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Differentiation induced by treatment with the Gsk3 inhibitor in hPSCs <t>is</t> <t>β-catenin</t> dependent. (A) H9-7TGP cells were treated with 12 μM CH in mTeSR1 for 4 d. Immunofluorescent staining for Oct4, Isl1, and Nkx2.5 was compared with GFP expression. (Scale bars, 50 μm.) (B and C) 19-9-11 shcat-2 and scramble cells were cultured on Matrigel with mTeSR1 medium containing 12 μM CH for 4 d. (B) RT-PCR analysis of pluripotent, mesendoderm, early mesoderm, and early cardiac gene expression was performed. (C) Oct4 expression on day 4 was analyzed by flow cytometry. Each colored line represents an independent replicate. n = 3. (D) 19-9-11 shcat-2 and scramble lines were cultured on Matrigel in mTeSR1 containing CH. After 2 d, the expression of T in the scramble relative to its expression in the shcat-2 line was quantified by quantitative PCR. (E) Flow cytometry analysis of brachyury expression in 19-9-11 shcat-2 and scramble cells exposed to CH for 4 d. Error bars represent SEM of three independent replicates. (F) 19-9-11 shcat-2 and scramble lines were cultured on Matrigel in mTeSR1 containing 12 μM CH. After 4 d, cells were immunostained for Nanog and Isl1. (Scale bar, 50 μm.)

β Catenin, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/cell+transcriptome+sequencing+analysis/pmc03390875-415-16-13?v=Addgene+inc
Average 93 stars, based on 1 article reviews

β catenin - by Bioz Stars, 2026-08

93/100 stars

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1) Product Images from "Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling"

Article Title: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1200250109

Differentiation induced by treatment with the Gsk3 inhibitor in hPSCs is β-catenin dependent. (A) H9-7TGP cells were treated with 12 μM CH in mTeSR1 for 4 d. Immunofluorescent staining for Oct4, Isl1, and Nkx2.5 was compared with GFP expression. (Scale bars, 50 μm.) (B and C) 19-9-11 shcat-2 and scramble cells were cultured on Matrigel with mTeSR1 medium containing 12 μM CH for 4 d. (B) RT-PCR analysis of pluripotent, mesendoderm, early mesoderm, and early cardiac gene expression was performed. (C) Oct4 expression on day 4 was analyzed by flow cytometry. Each colored line represents an independent replicate. n = 3. (D) 19-9-11 shcat-2 and scramble lines were cultured on Matrigel in mTeSR1 containing CH. After 2 d, the expression of T in the scramble relative to its expression in the shcat-2 line was quantified by quantitative PCR. (E) Flow cytometry analysis of brachyury expression in 19-9-11 shcat-2 and scramble cells exposed to CH for 4 d. Error bars represent SEM of three independent replicates. (F) 19-9-11 shcat-2 and scramble lines were cultured on Matrigel in mTeSR1 containing 12 μM CH. After 4 d, cells were immunostained for Nanog and Isl1. (Scale bar, 50 μm.)

Figure Legend Snippet: Differentiation induced by treatment with the Gsk3 inhibitor in hPSCs is β-catenin dependent. (A) H9-7TGP cells were treated with 12 μM CH in mTeSR1 for 4 d. Immunofluorescent staining for Oct4, Isl1, and Nkx2.5 was compared with GFP expression. (Scale bars, 50 μm.) (B and C) 19-9-11 shcat-2 and scramble cells were cultured on Matrigel with mTeSR1 medium containing 12 μM CH for 4 d. (B) RT-PCR analysis of pluripotent, mesendoderm, early mesoderm, and early cardiac gene expression was performed. (C) Oct4 expression on day 4 was analyzed by flow cytometry. Each colored line represents an independent replicate. n = 3. (D) 19-9-11 shcat-2 and scramble lines were cultured on Matrigel in mTeSR1 containing CH. After 2 d, the expression of T in the scramble relative to its expression in the shcat-2 line was quantified by quantitative PCR. (E) Flow cytometry analysis of brachyury expression in 19-9-11 shcat-2 and scramble cells exposed to CH for 4 d. Error bars represent SEM of three independent replicates. (F) 19-9-11 shcat-2 and scramble lines were cultured on Matrigel in mTeSR1 containing 12 μM CH. After 4 d, cells were immunostained for Nanog and Isl1. (Scale bar, 50 μm.)

