zeb2  (OriGene)

Journal: Molecular cancer research : MCR

Article Title: Palmitate-induced IRE1-XBP1-ZEB signaling represses desmoplakin expression and promotes cancer cell migration

doi: 10.1158/1541-7786.MCR-19-0480

Figure Lengend Snippet: A: Scatterplots showing correlation in basal expression levels between DSP, ZEB1, and ZEB2 across six cancer cell lines. The expression levels (Y-axis) of the three genes were normalized to GAPDH in each cell line and represented relative to the levels in HepG2 cells. B-C. Barplots showing results of the DSP promoter luciferase assay in response to siRNA-mediated knockdown of ZEB1 or ZEB2 in Hep3B (C) or MDA-MB-231 cells (C). D-F. Barplots displaying results of ZEB1 ChIP-qPCR assay in Hep3B (D), MDA-MB-231 (E) or HepG2 (F) cells. The Y-axis represents number of binding events detected per 1000 input cells. The binding events were quantified by qPCR for various segments along the DSP promoter and first DSP intron that contained putative ZEB binding sites, and compared with negative control. G, H. Loss of ZEB1 gene expression in IRE1−/− KO Hep3B (G) or MDA-MB-231 (H) cells. The barplots in each panel represent mean ±SE of three independent replicates with * indicating Student’s T-test P < 0.05, ** indicating P < 0.01 and ns indicating not significant.

Article Snippet: Primary antibody incubation was done overnight at 4°C with antibodies against IRE1 (Cell Signaling Tech, cat # 3294), DSP I + II (Abcam, cat # ab71690), E-cadherin (Cell Signaling Tech, cat # 3195), Vimentin (Cell Signaling Tech, cat # 3932), ZEB1 (ORIGENE, cat # TA802298), ZEB2 (ORIGENE, cat # TA802113) and GAPDH (GeneTex, cat # GTX100118).

Techniques: Expressing, Luciferase, Binding Assay, Negative Control

Journal: Molecular cancer research : MCR

Article Title: Palmitate-induced IRE1-XBP1-ZEB signaling represses desmoplakin expression and promotes cancer cell migration

doi: 10.1158/1541-7786.MCR-19-0480

Figure Lengend Snippet: A. Schematic diagram of the DSP promoter (−1000bp upstream of transcription start site) displaying putative ZEB binding sites B. Scatterplots showing the correlation between DSP, ZEB1 and ZEB2 expression levels across 10,022 samples in the TCGA PANCAN (pan-cancer) dataset C. Scatterplots showing the correlation between DSP methylation levels with DSP, ZEB1 and ZEB2 expression in the TCGA PANCAN dataset. PCC indicates Pearson’s correlation coefficient and the P values indicates significance of correlation

Article Snippet: Primary antibody incubation was done overnight at 4°C with antibodies against IRE1 (Cell Signaling Tech, cat # 3294), DSP I + II (Abcam, cat # ab71690), E-cadherin (Cell Signaling Tech, cat # 3195), Vimentin (Cell Signaling Tech, cat # 3932), ZEB1 (ORIGENE, cat # TA802298), ZEB2 (ORIGENE, cat # TA802113) and GAPDH (GeneTex, cat # GTX100118).

