Journal: bioRxiv

Article Title: Phenotypic CRISPR screening identifies ZBTB10 as a novel regulator of human trophoblast differentiation

doi: 10.64898/2026.06.29.734890

Figure Lengend Snippet: A) Sanger Sequencing of polyclonal ZBTB10 knockout BeWo cells with deletion and indel formation. B) Western blot of ZBTB10 in wild-type (WT) and ZBTB10 KO BeWo cells treated with DMSO or Forskolin for 48 hours (differentiated). C) hCG secretion measured by ELISA in supernatant from WT vs. ZBTB10 KO BeWos treated with DMSO or Forskolin for 48 hours (differentiated). Significance bars indicate Brown-Forsythe and Welch ANOVA test with adjusted p-value less than 0.0332 (*), 0.0021 (**), and 0.0002 (***). D) Example images of BeWo fusion assays in polyclonal TRAC KO (control) versus ZBTB10 KO mCherry and GFP BeWo co-cultures. GFP BeWos (blue) and mCherry BeWos (yellow) and Hoechst-labeled nuclei (magenta) are shown with white arrows indicating examples of fused regions. Quantified Fusion Index across individual wells from at least 3 independent biological experiments is shown where comparisons indicate a One-way ANOVA with mixed-effects analysis, Geisser-Greenhouse correction, and Dunnett’s multiple comparisons test and **** = adjusted p-value <0.0001. Scale bars are 200μm. E) RNA-Seq differential gene expression analysis comparing Forskolin-differentiated ZBTB10 KO BeWos versus Forskolin-differentiated WT BeWos. Significantly up-regulated genes (FDR<0.05, Log Fold-Change >0) are shown in blue, and significantly down-regulated genes (FDR<0.05, Log Fold-change <0) are shown in red. Unchanged genes are in black. F, G) Genome browser tracks for F) MFSD2A and G) the CGB cluster genes in BeWo RNA-seq data. Merged (summed) reads from 3 RNA-Seq replicates are shown.

Article Snippet: The antibodies used for western blotting were: hCGβ (1:1000; Abcam ab53087), ZBTB10 (Bethyl, A303-257A, Rabbit), Beta-actin (1:5000, Sigma, A2228-100UL), anti-rabbit HRP secondary (1:5000; Invitrogen 31462), and anti-mouse HRP secondary (1:5000; Invitrogen 31430).

Techniques: Sequencing, Knock-Out, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Labeling, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: Phenotypic CRISPR screening identifies ZBTB10 as a novel regulator of human trophoblast differentiation

doi: 10.64898/2026.06.29.734890

Figure Lengend Snippet: A) Immunofluorescence staining of ZBTB10 in negative control (NEG) and polyclonal ZBTB10 knockout hTSCs and quantified percent of nuclei with nuclear ZBTB10 expression shown at right. B) Cell growth curves for NEG and ZBTB10 KO hTSCs (6-8 wells per experiment in 3 independent biological experiments). Confluency is normalized to t=0 per well and the 95% confidence interval is shown as dashed lines. Brightfield images of cell morphology and confluency at 90hrs of growth shown at right. C) Immunofluorescence of NEG and ZBTB10 KO hTSCs differentiated for 6 days to STB. Quantified immunofluorescent intensity for 3 independent biological replicates and compared by paired t-test. D) hCG secretion measured by ELISA in supernatant from NEG vs. ZBTB10 KO differentiated STB compared by Welch’s t-test from six technical replicates across three biological replicates. E) Raw images of 2-color fusion assays performed in GFP/mScarlet co-cultured hTSCs treated with 0.1% DMSO (undifferentiated) or differentiated for 6 days into STB. Quantified Fusion Index for five biological replicates shown at right. The statistical comparison indicates a RM one-way ANOVA test with the Geisser-Greenhouse correction and Dunnett’s multiple comparisons test. F) F-actin (Phalloidin; magenta) and DAPI (blue) staining of 6-day STB differentiated NEG and ZBTB10 KO hTSCs. The quantified percentage of annotated unfused cells over total nuclei in each condition is shown at right and compared by paired t-test. G) Close-up overlay of Phalloidin (magenta) and ZBTB10 (green) immunofluorescence in 6-day STB differentiated ZBTB10 KO STBs. Nuclei within annotated unfused cells are shown with yellow outlines. The percent of ZBTB10+ nuclei in unfused versus syncytialized regions is quantified at right for 3 independent biological replicates compared by paired t-test. H) Immunofluorescence of 6-day EVT differentiated NEG control versus ZBTB10 KO hTSCs. Quantified intensity of HLA-G staining is shown at right for 3 independent biological replicates and compared by paired t-test. Scale bars are 200μm in panels A-H. I) Brightfield images of Day 7 hTSC trophoblast organoids (TO) in Matrigel domes. The total area occupied by organoids within the imaged domes is quantified at right and compared via Welch’s t-test. J) hCG ELISA measurements for supernatant sampled from Day 7 trophoblast organoids formed from NEG versus ZBTB10 KO hTSCs and compared via Welch’s t-test. K) Immunofluorescent staining in cleared trophoblast organoids (TOs) formed from NEG versus ZBTB10 KO hTSCs. Each image is a background-subtracted, maximum intensity projection of Z-stack confocal images taken through the organoid. L) Immunofluorescent staining of NEG versus ZBTB10 KO EVT-TOs with the percent of organoids demonstrating nuclear ZBTB10 signal quantified at right. Image is a background-subtracted, maximum intensity projection of Z-stack confocal images. Scale bars are 100μm in panel K-L. For all figures, error bars indicate standard deviation and adjusted p-values are indicated by asterisks as less than 0.0332 (*), 0.0021 (**), and 0.0002 (***).

