tcf4 knockdown plasmid  (Addgene inc)

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-495 Modulates Neuronal Layer Fate Determination by Targeting Tcf4

doi: 10.7150/ijbs.94739

Figure Lengend Snippet: TCF4 promotes the increased generation of deep-layer neurons. (A) Schematic diagram of TCF4 overexpression clone construction. (B) Western blot validation of TCF4 overexpression clone efficiency. β-actin is as the reference protein. (C-H) pCIG+pCAG (control) and pCIG- Tcf4 +pCAG (TCF4) were electroporated into embryonic brains from mice at E13.5. Brain sections at P3 were immunostained with cortical markers (CUX1 and SOX5). Scale bar: 500 µm (C, D) and 100 µm (E-H, E'-H'). (I) The sections labelled A' to B' were divided into 10 bins to count the distribution of EGFP-positive cells in the cortex. These sections were derived from the electroporated mouse brains, which were from different littermates (control: n=15 sections from 13 brains, TCF4: n=4). (J) Statistical analysis of the percentage of CUX1+/GFP+ cells in (E', F') and SOX5+/GFP+ cells in (G', H') (control: n = 15 sections from 13 brains, TCF4: n=4). Statistical significance was determined using an unpaired two-tailed Student's t-test. *P<0.05, ** P<0.01 and ***P<0.001

Article Snippet: The TCF4 knockdown plasmid was constructed using the pLL3.7 vector (Addgene).

Techniques: Over Expression, Western Blot, Biomarker Discovery, Control, Derivative Assay, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-495 Modulates Neuronal Layer Fate Determination by Targeting Tcf4

doi: 10.7150/ijbs.94739

Figure Lengend Snippet: TCF4 knockdown restrains the generation of deep-layer neurons. (A) Schematic diagram of TCF4 knockdown clone construction. The designations sh1, sh2, and sh3 refer to pLL3.7-sh1- Tcf4 , pLL3.7-sh2- Tcf4 , and pLL3.7-sh3- Tcf4 , respectively. (B) WB validation of TCF4 knockdown clone efficiency. β-actin is as the reference protein. (C-K) pLL3.7-Scr+pCAG, pLL3.7-sh1- Tcf4 + pCAG and pLL3.7-sh2- Tcf4 + pCAG were electroporated into the embryonic brains of mice at E13.5. Brain sections at P3 were immunostained with cortical markers (CUX1 and SOX5). Scale bar: 500 µm (C, D, E) and 100 µm (F-K, F'-K') . (L) The sections labelled F to H were divided into 10 bins to count the distribution of EGFP-positive cells in the cortex. These sections were derived from the electroporated mouse brains, which were from different littermates (Scr: n=3, sh1-Tcf4: n=3, sh2-Tcf4: n=3). (M) The percentage of CUX1+/GFP+ cells and SOX5+/GFP+ cells in (F-K) (Scr: n=3, sh1-Tcf4: n=3, sh2-Tcf4: n=3). Statistical significance was determined using an unpaired two-tailed Student's t-test. Results are expressed as the mean ±SD. P values are shown as *P<0.05, ** P<0.01.

Article Snippet: The TCF4 knockdown plasmid was constructed using the pLL3.7 vector (Addgene).

Techniques: Knockdown, Biomarker Discovery, Derivative Assay, Two Tailed Test

Journal: Nature communications

Article Title: Transcription Factor 4 loss-of-function is associated with deficits in progenitor proliferation and cortical neuron content.

