Journal: Leukemia

Article Title: CREB1 promotes expression of immune checkpoint HLA-E leading to immune escape in multiple myeloma.

doi: 10.1038/s41375-024-02303-w

Figure Lengend Snippet: Fig. 2 CD56 and CREB1 modulate HLA-E expression in MM. A Schema of CD56-CREB1 signaling in MM. CD56 induces phosphorylation of RPS6KA3 (also known as RSK2), which in turn phosphorylates CREB1, leading to gene transcription. B Pathway analysis of the upregulated IFN_driven gene sets in the MMRF CoMMpass and GSE4452 datasets. Patients are divided based on median cutoff of CREB1 expression. NES normalized enrichment score; FDR false discovery rate; gene count includes the number of significant genes in the pathway. C HLA-E log2 expression values in patients with low or high CREB1 expression based on median cutoff of CREB1 expression. CoMMpass MMRF dataset: n = 809, p < 0.0001, **** and GSE4452 dataset: n = 65, p = 0.0061, **. Dashed blue lines indicate the median values; black dotted lines represent the 25th and 75th percentile. D Regression studies to correlate HLA-E (probe 200904_at) as dependent variable to CREB1 (probe 204314_s_at). p = 0.0031; R = 0.13. E ChIP-sequencing tracks showing CREB1 signal on individual locus for HLA-E. The x-axis shows genomic coordinates. F Quantitative PCR of CREB1-ChIP enriched binding site to HLA-E promoter. n = 2, t test, two-tailed; p = 0.0317, *. G HLA-E MFI fold change in U266 cells overexpressing CREB1 compared with U266 control cells (CNT). n = 3, t test, two-tailed; p = 0.0001, ***. H HLA-E MFI fold change in U266 cells overexpressing CD56 compared with U266 control cells (CNT). n = 3, t test, two-tailed; p = 0.0010, **. I HLA-E MFI fold change in OPM-2 cells, H929 cells, and CD56+ CD138+ patient-derived MM cells treated with DMSO (D) or 666-15 (CRi) 0.3 μM for 48 h. n = 3, t test, two-tailed; OPM-2 p = 0.0153, *; H929 p = 0.04, *; MM patient samples (MM pts) p = 0.04, *. J HLA-E mRNA fold change in OPM-2 and H929 cells silenced for CREB1 (shCREB1) or with scrambled vectors, scr (n = 2, t test, two-tailed; p = 0.039, * and 0.0031, **) and in U266 cells overexpressing CREB1 or the control vector- CNT (n = 3, t test, two-tailed; p = 0.05, *). K HLA-E mRNA fold change in OPM-2 and H929 cells silenced for CD56 (shCD56) or with scrambled vectors, scr (n = 2, t test, two-tailed; p = 0.0008, *** and 0.0021, **) and in U266 cells overexpressing CD56 or the control vector-CNT (n = 3, t test, two-tailed; p = 0.04, *).

Article Snippet: The following antibodies were used: HLA-E-PE (BioLegend, San Diego, CA, USA, Cat. No. 342604), CD56-APC (BD Biosciences, Cat. No. 555518, RRID:AB_398601), CD56-PE (Miltenyi Biotec, Cat. No. 170-081-014, Clone REA196), CD138-V450 (BD Biosciences, Cat. No. 562098, RRID:AB_10894011), and CD38-PE (BD Biosciences, Cat. No. 555460, Clone HIT2, RRID:AB_395853).

Techniques: Expressing, Phospho-proteomics, ChIP-sequencing, Real-time Polymerase Chain Reaction, Binding Assay, Two Tailed Test, Control, Derivative Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: LSD1 inhibitors induce neuronal differentiation of Merkel cell carcinoma by disrupting the LSD1-CoREST complex and activating TGFβ signaling

doi: 10.1101/2020.04.14.041657

Figure Lengend Snippet: a , Bulk mouse skin labeled with IgM-PE mouse isotype control. b , Bulk mouse skin labeled with NCAM-PE. DAPI - cells were sorted. c , Merkel cells labeled with IgM-PE mouse isotype control. d , Merkel cells labeled with NCAM-PE. DAPI - /NCAM + cells were sorted. Each plot shows the subpopulation gated for in the preceding plot to the left.

