gcamp variants  (New England Biolabs)

Journal: Cell & Bioscience

Article Title: β-catenin mediates endodermal commitment of human ES cells via distinct transactivation functions

doi: 10.1186/s13578-024-01279-5

Figure Lengend Snippet: Generation of new CTNNB1-/- hESC clones and rescue of DE differentiation by ectopic expression of β-catenin. A Schematics of the donor and CRISPR/Cas9-based targeting strategy for insertional disruption of CTNNB1 gene via NHEJ mechanism. B Morphology and immunostaining of undifferentiated wt hESC (H1) and CTNNB1-/- clones (#3 and #7). Bright field images were shown with scale bar 100 μm. Immunostainings were performed using antibodies specific to β-catenin, pluripotency marker OCT4, NANOG, SOX2, and membrane protein E-cadherin, α-catenin and JUP. Scale bars = 50 μm in fluorescence images. C Western blot detection of β-catenin, JUP, E-cadherin, and β-catenin proteins in undifferentiated wt hESC (H1) and CTNNB1-/- clones (#3 and #7). D DE induction from wt hESC (H1) and CTNNB1-/- clones (#3 and #7). Shown are the workflow diagram for DE induction (upper) and results of DE differentiation (lower). Bright field images were taken at day 2 (d2) and day 4 (d4) post DE induction. Immunostaining for DE marker FOXA2 and SOX17 were performed at DE (d4). Nuclei were counterstained using Hoechst. Scale bars = 50 μm. E Lentivirus-delivered CTNNB1 expression in CTNNB1-/- clones. Shown are schematics of the Dox-inducible CTNNB1 cassette delivered by lentivirus (Lenti- CTNNB1 ) (upper) and qRT-PCR results from the transduced CTNNB1-/- clones (#3 and #7) (lower), which confirmed the Dox-induced expression of CTNNB1 transgene (CDS) and constant depletion of endogenous CTNNB1 mRNA (3’UTR). Data were normalized to wt hESC (H1) (red dashed line) and presented as mean ± SD. Student’s t-test was performed between Dox-induced and non-induced cells. *, p ≤ 0.05. F DE induction from Lenti- CTNNB1 transduced CTNNB1-/- clones (#3 and #7). Shown are the workflow diagram for Dox-based DE induction (upper) and results of DE differentiation in the absence (–) and presence (+) of Dox (lower). Bright-field images were taken at DE (d2) and immunostainings were performed for FOXA2 and SOX17 at DE (d4). Nuclei were counterstained using Hoechst (shown in small box). Scale bars = 50 μm

Article Snippet: The full-length coding sequences (CDS) of human CTNNB1 transcript variant 1 (NM_001904.3) (2346 bp) and CDH1 transcript variant 1 (NM_004360.5) (2649 bp) were amplified from H1 hESCs by RT-PCR and cloned into Fuw-tetO lentiviral vector carrying Dox-inducible CMV promoter (Addgene #20726) [ ].

Techniques: Clone Assay, Expressing, CRISPR, Disruption, Immunostaining, Marker, Membrane, Fluorescence, Western Blot, Quantitative RT-PCR

Journal: Cell & Bioscience

Article Title: β-catenin mediates endodermal commitment of human ES cells via distinct transactivation functions

