ta802113 (OriGene)
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ta802113
Ta802113, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ta802113/product/OriGene
Average 92 stars, based on 8 article reviews
Ta802113, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ta802113/product/OriGene
Average 92 stars, based on 8 article reviews
ta802113 - by Bioz Stars,
2026-02
92/100 stars
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1) Product Images from "Integrated proteogenomic characterization across major histological types of pituitary neuroendocrine tumors"
Article Title: Integrated proteogenomic characterization across major histological types of pituitary neuroendocrine tumors
Journal: Cell Research
doi: 10.1038/s41422-022-00736-5
Figure Legend Snippet:
Techniques Used:
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Figure 6 A. Representative immunofluorescence image showing ZEB2 expression in SOX2+ progenitor cells in day 10 human (H9) organoid. Scale bar: 50 μm. B. Representative immunofluorescence image showing a salt-and-pepper pattern of ZEB2 expression in the ventricular zone at day 25, after the onset of neurogenesis in human (H9) organoid. DCX (Doublecortin) stains newly born neurons. Scale bar: 100 μm. C. Representative immunofluorescence image of a mature day 60 human (H9) organoid revealing ZEB2 expression in CTIP2+ neurons and absence of ZEB2 staining in the ventricular zone. Scale bar: 100 μm. D. Representative immunofluorescence image of a day 25 human (H9) organoid showing a mutually exclusive pattern of expression between ZEB2 and the radial glia marker protein GLAST. Scale bar: 100 μm. E. Schematic representation of the CRISPR-Cas9n editing strategy, where the first coding exon of the ZEB2 gene (exon 2, NCBI ref sequence NM_014795.4:182-323) was targeted by two nickases (dashed lines) and screening was performed by assaying the drop-off frequency of a HEX-labeled probe, binding to one of the nick sites, relative to a FAM-labeled reference probe binding away from the disrupted region. The exon is marked in orange while introns are marked in purple. F. Example ddPCR 2D scatter-plots of a negative control sample (HEK293 cells), showing only a FAM-HEX double positive (red) and an empty droplet cluster (black) and a positive control sample (HEK293 cells expressing WT Cas9 and ZEB2 guides), showing a FAM-only cluster (blue) in the upper-left quadrant of the 2D plot corresponding to edited alleles. ddPCR 2D scatter-plot of the H9 ZEB2 +/− hESC edited line showing a 1:1 ratio between the WT and edited allele. (G). Representative chromatograms of the ZEB2 alleles in the H9 ZEB2 +/− hESC cells. The CRISPR-Cas9 target region was PCR amplified with a high-fidelity polymerase, the PCR product was blunt-end cloned into the pJET1.2 vector and following purification, plasmids from different colonies carrying the insert were sequenced. Sequencing reveals that the edited allele harbors a 23 bp deletion. H. DNA-PAGE analysis of a short PCR amplicon spanning the CRISPR-Cas9 ZEB2 target site in WT H9 and H9 ZEB2 +/− hESCs. The gel reveals the presence of two bands, corresponding to the WT and the edited allele in H9 ZEB2 +/− hESCs. I. Representative images of karyotype analysis on 20 G-banded metaphase spreads from the H9 ZEB2 +/− hESCs used to generate the stock. The cell line displays normal karyotype. J. RT-PCR analysis for expression of ZEB2, the key pluripotency markers SOX2, NANOG, OCT4 and DPPA5 and the loading control GAPDH . PCR shows that upon a ~50% reduction in ZEB2 mRNA levels the mutant stem cells retain expression of pluripotency markers at comparable levels to WT H9 hESCs. WT and ZEB2 +/− were run on the same gel but not adjacent to each other, the