Article Snippet: Merkel cells were stained with anti-PSA-NCAM-PE antibody (Miltenyi Biotec, Cat. 130-117-394) or with mouse IgM-PE isotype control (Miltenyi Biotec, Cat. 130-120-070) according to manufacturer’s instructions, for 10 min at 4°C.

Techniques: Labeling, Control

Journal: The EMBO Journal

Article Title: Human fetal kidney organoids model early human nephrogenesis and Notch-driven cell fate

doi: 10.1038/s44318-025-00504-2

Figure Lengend Snippet: ( A ) Flow cytometry analysis of composition of freshly dissociated hFKs containing NCAM1 + EPCAM - early epithelial progenitors as well as NCAM1 + EPCAM + committed early differentiated epithelium. Distribution of gestational age of hFKs received for research; weeks 17–19 were the most common. ( B ) Morphological difference between NCAM1 + PAX2 + early developing nephrons and EPCAM + mature differentiated epithelial. NCAM1 + structures are aggregates, while EPCAM + structures are more convoluted and tubular in nature. ( C ) hFKOs with EPCAM + VIM + cells undergoing mesenchymal-to-epithelial transition (MET) and also MEIS1 + fetal kidney stromal cells surrounding the epithelial structures. ( D ) Mature proximal tubule structures in hFKOs expressing HNF1B, CDH6, and LTL. ( E ) Left, human embryonic stem cell-derived kidney organoids stained for LTL and synaptopodin (SYNPO). Middle, P0 hFKOs with podocytes expressing SYNPO accompanied by LTL + proximal tubules. Left, P2 hFKOs expressing SYNPO. ( F ) ECAD + GATA3 + connecting tubules in hFKOs. ( G ) CD31 + endothelial cells form small blood vessels in EPCAM + hFKOs. ( h ) hFKOs consist of multiple lineages, including WT1 + CDH6 + proximal tubules and JAG1 + medial and distal lineages.

Article Snippet: hFKOs were grown for two passages (P2, 8 weeks of culture) and dissociated into single cells, which were isolated by positive selection of NCAM1 (CD56 Microbeads, 130-097-042, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing, Derivative Assay, Staining

Journal: The EMBO Journal

Article Title: Human fetal kidney organoids model early human nephrogenesis and Notch-driven cell fate

doi: 10.1038/s44318-025-00504-2

Figure Lengend Snippet: Similarly, to the native tissue, hFKOs harbor. ( A ) Nephron progenitor cells which are NCAM1 + SIX2 + , scale bars: fetal tissue—50 μm, hFKOs—20 μm. ( B ) Early nephron epithelium which expresses NCAM1 + PAX2 + , enabling the growth and proliferation of these organoids. Scale bars: fetal tissue—20 μm, hFKOs—20 μm. ( C ) The proximal tubule has a differentiation axis which involves early WT1 expression, followed by CDH6 and ultimately LTL. These markers are present in human fetal kidney organoids, suggesting that differentiation states are conserved in vitro. scale bars: fetal tissue—20 μm, hFKOs—20 μm. ( D ) In addition to proximal tubules, MUC1 + and ECAD + distal tubule structures are present in culture. Scale bars: fetal tissue—20 μm, hFKOs—20 μm. ( E ) The C-shape is a hallmark developmental structure in nephron differentiation, in which nephron segmentation begins. Scale bar: 20 μm. ( F ) The segmentation and patterning orchestrated by the proximal WT1, Medial JAG1 and Distal LHX1 markers are recapitulated in hFKOs. Scale bar: 20 μm. .

