Journal: STAR Protocols

Article Title: Protocol for validating computationally predicted splice-altering variants using full-length gene reporter assays

doi: 10.1016/j.xpro.2026.104433

Figure Lengend Snippet: Transient splicing reporter assay using the genomic locus sequence reproduces the endogenous TREM2 and APOE isoform patterns (A) Schematic representation of the genomic TREM2 and APOE sequences cloned into the pcDNA3.1(+) vector. (B) Human cell lines expressing endogenous TREM2 (THP-1, HMC3), non-expressing HEK293T cells, and mouse microglial BV2 cells were transfected with the full-length (FL) TREM2 reporter. (C) HMC3 and HEK293T cells expressing endogenous APOE were transfected with the FL APOE reporter. HB, endogenous TREM2 (B) and APOE (C) transcripts from human brain (frontal cortex). cDNA synthesis was primed with either oligo(dT) or a plasmid-specific RT primer (P) positioned downstream of the gene insert. Arrows mark RT-PCR primer positions. Amplified products were resolved by agarose gel electrophoresis.

Article Snippet: HEK293T , ATCC , CRL-3216.

Techniques: Reporter Assay, Sequencing, Clone Assay, Plasmid Preparation, Expressing, Transfection, cDNA Synthesis, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: Single-cell analysis identifies EpCAM + /CDH6 + /TROP-2 − cells as human liver progenitors

doi: 10.1101/294272

Figure Lengend Snippet: (A) Overview of FACS strategy in adult human liver to isolate cells for scRNA-seq. (B) 2D t-SNE visualisation of single cells isolated from adult human liver included in the study coloured by FACS parent population. (C) 2D t-SNE visualisation of single cells isolated from adult human liver t-SNE plot points coloured by K-mean cluster and labelled A1-A2. (D) Violin plots detailing expression levels of selected marker genes in each cluster identified in adult liver scRNA-seq analysis detailing distinct cell types. Scale is log10 transcripts per million (TPM). (E) Expression of selected enriched ‘Adult progenitor’ (A2) marker transcripts overlaid on the 2D t-SNE space of adult liver scRNA-seq analysis. (F) Schematic of known and novel markers expressed in adult clusters A1 and A2 labelled as ‘Adult hepatocyte and ‘Adult progenitor’ cell types respectively. (G) Duplex RNA-ISH for CDH6(red)/STAT1(blue) in adult human intra-hepatic bile ducts (BD) and limiting plate (LP), detail of the red/blue dots expanded in the square. Scale bars represent 50 μm, 25 μm in blown up squares. (I) Immunofluorescence (IF) staining on Endoscopic retrograde cholangiopancreatography procedure (ERCP) intra-hepatic biliary brushing primary human samples for EpCAM (purple) with CLDN3 (red) (i) and KRT7 (magenta) with CDH6 (green) (ii), after 2 days in Matrigel with liver expansion (LE) media. Scale bars represent 25 μm.

Article Snippet: Cells were resuspended in 3% B.S.A with 0.1mM EDTA (15575020, Gibco, Life technologies) and stained for the following conjugated primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324221, PE/Cy7, mouse, 1:100, Biolegend), Claudin-3 (130-110-834, PE, REA, 1:50, Miltenyl Biotech), CDH6 (130-113-099, APC, REA, 1:50, Miltenyl Biotech) and CD133 (372808, BV 421, mouse, 1:100, Biolegend) for 30 minutes at 4 ̊C.