dashed line indicates where the gel was spliced. K. Full length western blot for ZEB2 in WT and ZEB2 +/− organoids at day 15 – loading control was GAPDH L. Box and whiskers plot reporting the quantifications of the number of TBR2+ cells per unit area (TBR2+ cells/mm 2 ) in day 16 WT and ZEB2 +/− organoids. Quantifications were performed by manual counting on n = 52 WT and n = 68 ZEB2 +/− ventricles corresponding to 12 organoids from 2 distinct batches. A two-tailed Mann-Whitney U test was used for statistical comparison ( ∗∗∗∗ p < 0.0001). M. Representative immunofluorescence images of day 55 WT and ZEB2 +/− cerebral organoid buds used for quantifications shown in N. Scale bar: 200 μm. N. Box and whiskers plot reporting the quantifications of the number of TBR2+ cells per unit area (TBR2+ cells/mm 2 ) in day 55 WT and ZEB2 +/− organoids. Quantifications were performed using an automated cell segmentation pipeline on n = 17 WT and n = 17 ZEB2 +/− organoid regions from 3 distinct batches. A two-tailed Mann-Whitney U test was used for statistical comparison (ns, p = 0.1139). O. Plasmid maps of the CRISPR homology-directed repair (HDR) templates used to target the AAVS1 safe-harbor locus in H9 hESC cells – top is the CAG-lox-STOP-lox-ZEB2-GFP-Flag inducible expression construct and bottom is the construct encoding CRE recombinase under the control of a tetracycline responsive promoter and the reverse tetracycline transactivator (rtTA) driven by the CAG promoter. P. UCSC Genome Browser view of the AAVS1 locus and CRISPR-Cas9 targeting strategy of intron 1 of PPP1R12C . The promoter-less splice-acceptor (SA), T2A peptide-linked “gene trap” is such that expression of the promoter-less selection cassette is driven by the endogenous PPP1R12C gene, thus effectively eliminating false-positive background arising from random integration. The panel reports the PCR genotyping strategy – upon successful targeting of the AAVS1 locus, while amplicon 1 is lost due to the size increase following insert integration, amplicons 2 and 3 are gained - see A. Q. PCR gel showing successful genotyping of the two rescue clones used for the experiments shown. R. Representative brightfield images of day 15 ZEB2 +/− ; iZEB2 cerebral organoids treated with and without doxycycline. Scale bar: 100 μm. S. Representative immunofluorescence images of ZEB2 +/− ; iZEB2 treated with and without doxycycline stained for GFP, TBR2 and DAPI. Scale bar: 100 μm T. Box and whiskers plot reporting the quantifications done using an automated cell segmentation pipeline of the number of TBR2+ cells per unit area (TBR2+ cells/mm 2 ) in day 15 ZEB2 +/− ; iZEB2 organoids - colony 1: -Dox (n = 17 organoid regions), +Dox (n = 16 organoid regions); colony 2: -Dox (n = 13 organoid regions), +Dox (n = 13 organoid regions) from three independent batches. Mann-Whitney U tests, two-tailed ( ∗∗ p < 0.01). " width="100%" height="100%">
Figure 7 A. PCR gel showing successful genotyping of the Hum iZEB2 colony used for all experiments shown based on the PCR genotyping strategy outlined in 
Figure 2 E are included for comparison. Mean apical process volume:surface area ratio: Hum iZEB2 − Dox = 1.11; Hum iZEB2 + Dox = 0.76. ∗∗ p < 0.01, Mann-Whitney U, two-tailed, n (− Dox and + Dox) = 9 cells. Error bars are SD. (G) Quantification of the surface area of individual delineated ZO1 cell perimeters as shown in (E). Gorilla measurements from two cell lines (G1, G2) combined are shown for comparison. Mean apical surface area/cell: Hum iZEB2 − Dox = 9.62 μm 2 , Hum iZEB2 + Dox = 3.08 μm 2 , and gorilla (G1,G2) = 4.50 μm 2 . ∗∗∗∗ p < 0.0001, Mann-Whitney U, two-tailed, n (− Dox) = 180 cells from 8 organoids, n (+ Dox) = 199 cells from 8 organoids, both from 2 