Article Snippet: The antibodies used for western blotting were: hCGβ (1:1000; Abcam ab53087), ZBTB10 (Bethyl, A303-257A, Rabbit), Beta-actin (1:5000, Sigma, A2228-100UL), anti-rabbit HRP secondary (1:5000; Invitrogen 31462), and anti-mouse HRP secondary (1:5000; Invitrogen 31430).

Techniques: Immunofluorescence, Staining, Negative Control, Knock-Out, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Comparison, Control, Standard Deviation

Journal: bioRxiv

Article Title: Phenotypic CRISPR screening identifies ZBTB10 as a novel regulator of human trophoblast differentiation

doi: 10.64898/2026.06.29.734890

Figure Lengend Snippet: A, B) RNA-Seq differential gene expression analysis comparing A) day 6 STB-differentiated ZBTB10 KO versus NEG hTSCs or B) day 6 EVT-differentiated ZBTBl0 KO versus NEG hTSCs. Significantly up-regulated genes (FDR<0.05, Log Fold-Change >0) are shown in blue, and significantly down-regulated genes (FDR<0.05, Log Fold-change <0) are shown in red. Unchanged genes are in black. C) Heatmap of column-scaled log2(CPM) values from individual RNA-Seq replicates, grouped by marker genes for TSC, STB, EVT, and Fusion-related genes, normalized per gene column. D) Total number of called MACS2 peaks per replicate and condition for BeWo ZBTBlO ChIP. The number of peaks called in both Forskolin replicates is shown by the “intersection” at right. E) Top 3 sequence motifs from HOMER motif emichment of the Forskolin-treated consensus ZBTB10 ChlP peaks. F) Genome browser track of ZBTB10 ChlP in BeWo cells at the LEP gene. Consensus ZBTBlO peaks identified in both Forskolin replicates are shown with a black bar and the peak center indicated by a teal bar overlay. G) Volcano plot showing the overlay of ZBTB10 target genes identified by ChIP (consensus peak within+/- 2000bp of promoter) in BeWo (green) and differentially expressed genes in STB and EVT differentiated hTSCs, as in . The number of unique gene targets (by ChlP) that are significantly dysregulated upon ZBTB10 KO in each condition are indicated at the top of the plot. H) Annotated cell clusters from snATAC-Seq of first trimester human placenta (Wang, et al. 2024 ) are plotted by UMAP (left) and major cell groups are indicated (CTB, STB, and Fusion). Right, per-cell ATAC activity (chromVAR deviation z-scores) for the specified ZBTBlO ChlP motifs are overlaid on the snATAC-Seq UMAP.

Article Snippet: The antibodies used for western blotting were: hCGβ (1:1000; Abcam ab53087), ZBTB10 (Bethyl, A303-257A, Rabbit), Beta-actin (1:5000, Sigma, A2228-100UL), anti-rabbit HRP secondary (1:5000; Invitrogen 31462), and anti-mouse HRP secondary (1:5000; Invitrogen 31430).

Techniques: RNA Sequencing, Gene Expression, Marker, Sequencing, Activity Assay

Journal: bioRxiv

Article Title: Phenotypic CRISPR screening identifies ZBTB10 as a novel regulator of human trophoblast differentiation

doi: 10.64898/2026.06.29.734890

Figure Lengend Snippet: A) Annotated cell clusters from snRNA-Seq of first trimester human placenta (Wang, et al. 2024 ) are plotted by UMAP with cluster number indicated (top). Cell groups are labeled by major categories (CTB, STB, and Fusing, etc.). Bottom, ZBTB10 expression is shown on the same UMAP with log2-normalized counts per cell. Right, Bar charts quantify the mean expression and the percent of cells with >0 reads for ZBTB10 in each annotated cell cluster. B) The two mature STB clusters overlaid on the UMAP plot of first trimester placenta snRNA-Seq. Right, GSEA plots of the top 100 differentially expressed genes in eSTB mature 1 and mature 2 pathways, with genes ordered from enrichment in ZBTB10 KO STBs (left) to NEG STBs (right). Black bars indicate the presence of each pathway gene in the ordered gene set. NES = normalized enrichment score. C) Immunofluorescence of ZBTB10 in undifferentiated hTSCs versus day 6 EVT-differentiated or STB-differentiated hTSCs. Scale bars are 200μm in panel C. D,E) Immunofluorescence of D) first trimester (weeks 8-12) and E) term (weeks 36-40) human placental tissue. F) Left, quantification of ZBTB10 intensity in immunofluorescence of in vitro differentiated hTSCs. Comparison indicates a RM one-way ANOVA test with the Geisser-Greenhouse correction and Dunnett’s multiple comparisons test where * = adjusted p-value < 0.0332. Right, quantification of ZBTB10 intensity quantified in the indicated segmented marker-positive cells within first trimester placental tissue. Statistical comparison indicates a paired t-test comparing means between individual donors (n=4). G) Close-up overlay images of immunofluorescence staining shown in D,E shown at bottom right with example cell types indicated by white arrows. Scale bars are 100μm in panels D-G.

