Journal: Nature Communications

Article Title: Tissue-intrinsic beta-catenin signals antagonize Nodal-driven anterior visceral endoderm differentiation

doi: 10.1038/s41467-024-49380-0

Figure Lengend Snippet: a Experimental approach to determine clonal composition of AVE nests. b Expression of clonal labels (red, cyan) and Cer1:H2B-Venus reporter (yellow) in cultures differentiated as in a . Insets on the right show examples of Cer1:H2B-Venus-expressing nests with a single clonal label (top, 13/30 nests), or with multiple labels (bottom, 17/30 nests). One out of n = 2 independent experiments shown. c Immunostaining for OTX2 (magenta) and H2B-Venus (yellow) of Cer1:H2B-Venus reporter cells differentiated for 3 days after an extended doxycycline pulse with 50 ng/ml ActivinA (AA), together with 3 µM Chir99021 (Chi), 20 µM XAV939 (XAV), or 2 µM IWP2 as indicated. One out of n = 3 independent experiments shown. d Flow cytometry of cells differentiated and stained as in c . e Mean percentage of Cer1:H2B-Venus; OTX2 double-positive cells differentiated as in c . n = 4 independent experiments, error bars indicate SD. ** p < 0.001 for AA vs. AA + Chi and AA vs. AA + XAV, and p = 0.0045 for AA vs. AA + IWP2 (two-tailed, unpaired t -test). f Same as e but showing percentage of OTX2-positive cells. ** p < 0.001 for AA vs. AA + Chi and p = 0.0001 for AA vs. AA + XAV, * p = 0.0434 for AA vs. AA + IWP2 (two-tailed, unpaired t -test). Scale bars: 200 µm (( b ) overview); 20 µm (( b ) inset); 500 µm (( c ) overview); 50 µm (( c ) inset). Source data for e , f are provided in the Source Data file.

Article Snippet: To test the functionality of the putative AVE, we adopted a protocol to trigger mesoderm differentiation in ESC aggregates , consisting of a 24 hour treatment with 3 μM Chir99021 (Chi) on day 2, followed by analysis 24 hours after the end of the Chi pulse (Supplementary Fig. ).

Techniques: Expressing, Immunostaining, Flow Cytometry, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Disruption of The Psychiatric Risk Gene Ankyrin 3 Enhances Microtubule Dynamics Through GSK3/CRMP2 Signaling

doi: 10.1101/303990

Figure Lengend Snippet: Changes in EB3 expression and tubulin polymerization induced by brain-specific Ank3 repression are normalized by inhibition of GSK3. (a) Schematic representation of the experimental design. Mouse neuro-2a cells were transfected with pHAGE-EF1α-dCas9-KRAB repressor plasmid and sgRNA(MS2)_EF1α plasmid expressing either the non-targeting control sgRNA or the sgRNA targeting Ank3 exon 1b, followed by puromycin and zeocin selection, and treatment with lithium (1mM), CHIR99021 (1μM), or DMSO vehicle for 1hr prior to cell harvest for protein extraction, (b) Top: Representative Western blot of EB3 and GAPDH. Bottom: Quantification of EB3 expression normalized to GAPDH. Two-way ANOVA, drug effect F (2,30) =4.217 P =0.02, Ank3 repression effect F (1,30) =4.137 P =0.05, interaction F (2,30) =4.217, P =0.02. (c) Top: Representative Western blot of α-tubulin in soluble (S) and polymerized (P) protein fractions. Bottom: Quantification expressed as the ratio of α-tubulin in soluble and polymerized fractions. Two-way ANOVA, drug effect F (2,28) =2.460 P =0.10, Ank3 repression effect F (1,28) =3.281 P =0.08, interaction F (2,28) =5.463 P =0.01. (d) Top: Representative Western blot of GSK3α/β phosphorylation at serine 21 (GSK3α-pS21), serine 9 (GSK3β-pS9), tyrosine 279 (GSK3α-pY279), and tyrosine 216 (GSK3β-pY216), total GSK3α and GSK3β, and GAPDH. Bottom: Quantification of GSK3β-pS9 and GSK3β-pY216 normalized to total GSK3β, and GSK3α-pS21 and GSK3α-pY279 normalized to total GSK3α. (e) Top: Representative Western blot of CRMP2 phosphorylation at threonine 514 (CRMP2-pT514), total CRMP2, and GAPDH. Bottom: Quantification of CRMP2-pT514 normalized to total CRMP2. Two-way ANOVA, drug effect F (2,12) =4.137 P =0.05, Ank3 repression effect F (1,12) =3.281 P =0.08, interaction F (2,12) =4.217 P =0.02. Western blot data were averaged from three independent experiments with three biological replicates per group in each experiment. Control or C, non-targeting sgRNA; Ank3 or A, sgRNA targeting Ank3 exon 1b. Data were analyzed using two-tailed Student’s t -test or ANOVA and Bonferroni post hoc tests. Data are presented as mean ± s.e.m. * P <0.05, ** P <0.01. n.s. indicates not significant.

