wnt signaling inhibitor iwp2  (PeproTech)

Journal: Nature

Article Title: WNT signalling control by KDM5C during development affects cognition

doi: 10.1038/s41586-024-07067-y

Figure Lengend Snippet: a , Immunofluorescence for TBR2 and ( b ) CTIP2 and TUJI at day 60 of neuronal differentiation in patient Mutant and Corrected lines. Cells were counterstained with Hoechst. More than 3 independent experiments were performed with similar results. Scale bars, 100 µm. c , qPCR analysis of TBR2 , CTIP2 and TUJI mRNAs in Mutant (M) and Corrected lines (C1 and C2) at day 60. Data are represented as mean ± SD of 3 independent experiments. The p -values by two-tailed unpaired Student’s t-test are indicated. P < 0.05 was considered statistically significant. d , Immunofluorescence for CTIP2 and ( e ) SATB2 and TUJI at day 90 of neuronal differentiation in Mutant and Corrected lines. Cells were counterstained with Hoechst. More than 3 independent experiments were performed with similar results. Scale bars, 100 µm. f , qPCR analysis of CTIP2 , TUJI and SATB2 mRNAs in Mutant (M) and Corrected lines (C1 and C2) at day 90. Data are represented as mean ± SD of 3 independent experiments. The p -values by two-tailed unpaired Student’s t-test are indicated. P < 0.05 was considered statistically significant. g , Immunofluorescence analysis for TBR2 and ( h ) CTIP2 and TUJI in patient Mutant and Corrected lines at day 30 of neuronal differentiation, which have been treated transiently either with vehicle (Mutant (M), Corrected 1 and Corrected 2), IWP2 (1 mM (first pulse)/0.25 mM (second pulse)) or a combination of IWP2 (1 mM) and Wnt3a (200 ng/ml). More than 3 independent experiments were performed with similar results. i , Immunofluorescence for TUJI at day 11 of neuronal differentiation in Mutant and Corrected lines. Cells were counterstained with Hoechst. Corrected cells cultured with Wnt3a (200 ng/ml) show regions of greater axonal outgrowth similarly as observed in Mutant cells at this time of differentiation. More than 3 independent experiments were performed with similar results. This data is related to Extended Data Fig. . Abbreviations: C1=Corrected 1, C2=Corrected.

Article Snippet: Recombinant WNT3A protein (R&D Systems, 5036-WN-010/CF, 50 ng (for behavioural studies) and 33 ng (for lower dosage spine density analyses)) and the WNT signalling inhibitor IWP2 (9.34 ng; Sigma Aldrich, 10536) were dissolved in PBS.

Techniques: Immunofluorescence, Mutagenesis, Two Tailed Test, Cell Culture

Journal: Nature

Article Title: WNT signalling control by KDM5C during development affects cognition

doi: 10.1038/s41586-024-07067-y

Figure Lengend Snippet: a , Immunofluorescence for PAX6, NESTIN and ( b ) SOX2 at day 7 of neuronal differentiation in Mutant and two Corrected lines of brother 2. Cells were counterstained with Hoechst. 3 independent experiments were performed with similar results. c , qPCR analysis for PAX6 , NESTIN and SOX2 mRNAs at day 7 in the second brother. Data are represented as mean ± SD of 3 independent experiments. The p -values by two-tailed unpaired Student’s t-test are indicated. P < 0.05 was considered statistically significant. d , Immunofluorescence for PAX6, ZO1 and ( e ) TBR2 and KI67 at day 16 of neuronal differentiation in Mutant and two Corrected lines of brother 2. 3 independent experiments were performed with similar results. f , Rosette count in Mutant (M) and Corrected lines (C1 and C2) (top) and qPCR analysis for TBR2 and TUJI mRNAs at day 16 in the second brother. Data are represented as mean ± SD of 3 independent experiments. The p -values by two-tailed unpaired Student’s t-test are indicated. P < 0.05 was considered statistically significant. g , Immunofluorescence for TBR2 and ( h ) CTIP2 and TUJI at day 32 of neuronal differentiation in Mutant and two Corrected lines of brother 2. Cells were counterstained with Hoechst. 3 independent experiments were performed with similar results. i , qPCR analysis for TBR2, TUJI and CTIP2 mRNAs at day 32 in the second brother. Data are represented as mean ± SD of 3 independent experiments. The p -values by two-tailed unpaired Student’s t-test are indicated. P < 0.05 was considered statistically significant. j , Immunofluorescence for TBR2 and ( k ) CTIP2 and TUJI at day 60 of neuronal differentiation in Mutant and two Corrected lines of brother 2. Cells were counterstained with Hoechst. 3 independent experiments were performed with similar results. l , qPCR analysis for TBR2, TUJI and CTIP2 mRNAs at day 60 in the second brother. Data are represented as mean ± SD of 3 independent experiments. The p -values by two-tailed unpaired Student’s t-test are indicated. P < 0.05 was considered statistically significant. m , Western blotting of nuclear and cytoplasmatic fractions in Mutant and Corrected lines of brother 2 at day 7, 14 and 30 of neuronal differentiation. GAPDH and Histone H3 were used to mark the cytosolic and nuclear fraction respectively. β-Catenin expression in the cytoplasm and nucleus is indicated. At day 14, 3 independent experiments, and at day 7 and day 30, 2 independent experiments were performed. GAPDH, Histone H3 and β-Catenin were run on the same gel. For gel source data see Supplementary Fig. . n , q-PCR analysis of TBR2 , CTIP2 and TUJI mRNAs at day 30 of neuronal differentiation after treatment regime according to Fig. with the Wnt inhibitor IWP2 in Mutant cells and Wnt induction with recombinant Wnt3a in Corrected cells of brother 2. Data are represented as mean ± SD of 3 independent experiments. The p -values by two-tailed unpaired Student’s t-test are indicated. P < 0.05 was considered statistically significant. Abbreviations: M=Mutant; C1 and C2=Corrected 1 and Corrected 2.