Techniques: Isolation, Expressing, Marker, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Single-cell analysis identifies EpCAM + /CDH6 + /TROP-2 − cells as human liver progenitors

doi: 10.1101/294272

Figure Lengend Snippet: (A) RNA-ISH for CDH6 and STAT1 on human foetal liver ductal plate (DP) and bile ducts (BD) regions (detail of the red dots expanded in the squares). Scale bars represent 50 μm, 25 μm in blown up squares. (B) Immunohistochemistry (IHC) of EpCAM, CK19, CDH6 and STAT1 in BD and DP regions of human foetal liver. Scale bars represent 50 μm. (C) Immunofluorescence (IF) staining of CDH6 (yellow) and CLDN3 (magenta) (i) and CDH6 (yellow), CLDN3 (magenta) and STAT1 (grey) (ii) co-expression in human 2 nd trimester (15-21 pcw) foetal liver slides. Slides counterstained in DAPI (cyan). (D) Representative FACS plot of MAC-sorted total (left), EpCAM enriched(middle) and EpCAM depleted (right) cells from dissociated human foetal liver. All gates were set on fluorescence minus one (FMO) controls Gate percentages are representative of n = 3 isolations. (E) Phase-contrast and IF staining of foetal intra-hepatic organoids (f-IHOs) derived from EpCAM enriched foetal liver cells in liver expansion (LE) media. All structures are counterstained with DAPI (blue). P0 refers to cells isolated directly from primary tissue. All IF staining performed between passage 3-5. (F) IF staining of f-IHOs in LE media. All structures are counterstained with DAPI (blue). All IF staining performed between passage 3-5. (G) IF staining of f-IHOs for albumin (magenta) and KRT19 (red) cultured for 7 days after passage in LE media or Hepatic differentiation (HD) media. (H) IF staining of f-IHOs and foetal ex-hepatic organoids (f-EHOs) derived from foetal gallbladder for CDH6 (yellow) and biliary marker KRT7 (magenta). Counterstained with DAPI (blue). (I) Representative scatter plot of FAC-sorting strategy for isolation of EpCAM + /CLDN3 + /CDH6 + cells. Top plot displays EPCAM (X-axis) vs CLDN3 (y-axis) of Live/CD235a − /CD45 − negatively selected cells. Middle plot displays CDH6 (x-axis) and CD133 (y-axis) of EPCAM + /CLDN3 + cells. Bottom plot displays CDH6 (x-axis) and CD133 (y-axis) of EPCAM + /CLDN3 − cells. All plots gated on fluorescence minus one (FMO) staining controls. (J) Phase contrast and IF imaging of FAC-isolated EpCAM + /CLDN3 − /CDH6 − /CD133-(i) (scale bars represent 300 μm) and EpCAM + /CLDN3 + /CDH6 + /CD133 − (ii) (scale bars represent 200 μm) cells cultured for 10 days on RFP-HUVECs in LE media, with corresponding IF images. RFP negative regions circled in white. All scale bars represent 25 μm unless stated otherwise. FACS plots are representative of at least 3 independent experiments.

Article Snippet: Cells were resuspended in 3% B.S.A with 0.1mM EDTA (15575020, Gibco, Life technologies) and stained for the following conjugated primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324221, PE/Cy7, mouse, 1:100, Biolegend), Claudin-3 (130-110-834, PE, REA, 1:50, Miltenyl Biotech), CDH6 (130-113-099, APC, REA, 1:50, Miltenyl Biotech) and CD133 (372808, BV 421, mouse, 1:100, Biolegend) for 30 minutes at 4 ̊C.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Expressing, Fluorescence, Derivative Assay, Isolation, Cell Culture, Marker, Imaging

Journal: bioRxiv

Article Title: Single-cell analysis identifies EpCAM + /CDH6 + /TROP-2 − cells as human liver progenitors