Article Snippet: For drug experiments, cells were treated for 1 h with the GSK3 inhibitors lithium (Sigma-Aldrich, St. Louis, Mo) or CHIR99021 (LC Laboratories, Woburn, Ma), or for 24 h with the CRMP2 inhibitor lacosamide (Sigma-Aldrich, St. Louis, Mo).

Techniques: Expressing, Inhibition, Transfection, Plasmid Preparation, Selection, Protein Extraction, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Disruption of The Psychiatric Risk Gene Ankyrin 3 Enhances Microtubule Dynamics Through GSK3/CRMP2 Signaling

doi: 10.1101/303990

Figure Lengend Snippet: Rescue of altered EB3 expression and tubulin polymerization induced by brain-specific Ank3 repression is blocked by inhibition of CRMP2. (a) Schematic representation of the experimental design. Mouse neuro-2a cells were transfected with pHAGE-EF1α-dCas9-KRAB repressor plasmid and sgRNA(MS2)_EF1α plasmid expressing either the non-targeting control sgRNA or the sgRNA targeting Ank3 exon 1 b, followed by puromycin and zeocin selection. Cells were subsequently treated with 5μM lacosamide (LCM) for 24h, followed by treatment with lithium (1mM) or CHIR99021 (1μM) for 1h, and cell harvest for protein extraction, (b) Top: Representative Western blot of EB3 and GAPDH. Bottom: Quantification of EB3 expression normalized to GAPDH. Univariate ANOVA, LCM effect F (1,60) =2.00 P =0.163, lithium/CHIR99021 effect F (5,60) =3.586 P =0.03, Ank3 repression effect F (1,60) =21.389 P <0.001, LCM and lithium/CHIR99021 interaction F (2,60) =7.505 P =0.001. (c) Top: Representative Western blot of α-tubulin in soluble (S) and polymerized (P) protein fractions. Bottom: Quantification of the ratio of soluble:polymerized tubulin. Univariate ANOVA, LCM effect F (1,60) =17.561 P <0.001, lithium/CHIR99021 effect F (5,60) =0.203 P =0.817, Ank3 repression effect F (1,60) =68.479 P <0.001, LCM and lithium/CHIR99021 interaction F (2,60) =6.113 P =0.004. Western blot data were averaged from two independent experiments with three biological replicates per group in each experiment. C, control sgRNA; A, Ank3 -targeting sgRNA. Data were analyzed using two-tailed Student’s t -test or ANOVA and Bonferroni post hoc tests. Data are presented as mean ± s.e.m. * P <0.05, ** P <0.01. n.s. indicates not significant.

Article Snippet: For drug experiments, cells were treated for 1 h with the GSK3 inhibitors lithium (Sigma-Aldrich, St. Louis, Mo) or CHIR99021 (LC Laboratories, Woburn, Ma), or for 24 h with the CRMP2 inhibitor lacosamide (Sigma-Aldrich, St. Louis, Mo).

Techniques: Expressing, Inhibition, Transfection, Plasmid Preparation, Selection, Protein Extraction, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Disruption of The Psychiatric Risk Gene Ankyrin 3 Enhances Microtubule Dynamics Through GSK3/CRMP2 Signaling

doi: 10.1101/303990

Figure Lengend Snippet: Brain-specific Ank3 repression is associated with enhanced microtubule dynamics. Left: Under normal Ank3 expression, microtubules are stabilized by binding of microtubule-associated proteins (MAPs), such as EB3 and CRMP2, to the microtubule plus end where α- and β-tubulin heterodimers polymerize to facilitate elongation of the microtubule. As microtubules elongate, EB3 and CRMP2 move along the growing plus end tip to stabilize newly generated microtubule segments. Acetylation (red diamond) accumulates on α-tubulin within the microtubule due to low microtubule turnover. Motor proteins, such as kinesin, mediate transport of cellular cargo along the microtubule towards the plus end, which is oriented towards the distal axon. Right: Repression of brain-specific Ank3 reduces phosphorylation of GSK3 (pS9/pS21) and increases GSK3 activity, leading to an increase in CRMP2 phosphorylation (pT514) and impaired CRMP2 binding and stabilization of microtubules. Microtubules become more susceptible to catastrophes, as demonstrated by increased EB3 expression, reduced EB3 comet length and duration, and increased ratio of soluble:polymerized tubulin. The increased susceptibility to catastrophes increases microtubule turnover and decreases acetylation of α-tubulin. Inhibition of GSK3 activity by lithium or CHIR99021 reduces CRMP2 phosphorylation (pT514), thereby allowing CRMP2 to bind and stabilize microtubules. Pharmacological inhibition of CRMP2 by lacosamide reduces CRMP2 binding and stabilization of microtubules, which increases the amount of free tubulin and decreases acetylation of α-tubulin. Enhanced microtubule dynamics induced by brain-specific Ank3 repression may have a range of effects (e.g. axonal transport of synaptic vesicles, microtubule interaction with motor proteins) that alter neuronal function.

