assay determined the migrating capacity of glioma cells after various treatments. (G and H) Transwell assay was used to detect the invasion activity of glioma cells following different treatments. Both scale bars are 100 µm. All experiments repeated thrice and averaged (mean±SD). Unpaired Students's t-test was employed for all data analyses. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The overexpression plasmids (pcDNA3.1-circRNA Mini vector) of circCDK14 were acquired from Genscript (Nanjing, China).

Techniques: Migration, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, CCK-8 Assay, Wound Healing Assay, Transwell Assay, Activity Assay

Journal: Cell Death & Disease

Article Title: CircAGFG1 drives metastasis and stemness in colorectal cancer by modulating YY1/CTNNB1

doi: 10.1038/s41419-020-2707-6

Figure Lengend Snippet: a The overexpression efficiency of CTNNB1 in SW480 cells was examined by qRT-PCR and western blot. b – d Cell proliferation was detected by CCK-8 assay ( b ), colony formation assay ( c ) and EdU assay ( d ) in each group (scale bar = 100 μm). e , f Cell apoptosis was examined by flow cytometry ( e ) and TUNEL assay ( f ) in each group (scale bar = 50 μm). g , h Cell migration ( g ) and invasion ( h ) were assessed by transwell assay in each group (scale bar = 70 μm). i Sphere formation efficiency was detected in each group by sphere formation assay (scale bar = 30 μm). j CD133 + cell percentage was assessed in each group by flow cytometry assay. k The protein levels of Bax, Bcl-2, MMP2, CD44, SOX2, Oct4, and Nanog in each group were detected by western blot. In this figure, “a, b, c and d” represents the group of sh-NC, sh-circAGFG1#1, sh-circAGFG1#1+pcDNA3.1/YY1 and sh-circAGFG1#1+pcDNA3.1/CTNNB1, respectively. We repeated the experiments three times to ensure the accuracy of the experiments. ** P < 0.01.

Article Snippet: For mRNA or circRNA overexpression, the sequence of YY1 or CTNNB1 was cloned into pcDNA3.1 vector (Genechem), while circAGFG1 sequence was cloned into pcDNA3.1(+) circRNA Mini vector (Genechem), constructing overexpression plasmids pcDNA3.1/YY1, pcDNA3.1/CTNNB1, and pcDNA3.1/circAGFG1, with empty vectors as the controls.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Colony Assay, EdU Assay, Flow Cytometry, TUNEL Assay, Migration, Transwell Assay, Tube Formation Assay

Journal: Cell Death & Disease

Article Title: CircAGFG1 drives metastasis and stemness in colorectal cancer by modulating YY1/CTNNB1

doi: 10.1038/s41419-020-2707-6

Figure Lengend Snippet: a The protein expressions of β-catenin, cyclin D1 and c-myc in the group of sh-NC, sh-circAGFG1#1, sh-circAGFG1#1+pcDNA3.1/YY1 or sh-circAGFG1#1+pcDNA3.1/CTNNB1 were measured by western blot. b The nuclear translocation of β-catenin in each group was assessed by IF assay (scale bar = 20 μm). c The expression association between circAGFG1 and miR-4262, miR-185-5p, YY1 or CTNNB1 was revealed by Pearson’s correlation analysis. We repeated the experiments three times to ensure the accuracy of the experiments. ** P < 0.01.

Article Snippet: For mRNA or circRNA overexpression, the sequence of YY1 or CTNNB1 was cloned into pcDNA3.1 vector (Genechem), while circAGFG1 sequence was cloned into pcDNA3.1(+) circRNA Mini vector (Genechem), constructing overexpression plasmids pcDNA3.1/YY1, pcDNA3.1/CTNNB1, and pcDNA3.1/circAGFG1, with empty vectors as the controls.

Techniques: Western Blot, Translocation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Influence of RNA circularity on Target RNA-Directed MicroRNA Degradation

doi: 10.1093/nar/gkae094

Figure Lengend Snippet: Effect of artificial circRNA constructs on TDMD in HEK293T cells. ( A ) Illustration of the constructs expressing linear or circular RNA of different sizes from a backbone containing Drosophila laccase2 introns . ( B ) Northern blot analysis of samples from HEK293T cells expressing miR-218 constructs of the indicated length. n = 6 culture wells (from two independent experiments) of HEK293T cells for each condition. NT are non-transfected cells. ( C ) MiR-218-5p abundance upon transduction of the linear or circRNA-expressing constructs of different lengths carrying bulged (TDMD-compatible) or seed-mutant miR-218-5p sites. n = 6 culture wells (from two independent experiments) of HEK293T cells for each condition. ( D ) Northern blot analysis of samples from HEK293T cells expressing long miR-7 constructs. n = 3 culture wells (from one experiment) of HEK293T cells for each condition. ( E ) MiR-7-5p abundance upon transduction of the linear or circRNA-expressing constructs of different lengths carrying bulged (TDMD-compatible) or seed-mutant miR-7-5p sites. n = 6 culture wells (from two independent experiments) of HEK293T cells for each condition. Data are presented as mean ± SEM. Statistical significance was determined by unpaired Student's t tests (ns: P > 0.05, * P ⇐ 0.05, ** P ⇐ 0.01, *** P ⇐ 0.001, **** P ⇐ 0.0001).

Article Snippet: Constructs expressing artificial circRNAs of different sizes are based on the pcDNA3.1(+) Laccase2 MCS Exon Vector (Addgene 69893).

Techniques: Construct, Expressing, Northern Blot, Transfection, Transduction, Mutagenesis