Article Snippet: The antibodies used for western blotting were: hCGβ (1:1000; Abcam ab53087), ZBTB10 (Bethyl, A303-257A, Rabbit), Beta-actin (1:5000, Sigma, A2228-100UL), anti-rabbit HRP secondary (1:5000; Invitrogen 31462), and anti-mouse HRP secondary (1:5000; Invitrogen 31430).

Techniques: Labeling, Expressing, Immunofluorescence, In Vitro, Comparison, Marker, Staining

Journal: BMC gastroenterology

Article Title: ZBTB10 as a potential prognosis biomarker and correlates with the tumor immune microenvironment in stomach adenocarcinoma.

doi: 10.1186/s12876-025-04047-y

Figure Lengend Snippet: Fig. 3 The clinical characteristics of ZBTB10 expression in STAD tissues. Box-plot showing the ZBTB10 mRNA median expression level correlated with tumor grade (A), pathological stages (B), lymph node metastases (C), patient’s ages(D), TP53 mutation status (E), and H.pylori infection status (F) respectively. The median expression of ZBTB10 was analyzed using the Mann–Whitney U test for two-group comparisons and the Kruskal–Wallis (K-W) test for multiple independent groups respectively. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: The membranes were then blocked with 5% non-fat milk at room temperature for 1 h, followed by incubation with primary antibodies against ZBTB10 (1:1000, Proteintech, China) and GAPDH (1:1000, Abcam) overnight at 4 °C.

Techniques: Expressing, Mutagenesis, Infection, MANN-WHITNEY

Journal: BMC gastroenterology

Article Title: ZBTB10 as a potential prognosis biomarker and correlates with the tumor immune microenvironment in stomach adenocarcinoma.

doi: 10.1186/s12876-025-04047-y

Figure Lengend Snippet: Fig. 6 Correlation of ZBTB10 expression with immune characteristics. (A) Radar map displayed the association between ZBTB10 median expression and tumor mutational burden (TMB). (B) The radar map displayed the association between ZBTB10 median expression and microsatellite instability (MSI). (C) Comparison of immune score and stromal score based on ZBTB10 median expression. (D) 22 types of tumor-infiltrating immune cells were analyzed based on ZBTB10 median expression using the CIBERSORT algorithm. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: The membranes were then blocked with 5% non-fat milk at room temperature for 1 h, followed by incubation with primary antibodies against ZBTB10 (1:1000, Proteintech, China) and GAPDH (1:1000, Abcam) overnight at 4 °C.

Techniques: Expressing, Comparison

Journal: BMC gastroenterology

Article Title: ZBTB10 as a potential prognosis biomarker and correlates with the tumor immune microenvironment in stomach adenocarcinoma.

doi: 10.1186/s12876-025-04047-y

Figure Lengend Snippet: Fig. 7 The association of ZBTB10 expression with tumor immune microenvironment. (A) ZBTB10 median expression correlated with tumor-infiltrating lymphocytes. (B) ZBTB10 median expression correlated with immune stimulators. (C) ZBTB10 median expression correlated with immune inhibitors. (D) The correlation analysis of the top four targeting immunosuppressive and immuneactivative markers with ZBTB10 median expression in STAD. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: The membranes were then blocked with 5% non-fat milk at room temperature for 1 h, followed by incubation with primary antibodies against ZBTB10 (1:1000, Proteintech, China) and GAPDH (1:1000, Abcam) overnight at 4 °C.

Techniques: Expressing

Journal: BMC gastroenterology

Article Title: ZBTB10 as a potential prognosis biomarker and correlates with the tumor immune microenvironment in stomach adenocarcinoma.

doi: 10.1186/s12876-025-04047-y

Figure Lengend Snippet: Fig. 10 Multiplex immunohistochemistry (mIHC) staining for identification. Representative mIHC images of STAD tissues and adjacent normal tissues, a staining panel was developed to visualize CD45, EpCAM, ZBTB10 and DAPI simultaneously. CD45: leukocyte marker. EpCAM: epithelial cell marker. Scale bar 200 µm

Article Snippet: The membranes were then blocked with 5% non-fat milk at room temperature for 1 h, followed by incubation with primary antibodies against ZBTB10 (1:1000, Proteintech, China) and GAPDH (1:1000, Abcam) overnight at 4 °C.

Techniques: Multiplex Assay, Immunohistochemistry, Staining, Marker