Article Snippet: For drug experiments, cells were treated for 1 h with the GSK3 inhibitors lithium (Sigma-Aldrich, St. Louis, Mo) or CHIR99021 (LC Laboratories, Woburn, Ma), or for 24 h with the CRMP2 inhibitor lacosamide (Sigma-Aldrich, St. Louis, Mo).

Techniques: Expressing, Binding Assay, Generated, Activity Assay, Inhibition

Journal: Genes to Cells

Article Title: Neural cells play an inhibitory role in pancreatic differentiation of pluripotent stem cells

doi: 10.1111/gtc.12308

Figure Lengend Snippet: β‐cell differentiation of mouse embryonic stem ( ES ) cells is inhibited by co‐culture with neural cells and promoted by Wnt pathway activation. (a–c) β‐cell differentiation from ES cells was tested by co‐culture with neural cells derived from ES cells using a transwell culture. Schematic diagram for combining mycophenolic acid ( MPA ) (2 μ m ) with CHIR 99021 (1 μ m ) treatment and with the neural cell co‐culture (a), and a diagram showing the transwell culture (b). The proportion of insulin‐ and GFP (Pdx1) double‐positive β‐cells on day 12 ( n = 4) (c). (d,e) The Wnt pathway activator CHIR 99021 potentiated pancreatic differentiation, whereas MPA ‐mediated potentiation was inhibited by the Wnt pathway inhibitor XAV 939. The proportion of insulin‐ and GFP (Pdx1) double‐positive β‐cells on day 12 ( n = 3) after treatments with CHIR 99021 (1 μ m ) (d) or MPA (2 μ m ) and XAV 939 (0.02, 0.1, 0.5, 2.5 and 5 μ m ) (e). Data represent mean ± SEM . * P < 0.05, by Student's t ‐test. # P < 0.05, by Dunnett's test.

Article Snippet: On day 0, differentiation was initiated by replacing the culture with Medium A, consisting of DMEM with 4500 mg/L glucose supplemented with 3 μ m CHIR99021 (Merck KGaA, Darmstadt, Germany), 100 ng/mL recombinant human Activin‐A and 2% B27 supplement (Life Technologies).

Techniques: Cell Differentiation, Co-Culture Assay, Activation Assay, Derivative Assay

Journal: Genes to Cells

Article Title: Neural cells play an inhibitory role in pancreatic differentiation of pluripotent stem cells

doi: 10.1111/gtc.12308

Figure Lengend Snippet: β‐Cell differentiation of human induced pluripotent stem ( iPS ) cells is inhibited by co‐culture with neural cells and promoted by Wnt pathway activation. (a–d) Toe human iPS cell differentiation into β‐cells was inhibited by co‐culture with mouse embryonic stem‐derived neural cells from stage 2 (differentiation days 3–19). A schematic diagram of human iPS cell differentiation with the neural cell co‐culture (a), and a diagram showing the transwell culture (b). The proportion of insulin‐positive β‐cells on day 19 ( n = 6–9) (c). Immunostaining of insulin on day 19 (d). (e–g) Toe human iPS cell differentiation into β‐cells was enhanced when added with CHIR 99021, but inhibited when co‐cultured with the neural cells, during stage 5 (differentiation days 13–19). The effect of adding CHIR 99021 on the proportion of insulin‐positive β‐cells on day 19 ( n = 3–6) (e). The effect of neural cell co‐culture on the proportion of insulin‐positive β‐cells on day 19 ( n = 8) (f ). Immunostaining of insulin (green), somatostatin ( SS , red), and glucagon (cyan) on day 19 (g). Data represent mean ± SEM . * P < 0.05, by Student's t ‐test. Scale bar indicates 100 μm.

Article Snippet: On day 0, differentiation was initiated by replacing the culture with Medium A, consisting of DMEM with 4500 mg/L glucose supplemented with 3 μ m CHIR99021 (Merck KGaA, Darmstadt, Germany), 100 ng/mL recombinant human Activin‐A and 2% B27 supplement (Life Technologies).

Techniques: Cell Differentiation, Co-Culture Assay, Activation Assay, Derivative Assay, Immunostaining, Cell Culture