Techniques: Binding Assay, Expressing, Methylation

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet: The human neuroepithelium exhibits differential temporal dynamics of morphogenesis genes (A) Clustering genes by temporal expression dynamics shows species differences in GO:BP term enrichment. Columns from left to right: far left, TCseq clusters with genes in each cluster plotted with their temporal expression (z-scaled) and color-coded by membership value (degree to which data points of a gene belong to the cluster, pink represents high membership values). The 10 clusters are ordered (top to bottom) based on similarity in expression pattern. Middle left: representative GO:BP term from shared (purple), human-exclusive (black), or gorilla-exclusive (fuchsia) terms for each cluster. Middle right: histograms show the number of enriched (p < 0.05) GO:BP terms found in both species (purple), exclusively in human (black) or gorilla (fuchsia) per cluster. Axis range: 0–8 (cluster 2,5,7,8); 0–15 (cluster 9,10); 0–20 (cluster 1); 0–25 (cluster 3); 0–50 (cluster 4); 0–80 (cluster 6). Far right: weighted arc network graph visualizing interspecies differences in the enrichment/membership of specific GO:BP terms per cluster. The bases of the arc are aligned to both a human (black) and a gorilla (fuchsia) bar from the histogram in the adjacent panel, highlighting the species-specific shifts in expression patterns associated with specific GO:BP terms. Weight/thickness of the arc is dictated by the number of GO:BP terms enriched in a species-exclusive manner “moving” between clusters in the defined pattern. (B) Mean temporal expression pattern (z-scaled) of genes in clusters enriched for “cell morphogenesis”-related GO:BP terms (human clusters 1, 9, 10, and gorilla cluster 3). (C) Temporal expression pattern ( Z -scaled) of SHROOM3 , a gene involved in cell morphogenesis and apical constriction. (D) Immunofluorescent staining of day 5 organoids for SHROOM3 showing strong apical expression in gorilla (G1) neuroepithelium, but not human (H9) at this time point. Scale bar, 40 μm. (E) Immunofluorescent staining of day 5 organoids for OCLN showing expression spread along the apicobasal length of human (IMR-90) progenitor cells (white arrowheads) but more limited apically (yellow arrowheads) in gorilla (G1) progenitor cells. DAPI is shown in blue. Scale bar, 100 μm. (F) Venn diagram summarizing search for cell morphogenesis-related transcription factors with species-specific expression patterns. (G) Mean temporal expression pattern ( Z -scaled) of ZEB2 , showing peak expression earlier in gorilla (G1) than human (H9) organoids. Shaded error bars are SD. See also Figure S5 and .

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Expressing, Staining

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet: Decreased ZEB2 leads to expanded NE with delayed transition (A) Mean temporal expression pattern (log normalized transcripts per million) of ZEB2 across the entire time series, showing peak expression earlier in gorilla (G1) than human (H9) organoids. Shaded error bars are SD. (B) Western blot expression time course from PSCs to day 25 human (H9) and gorilla (G1) organoids reveals a premature onset and higher levels of ZEB2 protein expression in gorilla compared to human. This is accompanied by a premature expression of the radial glial marker vimentin, and premature downregulation of the epithelial markers CDH1 and EpCAM in gorilla relative to human. Bottom panel shows quantification of ZEB2 relative to GAPDH (AU, arbitrary units). (C) Immunofluorescent stain for ZEB2 and DAPI in human (H9) and gorilla (G1) organoids at days 3, 5, and 10 showing neuroepithelial buds with nuclear expression (yellow arrows). Note the interspecies difference at day 3 where gorilla organoids already display nuclear expression compared to a weaker stain in most human cells. Insets show higher magnification of the boxed regions. Scale bar, 40 μm. (D) Immunofluorescence image of a day 25 human (H9) organoid showing a mutually exclusively pattern of expression between ZEB2 and the committed radial glia marker, BLBP. Scale bar, 100 μm. (E) Western blot of H9 wild-type (WT) and ZEB2 +/− organoids at day 16 for ZEB2, the tight-junction protein OCLN, the junction components CDH1 and CDH2, the dorsal telencephalic marker EMX1, and the loading control β-actin. The blots show a sizeable increase in CDH1 and OCLN and a decrease in CDH2, whereas EMX1 levels, and thus dorsal telencephalic identity, appears to be largely unaffected. (F) Representative bright field images of day 12 WT and ZEB2 +/− . Insets show higher magnification of the boxed regions, dashed yellow are representative neuroepithelial bud perimeters quantified in (G), dashed turquois are representative neuroepithelial bud thicknesses quantified in (H). Scale bar, 500 μm. (G) Quantification of neuroepithelial bud perimeters of WT (n = 106) and ZEB2 +/− (n = 116) organoid buds from 27 WT and 28 ZEB2 +/− organoids at day 17, Mann-Whitney U test, two-tailed ( ∗∗∗ p = 0.0001) from 3 organoid batches. (H) Quantification of neuroepithelial bud thickness of WT (n = 80) and ZEB2 +/− (n = 119) organoid buds from 26 WT and 31 ZEB2 +/− organoids at day 12, Mann-Whitney U test, two-tailed ( ∗∗∗∗ p < 0.0001) from 3 organoid batches. (I) Quantification of neuroepithelial bud perimeters of two ZEB2 +/− ; iZEB2 colonies treated with and without doxycycline. Colony 1: − Dox (n = 108 buds from 25 organoids), + Dox (n = 70 buds from 18 organoids). Colony 2: − Dox (n = 62 buds from 17 organoids), + Dox (n = 84 buds from 19 organoids) across 3 organoid batches. Mann-Whitney U tests, two-tailed ( ∗∗∗ p = 0.0003 ∗∗∗∗ p < 0.0001). (J) Immunofluorescence images of day 15 WT and ZEB2 +/− organoids showing increased OCLN immunostaining (yellow arrowheads) along the apico-(ZO1) basal (dashed line) axis of progenitor cells and reduced numbers of TBR2 + cells in ZEB2 +/− organoids compared to WT. Scale bar, 100 μm. See also Figure S6 .