doi: 10.1101/294272

Figure Lengend Snippet: (A) 2D t-SNE representation of all single cells included in this scRNA-seq study. Cells differentially coloured by K-means cluster (adult or foetal). Adult and foetal hLPCs shading (blue) represents single K-means cluster. (B) Heat map showing the spearman correlation coefficient of pairwise comparison between all single cells in data set. The spearman correlation coefficient between samples was calculated based on Log10(TPM) of each gene. Columns are labelled by foetal and adult K-means clusters. (C) PCA plot of Monocle pseudo-time analysis on all cells of foetal and adult hLPC cell clusters, colored by Pseudo state. (D) Heat maps of significantly expressed genes between pseudo states 1,2,3 (i) and 1,2,4 (ii). Gene expression in Log10(TPM). (E) Schematic of hepatic and cholangiocyte liver progenitor lineage based on monocle-derived ‘pseudo state’. (F) Scatter and bar plots of Gene expression Vs Pseudo state for selected genes enriched in pseudo states 1 (i), 2 (ii), 3 (iii) and 4 (iv). (G) PCA plot of Monocle pseudo-time analysis on hLPC cells with overlay of TROP-2 and CDH6 transcript expression. Gene expression in Log10(TPM). (H) Duplex RNA-ISH for TROP-2(red)/STAT1(blue) and CDH6(red)/STAT1(blue) in foetal human bile duct (BD) and ductal plate (DP) structures, detail of the red/blue dots expanded in the square. Scale bars represent 50 μm, 25 μm in blown up squares. (I) Duplex RNA-ISH for TROP-2(red)/STAT1(blue) and CDH6(red)/STAT1(blue) in adult human intra-portal BD and limiting plate (LP) regions. Scale bars represent 25 μm.

Article Snippet: Cells were resuspended in 3% B.S.A with 0.1mM EDTA (15575020, Gibco, Life technologies) and stained for the following conjugated primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324221, PE/Cy7, mouse, 1:100, Biolegend), Claudin-3 (130-110-834, PE, REA, 1:50, Miltenyl Biotech), CDH6 (130-113-099, APC, REA, 1:50, Miltenyl Biotech) and CD133 (372808, BV 421, mouse, 1:100, Biolegend) for 30 minutes at 4 ̊C.

Techniques: Comparison, Gene Expression, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Single-cell analysis identifies EpCAM + /CDH6 + /TROP-2 − cells as human liver progenitors

doi: 10.1101/294272

Figure Lengend Snippet: (A) Hematoxylin and eosin (H&E) staining with RNA-ISH for CDH6 and TROP-2 transcript expression in tissue overlay of normal human adult liver tissue. Arrows indicate bile duct and limiting plate structures. (B) H&E staining with RNA-ISH for CDH6 and TROP-2 transcript expression in adult liver tumour samples from hepatocellular carcinoma (HCC) with dedifferentiated morphology and intra-hepatic cholangiocarcinoma (ICC). (C) H&E staining with RNA-ISH for CDH6 and TROP-2 transcript expression in tissue overlay of human adult subacute liver failure bile duct and ductular reactions. (D) H&E staining with RNA-ISH for CDH6 and TROP-2 transcript expression in tissue overlay of human cirrhotic liver bile duct and ductular reactions. All scale bars represent 50 μm.

Article Snippet: Cells were resuspended in 3% B.S.A with 0.1mM EDTA (15575020, Gibco, Life technologies) and stained for the following conjugated primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324221, PE/Cy7, mouse, 1:100, Biolegend), Claudin-3 (130-110-834, PE, REA, 1:50, Miltenyl Biotech), CDH6 (130-113-099, APC, REA, 1:50, Miltenyl Biotech) and CD133 (372808, BV 421, mouse, 1:100, Biolegend) for 30 minutes at 4 ̊C.

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Single-cell analysis identifies EpCAM + /CDH6 + /TROP-2 − cells as human liver progenitors

doi: 10.1101/294272

Figure Lengend Snippet: Schematic of human CDH6 and TROP-2 expression in normal, tumour and liver injury models.

Article Snippet: Cells were resuspended in 3% B.S.A with 0.1mM EDTA (15575020, Gibco, Life technologies) and stained for the following conjugated primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324221, PE/Cy7, mouse, 1:100, Biolegend), Claudin-3 (130-110-834, PE, REA, 1:50, Miltenyl Biotech), CDH6 (130-113-099, APC, REA, 1:50, Miltenyl Biotech) and CD133 (372808, BV 421, mouse, 1:100, Biolegend) for 30 minutes at 4 ̊C.