doi: 10.1186/s13578-024-01279-5

Figure Lengend Snippet: Dose-dependent nuclear translocation and E-cadherin sequestration of ΔN 148 C mutant affect DE rescue outcomes. A Nuclear translocation of ΔN 148 and ΔN 148 C β-catenin mutants. Immunostaining of the ΔN 148 , ΔN 148 C and ΔC clones were performed after Dox treatment for 24 h (upper panels) and at DE (d1) (lower panels). Antibodies used were specific to β-catenin or Flag-tag. Two individual rescue clones were analyzed for each β-catenin mutant. The Dox-induced expression of CTNNB1 mutant transgenes were verified by qRT-PCR (middle panel) using primers binding to N-terminal (N-ter), central (ARM), and C-terminal (C-ter) regions in the CTNNB1 CDS. The data values were normalized to wt hESCs (H1) (dashed red lines). B Dose-dependent nuclear translocation of ΔN 148 C (left) and corresponding DE differentiation (right). Left panels showed the immunostaining of ΔN 148 C-F2 clone cells treated with Dox at different concentrations (indicated) for 24 h. Antibodies used were specific to E-cadherin (yellow) and Flag-tag (red). Shown were images with merged signals and close views of selected areas inside the dashed line boxes were shown below. Right panel showed the qRT-PCR analysis of DE-related genes ( FOXA2, SOX17, GATA4, GATA6 and EOMES ) at day 4 post DE induction, using Dox at different concentrations. The data values were normalized to that in wt hESCs (H1). For statistical analysis, all data were compared to that of non-induced cells. C E-cadherin expression in CDH1-/- hESCs derived from ΔN 148 C-F2 clone. Immunostaining of ΔN 148 C-F2 clone and ΔN 148 C-F2; CDH1-/- clones (#6 and #13) were performed. Antibody used were specific to E-cadherin (yellow). qRT-PCR and western blot quantified CDH1 mRNA and E-cadherin protein levels, respectively (lower panels). D Nuclear translation of ΔN 148 C in CDH1-/- hESC. Immunostaining of ΔN 148 C-F2 clone and ΔN 148 C-F2; CDH1-/- clones (#6 and #13) were performed after Dox treatment for 24 h (upper panels) and at DE (d1) (lower panels). Antibodies used were specific to Flag-tag (red). E Relative expression (fold) of FOXA2 and SOX17 at DE (d4) in ΔN 148 C-F2 clone and ΔN 148 C-F2; CDH1-/- clones (#6 and #13). For statistical analysis, data of ΔN 148 C-F2; CDH1-/- clones were compared to that of ΔN 148 C-F2 cells. F E-cadherin levels in CDH1- transduced ΔN 148 C-F2 clone. Immunostaining of ΔN 148 C-F2 and ΔN 148 C-F2; CDH1 OE cells were performed before and after Dox treatment for 6 h (upper panels). Antibodies used were specific to E-cadherin (yellow). qRT-PCR and western blot analysis showed CDH1 mRNA (lower left) and E-cadherin protein levels (lower right), respectively. G Nuclear translation (fold) of ΔN 148 C in CDH1 OE cells. Immunostaining of ΔN 148 C-F2 clone and ΔN 148 C-F2;C DH1 OE cells were performed after Dox treatment for 24 h (upper panels) and at DE (d1) (lower panels), using antibody specific to Flag-tag (red). Areas surrounded by the dashed white lines were enlarged below. H Relative expression (fold) of FOXA2 and SOX17 at DE (d4) in ΔN 148 C-F2 and the ΔN 148 C-F2;C DH1 OE cells. For statistical analysis, ΔN 148 C-F2;C DH1 OE clones’ data were compared to that of ΔN 148 C-F2 cells. Nuclei were counterstained using Hoechst (blue) in A , B , C , D , F and G . Scale bars = 50 μm in all images. The qRT-PCR data in A , B , C , E , F , and H were presented as mean ± SD (n = 3). Student’s t-test were performed for statistical analysis. ns, not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001

Article Snippet: The full-length coding sequences (CDS) of human CTNNB1 transcript variant 1 (NM_001904.3) (2346 bp) and CDH1 transcript variant 1 (NM_004360.5) (2649 bp) were amplified from H1 hESCs by RT-PCR and cloned into Fuw-tetO lentiviral vector carrying Dox-inducible CMV promoter (Addgene #20726) [ ].

Techniques: Translocation Assay, Mutagenesis, Immunostaining, Clone Assay, FLAG-tag, Expressing, Quantitative RT-PCR, Binding Assay, Derivative Assay, Western Blot