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Expressing, Western Blot, Marker, Staining, Immunofluorescence, MANN-WHITNEY, Two Tailed Test, Immunostaining

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet: ZEB2 expression and targeting for loss of function, related to 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).

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Expressing, Immunofluorescence, Staining, Marker, CRISPR, Sequencing, Labeling, Binding Assay, Negative Control, Positive Control, Amplification, Clone Assay, Plasmid Preparation, Purification, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Western Blot, Two Tailed Test, MANN-WHITNEY, Construct, Selection

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet: Modulation of ZEB2 and SMAD signaling in human and gorilla cells, related to 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 S6 P. The asterisks mark unspecific bands. B. Transgene induction in Hum iZEB2 cells treated with and without doxycycline for 6 days and assayed by western blot for ZEB2, GFP and β-actin. C. Immunofluorescence images of 6-day induced and uninduced Hum iZEB2 cells stained for ZEB2 and DAPI, showing that doxycyline induction results in ZEB2 expression and nuclear translocation, without adverse effects on its localization due to tagging with GFP. Scale bar: 20 μm D. Immunofluorescence images of 6-day induced and uninduced Hum iZEB2 cells stained for DAPI, CDH1 and CDH2. The data reveal a reduction in CDH1 expression and an increase in CDH2 expression following induction. Scale bar: 50 μm. E. Immunofluorescence images of 6-day induced and uninduced Hum iZEB2 cells stained for GFP, Vimentin and EpCAM. The data reveal a reduction in EpCAM expression and an increase in Vimentin expression following expression of ZEB2-GFP. Scale bar: 50 μm. F. Brightfield images of induced (+ Dox) and uninduced (- Dox) Hum iZEB2 organoids and gorilla (G1) organoids at days 3, 5 and 10, showing indistinguishable tissue architecture between organoids at day 3, while day 5 and 10 organoids show neuroepithelial buds that are smaller and more rounded in gorilla and ZEB2 induced (+ Dox) versus uninduced (- Dox) organoids. Scale bar: 200 μm. G. Western blot of day 5 WT and ZEB2 +/− organoids revealing both decreased ZEB2 and SHROOM3 levels in ZEB2 +/− organoids compared to WT control β-Actin was used as loading control. H. Representative bright-field images of uninduced and induced Hum iZEB2 and gorilla neuroepithelial buds at day 5, used for quantification in Figure 7 H. Scale bar: 100 μm. I. Immunofluorescent staining for ZO1, SOX2, DCX and DAPI showing normal tissue morphology and onset of neurogenesis (DCX+ neurons) in uninduced (- Dox) and induced (+ Dox) Hum iZEB2 organoids at day 10. Scale bar: 100 μm. J. Western blot for ZEB2, CDH1, and CDH2, with β-Actin as loading control, of WT and ZEB2 +/− organoids treated with dual-SMAD inhibitors, or treated with vehicle, for 10 days and assayed at day 12. Note the rescued levels of junctional components CDH1 and CDH2. K. Morphological assessment of WT and ZEB2 +/− organoids treated with dual-SMAD inhibitors, or treated with vehicle, for 10 days and assayed at day 12 by brightfield imaging (left panels) and hematoxylin-eosin staining (right panels). Note the elongated neuroepithelial buds (arrows) in mutant organoids that appear rescued (arrowheads) upon SMAD inhibition. Scale bars: 1 mm (left panels), 50 μm (right panels).