independent batches, box and whisker plots show median with min-max values, data points represent individual cells. (H) Quantification of perimeters of neuroepithelial buds from bright field images at days 5 and 10, and overall organoid size at day 10. Gorilla (G1,G2) measurements were combined and included for comparison. Day 5 mean neuroepithelial bud perimeter: Hum iZEB2 − Dox = 272 μm, Hum iZEB2 + Dox = 237 μm, and gorilla (G1,G2) = 232 μm. ∗∗∗∗ p < 0.0001, Mann-Whitney U, two-tailed, n (− Dox) = 142 neuroepithelial buds from 41 organoids from 3 independent batches; n (+Dox) = 195 neuroepithelial buds from 38 organoids from 3 independent batches; n (G1,G2) = 555 neuroepithelial buds from 114 organoids from 16 independent batches. Day 10 mean neuroepithelial bud perimeter: Hum iZEB2 − Dox = 300 μm, Hum iZEB2 + Dox = 198 μm, and gorilla (G1,G2) = 227 μm. Day 10 mean organoid area: Hum iZEB2 − Dox = 201,434 μm 2 , Hum iZEB2 + Dox = 132,325 μm 2 , and gorilla (G1,G2) = 93,447 μm 2 . ∗∗∗∗ p < 0.0001, Mann-Whitney U, two-tailed, n (− Dox day 10) = 15 organoids and 106 neuroepithelial buds from 3 independent batches, n (+ Dox day 10) = 15 organoids and 149 neuroepithelial buds from 3 independent batches, error bars are SD. (I) Representative immunofluorescence images showing the effect of BMP4 on the morphology of neural progenitor cells revealed by sparse viral labeling with GFP on day 5 untreated (− BMP4) and treated (+ BMP4) gorilla (G1) organoids with staining for GFP, SOX2, and DAPI. Arrows indicate the apical process. Scale bar, 40 μm. (J) Immunofluorescent staining for ZO1 showing apical surface areas of individual progenitor cells from BMP4-treated (+ BMP4) and untreated (− BMP4) gorilla (G1) organoids at day 5. Perimeters of some individual progenitor cells are delineated in white. Scale bar, 10 μm. (K) Quantification of individual delineated ZO1 cell perimeters as shown in (J). Mean apical surface area/cell: gorilla − BMP4 = 2.72 μm 2 and gorilla + BMP4 = 4.13 μm 2 . ∗∗∗∗ p < 0.0001, Mann-Whitney U, two-tailed, n (− BMP4) = 301 cells from 8 organoids from 2 independent batches, n (+ BMP4) = 326 cells from 8 organoids from 2 independent batches, box and whisker plots are median with min-max values, data points represent individual cells. (L) Immunofluorescence images of human (H9) and gorilla (G1) day 5 organoids untreated (− LPA) and treated (+ LPA) with staining for OCLN, ZO1, and DAPI. LPA treatment in gorilla results in increased OCLN distribution along the apicobasal axis of cells (arrowheads) and expanded apical surfaces of cells (ZO1, bottom panel). Scale bar, 40 μm (upper panels), 10 μm (bottom panels). (M) Quantification of individual delineated ZO1 cell perimeters as shown in (L). Mean apical surface area/cell: human − LPA = 5.36 μm 2 , human + LPA = 5.25 μm 2 , gorilla − LPA = 2.44 μm 2 , and gorilla + LPA = 4.73 μm 2 . ∗ p < 0.05 ∗∗∗∗ p < 0.0001, Kruskal-Wallis and post hoc Dunn’s multiple comparisons test, n (human − LPA) = 146 cells from 3 organoids of 1 batch, n (human + LPA) = 200 cells from 3 organoids of 1 batch, n (gorilla − LPA) = 375 cells from 7 organoids and 2 independent batches, n (gorilla + LPA) = 457 cells from 10 organoids and 2 independent batches, box and whisker plots show median with min-max values, data points represent individual cells. (N) Schematic summarizing the morphological changes that occur in neural progenitor cells as they transition from NE to tNE cells (purple background). ZEB2 is highlighted as a driver, which acts through BMP-responsive SMADs to downregulate epithelial features, notably tight-junction proteins (TJs, green), and involves apical constriction through rearrangements in the actin cytoskeleton (actin, magenta). See also 