Article Snippet: Recombinant WNT3A protein (R&D Systems, 5036-WN-010/CF, 50 ng (for behavioural studies) and 33 ng (for lower dosage spine density analyses)) and the WNT signalling inhibitor IWP2 (9.34 ng; Sigma Aldrich, 10536) were dissolved in PBS.

Techniques: Immunofluorescence, Mutagenesis, Two Tailed Test, Western Blot, Expressing, Recombinant

Journal: Nature

Article Title: WNT signalling control by KDM5C during development affects cognition

doi: 10.1038/s41586-024-07067-y

Figure Lengend Snippet: a , Volcano plot showing differential gene expression (log2 fold change ≥ 1) between patient Mutant and Corrected cells at day 16 (top) and day 32 (bottom). P -values for a two-group comparison were calculated using a two-sided Student’s t-test. b , Gene Set Enrichment Analysis (GSEA) plots showing the enrichment of up and downregulated genes in the set of genes annotated as GO “Cerebral Cortex Neuron Differentiation” at day 16 and ( c ) day 32. Mutant versus Mutant+Inhibitor IWP2 (1 mM (first pulse)/0.25 mM (second pulse)) are shown at the top and Corrected versus Corrected+Wnt3a (200 ng/ml) are shown at the bottom. Genes are ordered based on the differential expression values obtained from DESeq2. d , Gene Set Enrichment Analysis (GSEA) plots showing the enrichment of up and downregulated genes in the set of genes annotated as GO “Central Nervous System Neuron Differentiation” at day 16 and ( e ) day 32. Mutant versus Mutant+Inhibitor (IWP2) are shown at the top and Corrected versus Corrected+Wnt3a are shown at the bottom. Genes are ordered based on the differential expression values obtained from DESeq2. f , Heatmaps showing gene expression for critical neuronal genes in Mutant (Mut), Mutant+Inhibitor (IWP2) (Mut+I), Corrected (Corr) and Corrected+Wnt3a (Corr+W) at day 16 and ( g ) day 32. h , Heatmaps for differential gene expression (q = 0.01) of transcripts for signaling pathways, growth factors (GF) and genes that are members of the GO enrichment term “small GTPase mediated signal transduction” at day 32 of differentiation that are sensitive to Wnt signaling perturbations. Abbreviations: PDGF = PDGF growth factors; INS=insulin; NON.C.W= non canonical WNT. i , Heatmaps for differential gene expression (q = 0.01) (log2fold change, q = 0.01) of transcripts between the disease group (Mutant and Corrected+Wnt3a) and the Corrected group (Corrected and Mutant+Inhibitor (IWP2)) at day 16 and ( j ) day 32 of neuronal differentiation. k , Gene ontology (GO) analysis of genes differentially expressed (q ≤ 0.01) between the Corrected group (Corrected and Mutant+Inhibitor) and the disease group (Mutant and Corrected+Wnt3a) at day 16 (top) and ( l ) day 32 (bottom). Extracted from i and j .