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Translocation Assay, Imaging, Mutagenesis, Inhibition

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet: ZEB2-driven junctional remodeling and apical constriction dictate species-specific timing of NE transition (A) Immunofluorescent staining of uninduced (− Dox) and induced (+ Dox) Hum iZEB2 organoids for GFP and SHROOM3. Note the expression of ZEB2-GFP and apical accumulation of SHROOM3 in induced organoids. Scale bar, 50 μm. (B) Representative bright field images of day 5 Hum iZEB2 and gorilla organoids. Induced (+ Dox) Hum iZEB2 organoids show smaller neuroepithelial buds (arrowheads) that are more round in shape, similar to gorilla (G1), while uninduced (− Dox) show more elongated structures typical of human. Scale bar, 200 μm. (C) Immunofluorescence images through day 5 whole mount Hum iZEB2 uninduced (− Dox), induced (+ Dox) and gorilla (G1) organoids stained for GFP, ZO1, and SOX2. Sparse labeling with viral GFP shows ZEB2 induction triggers the constriction of apical processes (arrows) in progenitor cells, similar to gorilla at day 5. Scale bar, 50 μm. (D) Representative immunofluorescence images through whole mount day 5 uninduced (− Dox), induced (+ Dox) Hum iZEB2 and gorilla (G1) organoids with superimposed individual segmented GFP+ progenitor cells (white) showing their 3D morphology. Note the thinning of apical processes observed upon ZEB2 induction. Scale bar, 10 μm. (E) Immunofluorescent staining for ZO1 on the surface of apical lumens showing the apical surface areas of individual progenitor cells in day 5 Hum iZEB2 uninduced (− Dox), induced (+ Dox) and gorilla (G1) organoids. Perimeters of some individual progenitor cells of day 5 organoids are delineated in white. Scale bar, 10 μm. (F) Quantification of the volume as normalized to surface area of the apical processes of induced (+ Dox) versus uninduced (− Dox) Hum iZEB2 neural progenitor cells on day 5. The apical processes of segmented cells directly below the cell body were used for quantification. Gorilla day 5 measurements from 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 Figure S7 .

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Staining, Expressing, Immunofluorescence, Labeling, MANN-WHITNEY, Two Tailed Test, Whisker Assay

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet:

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Fluorescence, Recombinant, Knock-Out, Protease Inhibitor, Multiplex Assay, Software

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet:

Article Snippet: mouse anti-ZEB2 , Origene , Cat# TA802113, RRID: AB_2616296.

Techniques: Virus, Fluorescence, Recombinant, Knock-Out, Protease Inhibitor, Multiplex Assay, Membrane, Software

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: Primer and siRNA sequences.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Sequencing, Control, Chromatin Immunoprecipitation