Article Snippet: hFKOs were grown for two passages (P2, 8 weeks of culture) and dissociated into single cells, which were isolated by positive selection of NCAM1 (CD56 Microbeads, 130-097-042, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Expressing, In Vitro

Journal: The EMBO Journal

Article Title: Human fetal kidney organoids model early human nephrogenesis and Notch-driven cell fate

doi: 10.1038/s44318-025-00504-2

Figure Lengend Snippet: ( A ) heatmap comparing expression of key NPC, Renal vesicle/comma-shape body (RVCSB), proximal (SSBpr), medial-distal (SSBm_d) and podocyte (SSBpod) compartments of s-shaped bodies (SSBs) and ureteric bud/collecting duct (UBCD) markers in P0 (Freshly dissociated, embedded and cultured for 2–3 weeks) and P2 hFKOs (Organoids which were passaged twice and were cultured for 8 weeks in total). ( B ) Ontology analysis of P0 (Red) and P2 (Blue) hFKOs, processes of nephron morphogenesis and renal system development are evident in P0, while P2 is characterized by tube development and morphogenesis, in line with expected maturation and differentiation of the hFKO culture. ( C ) hFKOs cultured from week 18 of gestation for 5 months to p6 (aligning in time with the antepartum kidney, prior to birth), exhibit a maturation process commencing with a high expression of developmental genes such SIX2, PAX2, LHX1, HNF1B , and WT1 and progressing to a mature tubular kidney epithelium expressing PAX8 and CDH1 , as well as segment-specific markers such as CUBN (proximal) and MUC1, KRT8 (distal). While developmental are highly expressed in earlier passages in contrast to mature genes and vice versa, these genes are still expressed in higher passages, hence the trapezoid shape of the expression gradient across time. ( D ) Heatmap of the developmental program undergoing in hFKOs. P0 hFKOs show high expression levels of developmental genes such as JAG1, CDH6, WT1, EYA1, NCAM1, SIX2, SIX1 . Late P6 hFKOs exhibit higher levels of maturation markers such as PAX8 , CHD1, EPCAM , as well as segment-specific markers of proximal ( CUBN ) and distal ( KRT18, IRX2, MUC1 ) segments. ( E ) Ontology analysis of Early (P0) and Late (P6) hFKOs: kidney and renal system development are evident at P0, whereas P6 is characterized by epithelial cell development and renal absorption, in line with the expected maturation and differentiation of the hFKO culture. ( F ) P2 hFKOs, contain NCAM1 + PAX2 + tubular epithelium progenitors (Scale bar 20um), EPCAM + VIM + (Vimentin) tubular epithelium with ongoing differentiation and MET. CDH6 + WT1 + structures containing early proximal tubules and hFKOs with early distal, LHX1 + , and proximal, WT1 + , compartments of the SSB. Scale bars, 50 μm. ( G ) Scheme of isolation of NCAM1 + cells, by magnetic cell sorting (MACS) from P2 hFKOs, reveal the ability of these cells to form organoids ( H ) Brightfield images of NCAM1-negative fraction, devoid of newly grown hFKOs (left, scale bar 100um), Widefield NCAM1-positive fraction, giving rise to hFKOs (middle, scale bar 200 μm, n = 4), Zoomed image of a NCAM1 + derived kidney organoids (right, scale bar 100 μm). ( I ) IF of NCAM1 + -derived kidney organoids containing LTL + MUC1 + HNF1B + proximal–distal tubular epithelium (left, scale bar 50 μm), additional NCAM1 + PAX2 + epithelial progenitors (Middle, scale bar 20 μm) and EPCAM + NCAM1 + nascent nephron epithelium (right, scale bar 20 μm). ( J ) Heatmap of developmental genes, comparing expression between iPSC-KOs at early (D10) and late (D18) stages of differentiation, to hFKOs at P0 and P6. ( K ) Ontology analysis of Early hFKOs (P0, blue) vs differentiated iPSC-KOs (iPSCBD18, red): enrichment of genes correlating to urogenital and renal system development as well as epithelial cell proliferation and kidney development, while mitosis-related processes are evident in iPSC-KOs. ( L ) Ontology analysis of Late hFKOs (P5 and P6, blue) vs differentiated iPSC-KOs (iPSCBD18, Red): After 5 months of culture, P6 hFKOs are enriched with genes governing epithelial cell development as well as metanephric nephron epithelium development. Similar to the comparison to early hFKOs, iPSC-KOs are enriched with proliferation-related genes that regulate cell division and growth. .