Article Snippet: Recombinant WNT3A protein (R&D Systems, 5036-WN-010/CF, 50 ng (for behavioural studies) and 33 ng (for lower dosage spine density analyses)) and the WNT signalling inhibitor IWP2 (9.34 ng; Sigma Aldrich, 10536) were dissolved in PBS.

Techniques: Expressing, Mutagenesis, Comparison, Transduction

Journal: Nature

Article Title: WNT signalling control by KDM5C during development affects cognition

doi: 10.1038/s41586-024-07067-y

Figure Lengend Snippet: a , b , PCA plots of gene expression data from mutant cells (Mut), mutant cells treated with the WNT inhibitor IWP2 (1 µM (first pulse) and then 0.25 µM (second pulse)) (Mut+Inh), corrected cells (Corr) and corrected cells treated with WNT3A (200 ng ml –1 ) (Corr+W) using all 26,830 genes (iGenome UCSC hg38) in our dataset at day 16 ( a ) and day 32 (d32) (day 16 (d16) results also included) ( b ) of neuronal differentiation. c , Mutant cells exhibit a global transcriptomic change, referred to as reprogramming, towards the profile of corrected cells after brief WNT inhibitor treatment. Conversely, corrected cells undergo transcriptional reprogramming to mirror the mutant cell transcriptome following transient treatment with recombinant WNT3A protein. d , e , Volcano plots at day 16 ( d ) and day 32 ( e ) (log 2 (fold change) ≥ 1 and P ≤ 0.01) for transcripts detected by RNA-seq analysis. Top, mutant lines are compared with either mutant + inhibitor cells (left) or corrected + WNT3A cells (right). Bottom, corrected cells are compared either to corrected + WNT3A cells or mutant + inhibitor cells. Cut-off at P value = 0.05 and log 2 (fold change) = ±1. P values for two-group comparison were calculated using two-sided Student’s t -test. f , PCA of ATAC–seq data from mutant cells, mutant cells treated with the WNT inhibitor IWP2 (1 µM (first pulse) then 0.25 µM (second pulse)), corrected cells and corrected cells treated with WNT3A (200 ng ml –1 ) using all peaks in the genome at days 8, 12, 16 and 32 of neuronal differentiation. g , h , Heatmaps representing open chromatin regions (false discovery rate (FDR) ≤ 0.05) between the indicated cell lines at day 16 ( g ) and day 32 ( h ). i , IGV snapshot of open chromatin peaks (ATAC–seq) between the indicated cell lines at days 16 and 32 of differentiation.

Article Snippet: Recombinant WNT3A protein (R&D Systems, 5036-WN-010/CF, 50 ng (for behavioural studies) and 33 ng (for lower dosage spine density analyses)) and the WNT signalling inhibitor IWP2 (9.34 ng; Sigma Aldrich, 10536) were dissolved in PBS.

Techniques: Expressing, Mutagenesis, Recombinant, RNA Sequencing Assay, Comparison

Journal: Nature

Article Title: WNT signalling control by KDM5C during development affects cognition

doi: 10.1038/s41586-024-07067-y

Figure Lengend Snippet: a , Correlation heatmap of open chromatin regions (i.e. ATAC-seq peaks) at day 8, 12, 16 and 32. b , Principal component analysis (PCA) of ATAC-seq signals using all peaks in the genome at day 8, 12, 16 and 32 of neuronal differentiation representing all samples from the second biological replicate. c , Pie charts showing the genomic distribution of open chromatin regions (i.e. ATAC-seq peaks) in patient Mutant and Corrected cells at day 8 and ( d ) day 12. e , Pie charts showing the genomic distributions of open chromatin regions in Mutant, Mutant+Inhibitor (IWP2) (1 mM (first pulse)/0.25 mM (second pulse)), Corrected and Corrected+Wnt3a (200 ng/ml) cells at day 16 and ( f ) day 32. g , FPKM expression values for selected genes as extracted from RNA-seq data at day 16 and day 32. Data are represented as mean ± SD of 3 independent experiments. Abbreviations in the Figure: M or Mut=Mutant; C or Corr=Corrected; I or Inh=Wnt Inhibitor; W=Wnt3a.

Article Snippet: Recombinant WNT3A protein (R&D Systems, 5036-WN-010/CF, 50 ng (for behavioural studies) and 33 ng (for lower dosage spine density analyses)) and the WNT signalling inhibitor IWP2 (9.34 ng; Sigma Aldrich, 10536) were dissolved in PBS.