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: SIP1 is crucial in the HBx-induced epigenetic silencing of E-cadherin. (A) Epithelial-mesenchymal transition-related protein levels in HepG2 cells transfected with pcDNA3.1 and pHBx were examined by western blotting. (B) mRNA levels of SIP1 in HBx-expressing HepG2 cells were verified by reverse transcription-quantitative PCR analysis ( * P<0.05, ** P<0.01). (C) Expression levels of SIP1 and E-cadherin in HepG2-X and HepG2 cells were examined by western blotting. (D) Western blot analysis of E-cadherin and SIP1 in HepG2-X cells transfected with siHBx or siCont. (E) Western blotting results. shRNA reduced the expression of SIP1 in HBx-expressing HepG2 cells, with nonspecific shRNA serving as a negative control. (F) Transwell assay of HepG2 cells transfected with pcDNA3.1 or pHBx and treated with shSIP1 or scramble control. (G) Immunofluorescence analysis. The knockdown of SIP1 restored the epigenetic repression of E-cadherin induced by ectopic HBx. Vimentin concomitantly exhibited an inverse change in expression. DAPI was used to visualize nuclei. Scale bar, 10 µ m. Magnification, ×200. SIP1, Smad-interacting protein 1; HBx, hepatitis B virus X; pHBx, pcDNA3.1-HBx; si, small interfering RNA; sh, short hairpin RNA.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Transfection, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Negative Control, Transwell Assay, Control, Immunofluorescence, Knockdown, Virus, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: HBx recruits endogenous SIP1 to the E-cadherin promoter. (A) Dual-luciferase assay of human E-cadherin promoters in Mock treated, pcDNA3.1 or pHBX transfected HepG2 cells and HepG2-X cells. Data were normalized to the luciferase activity of cells treated with pcDNA3.1 and pGL3-Basic. Data are representative of at least three independent experiments. * P<0.05 HepG2 + pcDNA3.1 vs. HepG2 + pHBx; # P<0.05 HepG2 + pcDNA3.1 vs. HepG2-X. (B) ChIP primers were designed on E-cadherin gene regulatory regions. Distal primers correspond to downstream regulatory regions of -1895 to -1707 nt of the E-cadherin gene. The ChIP primers were designed adjacent to the TSS locations across the E-box region of the human E-cadherin promoter. pHBx-transfected HepG2 cell lysates were subjected to ChIP using an anti-SIP1 antibody. PCR was conducted using the indicated primer pairs. An empty vector and IgG served as external and internal negative controls. (C) Sequence homology of the consensus E-box in the E-cadherin promoter of mammalian. E-boxes 1, 3 and 4, CCAAT box and GC box are conserved regulatory elements, as shown in the diagram. The arrow indicates the putative TSS. (D) Mutations generated in the E-boxes carried the E-box 1 mutation CAGGTG → AAGGTA and E-box 3 mutation CACCTG → AACCTA. The wild-type E-cadherin promoter and promoter comprising two mutated E-boxes were cloned into a luciferase vector to construct the proE-cad-Luc and proE-cad-Luc-mEbox plasmids. (E) Dual-luciferase assay of E-cadherin promoter constructs with proE-cad-Luc or proE-cad-Luc-mEbox in pHBx- or pcDNA3.1-transfected HepG2 cells. ** P<0.01. (F) Relative E-cadherin promoter activities inshSIP1/shCont and pcDNA3.1/pHBX treated HepG2 cells. * P<0.05; (shSIP1 + WT, vs. shCont + WT). (G) Co-immunoprecipitation in protein extracts of pcDNA3.1-transfected HepG2 cells and HBx-expressing HepG2 cells with anti-SIP1 or anti-HBx antibodies and western blot detection of HBx and SIP1, respectively. (H) Immunofluorescent staining of HepG2-X cells with anti-HBx and anti-SIP1 to show the subcel-lular co-localization of HBx and SIP. DAPI was used to visualize nuclei. Scale bar=10 µ m. SIP1, Smad interacting protein 1; HBx, hepatitis B virus X; pHBx, pcDNA3.1-HBx; sh, short hairpin RNA. TSS, transcription start site; WT, proE-cad-Luc; Mut, proE-cad-Luc-mEbox; ChIp, chromatin immunoprecipitation .

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Luciferase, Transfection, Activity Assay, Plasmid Preparation, Sequencing, Generated, Mutagenesis, Clone Assay, Construct, Immunoprecipitation, Expressing, Western Blot, Staining, Virus, shRNA, Chromatin Immunoprecipitation