Article Snippet: hFKOs were grown for two passages (P2, 8 weeks of culture) and dissociated into single cells, which were isolated by positive selection of NCAM1 (CD56 Microbeads, 130-097-042, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Isolation, FACS, Derivative Assay, Comparison

Journal: The EMBO Journal

Article Title: Human fetal kidney organoids model early human nephrogenesis and Notch-driven cell fate

doi: 10.1038/s44318-025-00504-2

Figure Lengend Snippet: ( A ) Immunofluorescent imaging of human embryonic stem cell-derived kidney organoids (hESC-KOs), expressing many NCAM1+ and PAX2+ structures, containing nephron progenitor cells at Day 14 of differentiation. These structures become less abundant as the culture progresses, even after an additional two days (D16). As the differentiation progresses, PAX2+ cells become organized into tubular structures and decrease in number. scale bar 50 μm. ( B ) IF imaging of hiPSC-derived kidney organoids. NPC populations co-expressing NCAM1 + PAX2+ diminish as the organoid differentiates and NCAM1 cells become unorganized and are more abundant in the stroma of the organoid. ( C ) in P2 hFKOs, after 8 weeks of culture, NCAM1 + PAX2+ co-expression is evident in epithelial progenitors. Scale bar 20 μm. ( D ) Flow cytometry of positive and negative fractions of NCAM1+ after magnetic sorting.

Article Snippet: hFKOs were grown for two passages (P2, 8 weeks of culture) and dissociated into single cells, which were isolated by positive selection of NCAM1 (CD56 Microbeads, 130-097-042, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Imaging, Derivative Assay, Expressing, Flow Cytometry

Journal: The EMBO Journal

Article Title: Human fetal kidney organoids model early human nephrogenesis and Notch-driven cell fate

doi: 10.1038/s44318-025-00504-2

Figure Lengend Snippet: ( A ) UMAP clustering of P0 hFKOs reveal 16 clusters: nephron progenitors (NPCs), renal vesicles (RVs), renal vesicles and comma-shaped bodies (RVs-CSBs), early podocytes (Early Pod), podocytes (Pod-1, Pod-2), proximal tubules (PT), early distal tubule/connecting tubules (Early DT/CNT), distal/connecting tubules (DT/CNT), TOP2A + KI-67 + cells (Proliferating), stressed cells (Stress), stromal cells (Stroma 1, Stroma 2, Stroma 3), collecting ducts (CD), and endothelial cells (Endo). ( B ) Violin plots of the expression of key markers used to characterize clusters. ( C ) Feature plots of markers depicting development and regeneration states, NPCs ( LYPD1 , PAX2 , NCAM1), differentiating epithelium ( PAX8 , LHX1 , JAG1 ) differentiated epithelium ( EPCAM ), and cells with regenerative capacity ( CD24 ). ( D ) Projection of hFKOs on PDX-Wilms tumors, NPCs, and proliferating cells from hFKOs match blastema tumor cells. ( E ) UMAP clustering of the nephrogenic compartment of hFKOs after sub-setting. ( F ) Pseudotime plot of nephrogenic compartment, starting at NPCs and bifurcating into podocytes and tubular lineages. ( G ) UMAP clustering of developmental markers across pseudotime axis in nephrogenic compartment of hFKOs. .

Article Snippet: hFKOs were grown for two passages (P2, 8 weeks of culture) and dissociated into single cells, which were isolated by positive selection of NCAM1 (CD56 Microbeads, 130-097-042, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Expressing