Techniques Used: Staining, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Real-time Polymerase Chain Reaction

Temporal regulation of Wnt/β-catenin signaling promotes cardiac differentiation induced by serum or growth factors. (A) H9 cells on MEFs were treated with CH in hESC medium for 3 d before forming EBs. EBs were cultured in suspension using serum containing medium for 4 d before being transferred to 0.1% (wt/vol) gelatin-coated plates. The percentage of contracting EBs was determined visually. (B) H9 cells were cultured on Matrigel and treated with DMSO, 1 μM CH, or 1 μM BIO for 3 d before exposure to 100 ng/mL activin A at day 0 and 5 ng/mL BMP4 at day 1 in RPMI/B27-insulin medium using monolayer-directed differentiation. At day 15, the percentage of cTnT+ cells in culture was assessed by flow cytometry. #P < 0.005, CH versus DMSO or BIO versus DMSO; Student’s t test. (C) Schematic of the inducible shRNA construct for β-catenin knockdown and shRNA sequences targeting β-catenin. PH1TetO represents the human H1 promoter with Tet operator sequences. Red and green sequences are forward and reverse shRNA sequences of β-catenin, respectively; the loop sequence is shown in blue. (D) Representative phase-contrast and mCherry epifluorescence images of 19-9-11 cells transduced with lentiviral vectors containing the constructs described in C and selected by puromycin treatment. (E) 19-9-11 ishcat-1 and ishcat-2 cells were cultured in mTeSR1 containing 2 μg/mL dox. After 3 d, mRNA was collected, and β-catenin expression was evaluated by quantitative PCR. Error bars represent SEM of three samples. #P < 0.005, ishcat-1 versus iscramble or ishcat-2 versus iscramble; Student’s t test. (F) 19-9-11 ishcat-1 cells were cultured in mTeSR1 medium and were treated with BIO before exposure to 100 ng/mL activin A at day 0 and 5 ng/mL BMP4 at day 1, with 2 μg/mL dox added at the indicated times. Cells were analyzed for cTnT expression by flow cytometry 15 d after initiation of differentiation. Error bars represent SEM. of three independent experiments. #P < 0.005, for each time point versus no dox; Student’s t test.

Figure Legend Snippet: Temporal regulation of Wnt/β-catenin signaling promotes cardiac differentiation induced by serum or growth factors. (A) H9 cells on MEFs were treated with CH in hESC medium for 3 d before forming EBs. EBs were cultured in suspension using serum containing medium for 4 d before being transferred to 0.1% (wt/vol) gelatin-coated plates. The percentage of contracting EBs was determined visually. (B) H9 cells were cultured on Matrigel and treated with DMSO, 1 μM CH, or 1 μM BIO for 3 d before exposure to 100 ng/mL activin A at day 0 and 5 ng/mL BMP4 at day 1 in RPMI/B27-insulin medium using monolayer-directed differentiation. At day 15, the percentage of cTnT+ cells in culture was assessed by flow cytometry. #P < 0.005, CH versus DMSO or BIO versus DMSO; Student’s t test. (C) Schematic of the inducible shRNA construct for β-catenin knockdown and shRNA sequences targeting β-catenin. PH1TetO represents the human H1 promoter with Tet operator sequences. Red and green sequences are forward and reverse shRNA sequences of β-catenin, respectively; the loop sequence is shown in blue. (D) Representative phase-contrast and mCherry epifluorescence images of 19-9-11 cells transduced with lentiviral vectors containing the constructs described in C and selected by puromycin treatment. (E) 19-9-11 ishcat-1 and ishcat-2 cells were cultured in mTeSR1 containing 2 μg/mL dox. After 3 d, mRNA was collected, and β-catenin expression was evaluated by quantitative PCR. Error bars represent SEM of three samples. #P < 0.005, ishcat-1 versus iscramble or ishcat-2 versus iscramble; Student’s t test. (F) 19-9-11 ishcat-1 cells were cultured in mTeSR1 medium and were treated with BIO before exposure to 100 ng/mL activin A at day 0 and 5 ng/mL BMP4 at day 1, with 2 μg/mL dox added at the indicated times. Cells were analyzed for cTnT expression by flow cytometry 15 d after initiation of differentiation. Error bars represent SEM. of three independent experiments. #P < 0.005, for each time point versus no dox; Student’s t test.