Techniques: Mutagenesis, Expressing, RNA Sequencing Assay

Journal: Nature

Article Title: WNT signalling control by KDM5C during development affects cognition

doi: 10.1038/s41586-024-07067-y

Figure Lengend Snippet: a and b , KEGG and Gene ontology (GO) analysis in patient Mutant and Corrected cells at day 8 and c and d , day 12. Abbreviations: BP=Biological Processes; CC=Cellular Components; MF=Molecular Function. e , Most enriched Gene ontology (GO) terms for Biological Process, f , Cellular Components and g , Molecular Function for differentially accessible chromatin regions between three cellular comparisons (Corrected versus Mutant, Corrected versus Corrected+Wnt3a (+ W) (200 ng/ml) and Mutant versus Mutant+Inhibitor (+ Inh) cells (IWP2, 1 mM (first pulse)/0.25 mM (second pulse)) at day 16 and day 32 (FDR ≤ 0.05). h-j , KEGG analysis at day 16 and, k-m , day 32 between the same three cellular comparisons as in e-g (FDR ≤ 0.05). a-m , One-sided Fisher exact tests with multiple comparison correction were performed.

Article Snippet: Recombinant WNT3A protein (R&D Systems, 5036-WN-010/CF, 50 ng (for behavioural studies) and 33 ng (for lower dosage spine density analyses)) and the WNT signalling inhibitor IWP2 (9.34 ng; Sigma Aldrich, 10536) were dissolved in PBS.

Techniques: Mutagenesis, Comparison

Journal: Nature

Article Title: WNT signalling control by KDM5C during development affects cognition

doi: 10.1038/s41586-024-07067-y

Figure Lengend Snippet: a , Schematic of the experiment. PBS or recombinant WNT3A (50 ng) was injected into the lateral ventricle of WT E13.5 embryos. b , Elevated-plus maze anxiety test. n = 14 (PBS treated) and n = 16 (WNT3A treated) mice. c , Marble-burying test in PBS-injected mice ( n = 12) and WNT3A-injected mice ( n = 11). Data for b and c represent the mean ± s.e.m. P values calculated using one-tailed unequal variance t -test. d , Morris water maze escape latency test. One-tailed unequal variance t -test for results from the visible platform condition (V1 and V2) and two-way analysis of variance (ANOVA) for the hidden platform (H1–H5) and reversal (R1–R3) conditions were performed. P values are indicated. Data are presented as the mean ± s.e.m. n = 15 (PBS injected) and n = 17 (WNT3A injected) mice. e , At E13.5, WT embryos were injected with PBS, a low dose of WNT3A (33 ng; LW) or a high dose of WNT3A (50 ng; HW). Spine density in the basolateral amygdala (BLA), prefrontal cortex (PFC) and CA1 of the hippocampus was analysed (number of spines per µm). One-way ANOVA followed by Tukey’s multiple comparison test is presented based on single measurements averaged for each group. P values are indicated. Data are expressed as the mean ± s.e.m. of average values for each mouse. n = 7 (PBS) mice for basal spine density, n = 6 (PBS) mice for apical spine density, n = 4 mice for LW treatment and n = 3 mice for HW treatment. f , Representative dendritic segments from BLA, PFC, and CA1 regions in mice treated with PBS or with a high or low dose of WNT3A. g , Schematic of injections in E13.5 WT (PBS) and Kdm5c KO embryos (PBS or the WNT inhibitor IWP2 (9.34 ng)). h , Elevated plus maze anxiety test. Number of mice analysed: 8 WT + PBS (W+P), 9 KO + PBS (K+P) and 9 KO + IWP2 (K+I). i , Marble-burying test. Number of mice analysed: 13 WT + PBS, 7 KO + PBS and 9 KO + IWP2. Data for h and i are presented as the mean ± s.e.m. P values were calculated using one-tailed unequal variance t -test. j , Morris water maze escape latency test. Two-way ANOVA for V1–V2, H1–H5 and R1–R3 was performed. P values are indicated. Data are expressed as the mean ± s.e.m. n = 12 WT + PBS, 7 KO + PBS and 9 KO + IWP2 mice. P < 0.05 was considered significant. Details provided in the . Illustrations in a and g were created using BioRender ( https://www.biorender.com ).

Article Snippet: Recombinant WNT3A protein (R&D Systems, 5036-WN-010/CF, 50 ng (for behavioural studies) and 33 ng (for lower dosage spine density analyses)) and the WNT signalling inhibitor IWP2 (9.34 ng; Sigma Aldrich, 10536) were dissolved in PBS.

Techniques: Recombinant, Injection, One-tailed Test, Comparison