doi: 10.1038/s41467-022-29942-w

Figure Lengend Snippet: Fig. 8 Reversal of abnormal phenotypes in PTHS organoids after trans-epigenetic correction of TCF4 expression. a Schematic representation of CRISPR-based trans-epigenetic correction of TCF4 expression using constructs for guide RNA (gRNA), transcriptional activation module MPH, and dead Cas9 (see “Methods” for details). b Top: Virus application regimen. Bottom: Brightfield images of PTHS brain organoids at 4 weeks in vitro after correction of TCF4 expression (PTHS + TCF4 gRNA), compared with controls transduced with scrambled gRNA (scr gRNA). c Fluorescence microscopy images of transduced organoids at 2 weeks in vitro after immunostaining for TCF4 (green). Clustered TCF4+ cells (arrowhead) can be seen in aberrant outgrowth in image on the right in PTHS + scr gRNA condition. See Supplementary Fig. 12d, e for quantification of number of TCF4+ cells and mean TCF4 staining pixel intensity in transduced organoids. d Increase in TCF4 expression levels after CRISPR-mediated trans-epigenetic TCF4 correction in organoids at 2 weeks in vitro. N = 3 replicates per group (circles). e–g Expression levels of GADD45G (e), CDKN2A (f), and MAP2 (g) in organoids at 4 weeks in vitro after trans- epigenetic TCF4 expression correction. N = 3 replicates per group (circles). Organoids are from parent–patient pair #4. h Transduced organoids stained for MAP2 (magenta) and SOX2 (green), at two developmental time points. Arrowheads in middle panels: polarized PTHS organoids. High mag insets: clustered abnormally shaped MAP2+ cells in polarized organoid outgrowth. Arrowhead in right panel: neural rosettes. Experiments were conducted with organoids from parent-patient pair #4 (circle symbols in bar graphs). Colors in bar graphs represent parents (orange), PTHS (blue), or genetically manipulated PTHS (light blue) groups. Error bars represent SEM. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA followed by Tukey’s HSD post-hoc test in bar plots. Scale bars are 100 μm. DAPI nuclear staining in blue. See Supplementary Data 1 for sample and effect sizes and exact p-values. Attribution of DNA image in a: Ioana Davies/Shutterstock.com.

Article Snippet: To evaluate the efficiency of the designed TCF4 gRNA sequences at increasing the endogenous expression of the TCF4 gene via trans-epigenetic activation, we transfected SH-SY5Y cells with the pLentiSAMv2 and pLentiMPHv2 (Addgene #89308; http://n2t.net/addgene:89308; RRID:Addgene_89308) plasmids, followed by RT-qPCR to verify the levels of TCF4 transcripts.

Techniques: Expressing, CRISPR, Construct, Activation Assay, Virus, In Vitro, Transduction, Fluorescence, Microscopy, Immunostaining, Staining

Journal: Nature communications

Article Title: Transcription Factor 4 loss-of-function is associated with deficits in progenitor proliferation and cortical neuron content.

doi: 10.1038/s41467-022-29942-w

Figure Lengend Snippet: Fig. 10 Model of dysregulated pathways underlying PTHS pathophysiology. Mechanistic model to explain aberrant cellular phenotypes in PTHS neural structures. Due to TCF4 loss-of-function in PTHS, Wnt signaling activity diminishes, in turn leading to decreased SOX3 expression in NPCs, impairing proliferation. Moreover, we observed that SOX4 is also downregulated in PTHS cells, which we suggest impairs neuronal differentiation and content in the PTHS neural tissue.

Article Snippet: To evaluate the efficiency of the designed TCF4 gRNA sequences at increasing the endogenous expression of the TCF4 gene via trans-epigenetic activation, we transfected SH-SY5Y cells with the pLentiSAMv2 and pLentiMPHv2 (Addgene #89308; http://n2t.net/addgene:89308; RRID:Addgene_89308) plasmids, followed by RT-qPCR to verify the levels of TCF4 transcripts.

Techniques: Activity Assay, Expressing

Journal: Cancer research

Article Title: Hotspot ESR1 mutations are multimodal and contextual modulators of breast cancer metastasis