Techniques Used: Cell Culture, Flow Cytometry, shRNA, Construct, Sequencing, Transduction, Expressing, Real-time Polymerase Chain Reaction

Modulating regulatory elements of Wnt signaling is sufficient for efficient and reproducible generation of human cardiomyocytes in the absence of growth factors. (A) Schematic of protocol for defined, growth factor-free differentiation of hPSCs expressing dox-inducible β-catenin shRNA to cardiomyocytes via treatment with small molecules. (B and C) 19-9-11 ishcat-1 cells were cultured as indicated in A with dox added 36 h after treatment with 12 μM CH. At day 15, cells were analyzed for cTnT expression by flow cytometry (B) or immunofluorescence (C). In B, the green histogram represents cTnT expression, and the red histogram is an isotype control. (Scale bar in C, 50 μm.) (D) 19-9-11 ishcat-2 cells were cultured as indicated in A, with dox added at different time points after treatment with 12 μM CH. At day 15, cells were analyzed for cTnT expression by flow cytometry. Error bars represent SEM of three independent experiments. *P < 0.05 and #P < 0.005, each time point versus no dox; Student’s t test. (E and F) 19-9-11 ishcat-1 cells were differentiated as described in A, with dox added 36 h after treatment with 12 μM CH. (E) At different time points, mRNA was collected, and RT-PCR analysis of pluripotent, mesendoderm, mesoderm, and cardiac gene expression was performed. (F) Day 7 cells were analyzed for Isl1 and Nkx2.5 expression by immunofluorescence. (Scale bar, 100 μm.)

Figure Legend Snippet: Modulating regulatory elements of Wnt signaling is sufficient for efficient and reproducible generation of human cardiomyocytes in the absence of growth factors. (A) Schematic of protocol for defined, growth factor-free differentiation of hPSCs expressing dox-inducible β-catenin shRNA to cardiomyocytes via treatment with small molecules. (B and C) 19-9-11 ishcat-1 cells were cultured as indicated in A with dox added 36 h after treatment with 12 μM CH. At day 15, cells were analyzed for cTnT expression by flow cytometry (B) or immunofluorescence (C). In B, the green histogram represents cTnT expression, and the red histogram is an isotype control. (Scale bar in C, 50 μm.) (D) 19-9-11 ishcat-2 cells were cultured as indicated in A, with dox added at different time points after treatment with 12 μM CH. At day 15, cells were analyzed for cTnT expression by flow cytometry. Error bars represent SEM of three independent experiments. *P < 0.05 and #P < 0.005, each time point versus no dox; Student’s t test. (E and F) 19-9-11 ishcat-1 cells were differentiated as described in A, with dox added 36 h after treatment with 12 μM CH. (E) At different time points, mRNA was collected, and RT-PCR analysis of pluripotent, mesendoderm, mesoderm, and cardiac gene expression was performed. (F) Day 7 cells were analyzed for Isl1 and Nkx2.5 expression by immunofluorescence. (Scale bar, 100 μm.)

Techniques Used: Expressing, shRNA, Cell Culture, Flow Cytometry, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction

Differentiation of hPSCs to cardiomyocytes via small-molecule modulation of regulatory elements of canonical Wnt signaling. GSK-3 inhibition stimulates mesoderm commitment of undifferentiated hPSCs. In the later stages of differentiation, expression of β-catenin shRNA or inhibition of Wnt ligand production stimulates cardiomyocyte differentiation.

Figure Legend Snippet: Differentiation of hPSCs to cardiomyocytes via small-molecule modulation of regulatory elements of canonical Wnt signaling. GSK-3 inhibition stimulates mesoderm commitment of undifferentiated hPSCs. In the later stages of differentiation, expression of β-catenin shRNA or inhibition of Wnt ligand production stimulates cardiomyocyte differentiation.

Techniques Used: Inhibition, Expressing, shRNA

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