Techniques: Expressing

Journal: iScience

Article Title: Differential conservation analysis identifies residues defining constitutive internalization in beta-adrenergic receptors

doi: 10.1016/j.isci.2026.115033

Figure Lengend Snippet: B2AR V34A 1.33 and S41A 1.40 mutations alter the organization of receptor homomers (A) Schematic showing the experimental design of BRET for receptor-receptor associations. HEK 293 cells were transiently transfected with equal amounts of Rluc8-tagged receptor, either wild-type or with mutations, and increasing amounts of Venus-tagged receptor, either wild-type or with mutations. BRET saturation curves were used to determine the (B) BRET max and (C) the BRET 50. Data are the mean ± SEM; one-sample t test: ∗ p < 0.05. (D) Curve plotted using one-site specific binding saturation equation, representative curves shown, n = 6–8.

Article Snippet: HEK 293 , ATCC , CRL-1573; RRID:CVCL_0045.

Techniques: Transfection, Binding Assay

Journal: iScience

Article Title: Differential conservation analysis identifies residues defining constitutive internalization in beta-adrenergic receptors

doi: 10.1016/j.isci.2026.115033

Figure Lengend Snippet: The effect of B2AR mutations on basal and ligand-induced cAMP (A) cAMP accumulation in HEK 293 cells transiently transfected with HA-B2AR WT and mutants with isoproterenol treatment (1 pM-10 μM, 5-min). (B) cAMP production at the maximum isoproterenol dose (10 μM) and (C) basal cAMP production. Data are the mean ± SEM from n = 5–6 independent experiments. One-sample t test: ∗∗∗ p < 0.001. (D) EC 50 values for curves in A.

Article Snippet: HEK 293 , ATCC , CRL-1573; RRID:CVCL_0045.

Techniques: Transfection

Journal: iScience

Article Title: Differential conservation analysis identifies residues defining constitutive internalization in beta-adrenergic receptors

doi: 10.1016/j.isci.2026.115033

Figure Lengend Snippet: Double mutation decreases basal activity by increasing constitutive internalization (A and B) Cell surface expression of HEK 293 cells transiently transfected with HA-B2AR WT or mutant receptors. (A) Percent cells expressing receptor shown as fold change to the WT and (B) the amount of receptor at the plasma membrane shown as fold change to the WT. n = 5, one sample t test: ∗ p < 0.05, ∗∗ p < 0.01. (C) Whole-cell expression of B2AR-Rluc8 mutant receptors shown as fold change to the WT receptor, n = 6–10. One-sample t test: ∗∗ p < 0.01. (D and E) cAMP production in (D) basal and (E) isoproterenol (1 pM-10 μM, 5-min) treated HEK 293 cells transiently transfected with WT and V34A-S41A B2AR and matched for receptor expression at the plasma membrane . (F) Schematic demonstrating antibody conditions for labeling endocytic/constitutively internalized receptor pools and the total pool of receptor (endocytic + biosynthetic). (G) Confocal microscopy images of HEK 293 cells transiently transfected with HA-B2AR WT and mutant receptors, either fed live with anti-HA antibody (endocytic imaging) or incubated with anti-HA antibody post-fixation and permeabilization (endocytic + biosynthetic imaging). Representative images shown, n = 10 cells. Scale bars, 10 μm; inset = 3 μm. (H) Constitutive internalization measured in HEK 293 cells transiently transfected with HA-B2AR, HA-V34A-S41A B2AR, or HA-B1AR live labeled with anti-HA antibody at 4°C with/without 1-h incubation at 37°C. Cell surface expression measured via flow cytometry. Data shown as percentage change from surface expression in cells kept at 4°C, mean ± SEM. n = 4. One-way ANOVA with Šídák’s multiple comparisons test: ∗ p < 0.05; ∗∗ p < 0.01. See also .

Article Snippet: HEK 293 , ATCC , CRL-1573; RRID:CVCL_0045.

Techniques: Mutagenesis, Activity Assay, Expressing, Transfection, Clinical Proteomics, Membrane, Labeling, Confocal Microscopy, Imaging, Incubation, Flow Cytometry