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: HBx recruits SIP1 and HDAC1 to the E-cadherin promoter to repress its expression. (A) Western blot analysis of E-cadherin and HDAC1 in pcDNA3.1 or pHBX transfected HepG2 cells +/- TSA. E-cadherin promoter activities in TSA-treated cells following transfection with (B) shSIP1 or (C) SIP1 expression plasmids. Results are reported as the relative luciferase activity, vs. activity of pGL3-Basic. * P<0.05, ** P<0.01 (mean ± SD). (D) ChIP of lysates from HepG2 cells transfected with pHBx using anti-HDAC1 antibody. An empty vector pcDNA3.1 and protein G beads served as external and internal controls, respectively. (E) ChIP performed using HDAC1 antibody on the lysates of HepG2 cells treated with shSIP1/shCont and pcDNA3.1/pHBX. (F) Co-immunoprecipitation of HBx-expressing HepG2 cell-protein extracts with anti-SIP1 or anti-HBx antibodies and western blot detection of HDAC1, HBx and SIP1, respectively. (G) Immunofluorescent staining of HepG2-X cells with anti-HDAC1, anti-SIP1 and DAPI. The merged image showed HDAC1 and SIP1 co-localization in the nucleus. Scale bar=10 µ m. SIP1, Smad-interacting protein 1; HBx, hepatitis B virus X; pHBx, pcDNA3.1-HBx; sh, short hairpin RNA; Cont, control; HDAC1, histone deacetylase 1; ChIp, chromatin immunoprecipitation; TSA, trichostatin A.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Expressing, Western Blot, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Immunoprecipitation, Staining, Virus, shRNA, Control, Histone Deacetylase Assay, Chromatin Immunoprecipitation

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: SIP1 mediates cell proliferation and apoptosis affected by ectopic expression of HBx in HepG2 cells. (A) HepG2 and HepG2-X cells were trans-fected with shSIP1 or shCont. Cell counting kit-8 assays were performed to determine cell proliferation ability. (B) Colony formation assay in HepG2 and HepG2-X cells transfected with shSIP1 or shCont. The colonies were stained with crystal violet and counted. Images are representative of three replicate experiments. (C) Apoptosis of HepG2 and HepG2-X cells were analyzed by flow cytometry with Annexin V-FITC/propidium iodide. * P<0.05, ** P<0.01. SIP1, Smad-interacting protein 1; HBx, hepatitis B virus X; sh, short hairpin RNA; Cont, control.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Expressing, Cell Counting, Colony Assay, Transfection, Staining, Flow Cytometry, Virus, shRNA, Control

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: HBx accelerates tumor growth through SIP1 in vivo . (A) Representative images of nude mice implanted with HepG2 and HepG2-X cells treated with shSIP1 or shCont. (B) Growth curves of tumors from nude mice implanted with the indicated cells (xenograft mice). (C) Images of tumors from xenograft mice. (D) Average tumor weights from xenograft mice. (E) Expression of SIP1 and E-cadherin in tumor tissues of mice was detected using western blotting. (F) Representative images of liver tissues and H&E staining of intrahepatic metastasis tumors from each group of orthotopic transplantation mice were shown. The arrows indicate visible intrahepatic metastatic tumors. (G) Immunohistochemical analysis of the transplanted tumors from each group of orthotopic transplantation mice. (H) H&E staining of the diaphragm metastases was detected only in the shCont-treated HepG2-X cells group of mice. Scale bar, 150 µ m. SIP1, Smad-interacting protein 1; HBx, hepatitis B virus X; si, small interfering RNA; sh, short hairpin RNA; Cont, control; H&E hematoxylin and eosin.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: In Vivo, Expressing, Western Blot, Staining, Transplantation Assay, Immunohistochemical staining, Virus, Small Interfering RNA, shRNA, Control

Journal: International Journal of Oncology

Article Title: SIP1 serves a role in HBx-induced liver cancer growth and metastasis

doi: 10.3892/ijo.2019.4884

Figure Lengend Snippet: Schematic diagram showing how HBx regulates E-cadherin via SIP1 and histone deacetylation. (A) Abundant enrichment of CAC and TFs are recruited to E-cadherin promoter region in the absence of HBx protein, leading to the transcription of E-cadherin. (B) HBx protein increases the levels of SIP1 and HDAC1. The three factors form a repressive triple complex locates at the E-cadherin promoter, and then induces the epigenetic silencing of E-cadherin. SIP1, Smad-interacting protein 1; HBx, hepatitis B virus X; TF, transcription factor; Pol II, RNA polymerase II.

Article Snippet: The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627).

Techniques: Virus