doi: 10.1158/0008-5472.CAN-21-2576

Figure Lengend Snippet: A & B. Representative images (A) and quantification (B) of wound scratch assay of T47D WT and ESR1 mutant cells performed using IncuCyte living imaging system over 72 hours. The migratory region normalized to T0 are labelled in blue. Images were taken under 10x magnification. Cell migration rates were quantified based on relative wound densities with 8 biological replicates. Representative experiment from 11 independent repeats is shown. Pairwise two-way ANOVA between WT and each mutant was performed. (** p<0.01) C. Representative magnified images of the migratory edge of each group in wound scratch assays in A. D & E. Representative images (D) and quantification (E) of spheroid collective migration assays in T47D mutant cells. T47D cells were initially seeded into round bottom ULA plates to form spheroids, which were then transferred onto collagen I coated plates. Collective migration was measured after 4 days. The migratory edge of each spheroid is circled with a white line. Migratory distances were calculated based on the mean radius of each spheroid normalized to corresponding original areas. Representative experiment from three independent repeats is shown. Dunnett’s test was used for statistical analysis. (** p<0.01) F. Dot plots representing the enrichment distribution of the 50 MSigDB curated Hallmark gene sets in T47D-Y537S and T47D-D538G models normalized to WT cells. Significantly enriched gene sets (FDR<0.25) are highlighted in red, with names labeled in the venn diagram plot on the right panel. Gene sets enriched in Y537S and D538G cell models are in green and blue circles respectively. G. Immunoblot detection of β-catenin, phospho-GSK3β (Ser9), phospho-GSK3α (Ser21) total GSK3β and total GSK3α levels in T47D WT and mutant cells after hormone deprivation. Tubulin was blotted as a loading control. Representative blots from three independent repeats is displayed for each protein. H. Quantification of IncuCyte wound scratch assay with or without 5μM LGK974 treatment for 72 hours. The migratory region normalized to T0 are labelled in blue. Images were taken under 10x magnification. Cell migration rates were quantified based on relative wound densities with eight biological replicates. Representative experiment from three independent repeats is shown. Pairwise two-way ANOVA between WT and each mutant was performed. (** p<0.01) I. IncuCyte migration assay with combination treatment of four different doses of LGK974 and Fulvestrant in T47D-D538G cells. Inhibition rates were calculated using the wound density at 48 hours normalized to vehicle control with values labelled using color scales in the heatmap. Positive Bliss scores are considered a synergistic combination. Representative experiment from three independent repeats is shown. J. Dot plot representing the fold changes of all Wnt signaling component genes in both T47D ESR1 mutant cell models normalized to WT cells. The blue dotted frame highlights the unique T47D-D538G enriched genes as well as genes that are enriched in both mutants, but with a larger magnitude of enrichment in the T47D-D538G cells. K & L. Immunoblot validation of Fulvestrant-induced ER degradation (K) and FOXA1 knockdown (L). Cell lysates were subjected to ER and FOXA1 detection. Tubulin was blotted as a loading control. These validation experiments were performed once. M & N. Wound scratch assay in T47D-D538G and WT cells with 1μM of Fulvestrant treatment (M) or knockdown of FOXA1 (N) for 72 hours. Cell migration rates were quantified based on wound closure density. For fulvestrant treatment, data were merged from 3 (WT) or 6 (D538G) independent experiments. For FOXA1 knockdown, representative result from three independent repeats is displayed. Pairwise two-way ANOVA between siScramble/siFOXA1 or vehicle/Fulvestrant conditions in each cell type was performed. (* p<0.05, ** p<0.01) O. PCA plot showing the FOXA1 peak distribution of T47D WT, WT+E2, T47D-Y537S and T47D-D538G groups. P. Heatmaps representing the comparison of FOXA1 binding intensities in T47D-D538G mutants to FOXA1 binding in WT cells. Displayed in a horizontal window of ± 2kb from the peak center. The pairwise comparison between WT and mutant samples was performed to calculate the fold change (FC) of intensities. Binding sites were sub-classified into sites with increased intensity (FC>2), decreased intensity (FC<−2), and non-changed intensity (−22, FDR<0.005 in D538G vs WT excluding genes with |fold change|>1.5, FDR<0.01 in WT+E2 vs WT groups). TCF4 is highlighted. S. Wound scratch assay in T47D-WT and T47D-D538G cells with or without prior transfection of a dominant negative TCF4 plasmid for 72 hours. Pairwise two-way ANOVA between vehicle and treatment conditions was performed. Data from one representative experiment of three independent experiments (each with six biological repeats) is shown. (** p<0.01)

Article Snippet: For the dominant negative TCF4 overexpression experiment, Myc-tagged DN TCF4 plasmids (Addgene, #32729) were transiently transfected into targeted cells for a total of 24 hours before being subjected to the wound scratch assay. . Aggregation rate assay.

Techniques: Wound Healing Assay, Mutagenesis, Imaging, Migration, Labeling, Western Blot, Control, Inhibition, Biomarker Discovery, Knockdown, Comparison, Binding Assay, RNA Sequencing, Transfection, Dominant Negative Mutation, Plasmid Preparation