CITED1 monoclonal antibody (M03), clone 5H6 H00004435-M03 from Abnova Corporation

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PloS one
January 01 2017

Fig 1: Molecular characteristics in metanephrogenic epithelial structures in FK and those in the equivalent metanephrogenic epithelial structures in WT.1. Metanephric specification and in FK and WT. (A, B) FK (A): HOXD-11 (red) is localized in the nuclei of CM (surrounded by broken lines) as well as in those of stroma cells. WT (B): HOXD-11 (red) is expressed in the nuclei of CM-like blastemal aggregates and of stromal cells (Str). 2. Metanephric condensation in FK and WT. (C, D) FK (C): WT1 (green) and SIX2 (red) co-localized in nuclei of CM (orange nuclear signals); RVs lack expression of SIX2. WT (D): Expression of WT1 (green) and SIX2 (red) overlaps in nuclei of CM-like structures. (E, F) FK (E): Cells with nuclear SIX2 positivity (red) show nuclear and cytoplasmic expression of CITED1 (green) within the CM. WT (F): Both SIX2 (red) and CITED1 (green) positive cells are similarly present within CM-like structures. Note that the CM-like blastemal aggregates surround the WT1-negative UB-like structures and show parallel expression patterns to the CM in FK. 3. Transition from cap mesenchyme to renal vesicle in FK and their equivalent structures in WT. (G, H) Beta-catenin (red) in the cytoplasm and cell membrane of the CM and RV in FK (G) and in those of CM-like and RV-like epithelial structures in WT (H). (I, J) FK (I): WT1 and Pax-8 are co-expressed in the nuclei of RV, eGE, and lower part of S-shaped bodies (SSB, orange nuclear signals). Nuclear Pax-8 staining (red) is solely detected in UBs and maturing distal nephrons in FK. WT (J): Both WT1 and Pax-8 are expressed in RV-like epithelial structures (orange nuclear signals). Note the UB-like epithelial structure lacking nuclear WT1 (arrow). (K, L) FK (K): Jagged-1 (red) is highly expressed in the upper segment of the CSB (the proximal tubule-committed region), while its expression is weak in the apical membrane of renal lower part of the CSB. Membranous localization of Jagged-1 is noted in UBs. WT (L): Jagged-1 (red) is highly expressed in the cell membrane of RV-like structures and their derivatives where WT1 (green) is expressed. Jagged-1 is expressed in UB-equivalent epithelial structures, but its expression level is much weaker than E-cadherin and Beta-catenin. (M, N) FK (M): Integrin alpha 8 (red) is highly expressed in the cytoplasm and extracellular membrane of CM and RVs where WT1 (green) is localized in the nuclei. WT (N): Integrin alpha 8 (red) is expressed in the extracellular matrix of CM and RV-like structures, both of which express WT1. Integrin alpha 8 is also highly expressed in the stroma. Note that Beta-catenin, Jagged-1 and Integrin alpha 8 show a similar expression pattern recapitulating transition from cap mesenchyme to renal vesicle surrounding the WT1-negative UB-like structures. 4. Segmentation of GL (O), PT (R), and DT (T) in FK and their-equivalent structures (P, Q, S, U, V) in WT. (O, P, Q) FK (O): A GL marker, Synaptopodin (red) in the glomerular loop. WT (P, Q): Synaptopodin is localized to podocytes and tufts of glomeruloid structures (P) where WT1 (green) is highly expressed in WT (Q). (R, S) FK (R): A PT marker, NBAT (red) in the cytoplasm and lumen of PT. Nuclear positivity of WT1 (green) highlights the relationship between PT and GL. WT (S): NBAT (red) is positive in the epithelial structures (PT) connected to GL-like structures. (T, U, V) FK (T): A DT marker, KCNJ1 (red) in the apical membrane of DT (arrows). WT (U, V): KCNJ1 (red, indicated by arrows) in the epithelial structures adjacent to early glomeruloid epithelial structures (eGE). Co-localization of MUC-1 (green) confirmed the formation of DT (indicated by arrows). Nuclear counterstain with DAPI (O, P, T, U, V). B, D, F, H, L, N, P, S, U, V are tumours with triphasic histology group and J is a tumour from epithelial-predominant histology group. Bright signals for WT1 in glomerular tufts (the nucleus of podocytes and the cytoplasm of endothelial cells of capillaries) and the cytoplasm of some stromal cells indicate that WT1 is expressed at very high levels. (A, B, C, E, G, H, I, K, L, M, N), original magnification x400; (D, E, J), x200.
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Advanced science (Weinheim, Baden-Wurttemberg, Germany)
July 01 2023

Fig 2: Spatial transcriptomics (ST) analysis of SIX2+CITED1+ spots from hFK and WT reveals molecular heterogeneity of histologically similar structures in different WT. A. ST visualization of SIX2+CITED1+ spots in hFK, WT#12, and WT#3 obtained by unsupervised clustering of ST performed on integrated data. The hFK SIX2+CITED1+ spots are localized in the nephrogenic zone (identified by cap mesenchyme as shown by the H&E, black box). Zoomed in H&E representing the cap mesenchyme in hFK and the blastema component in WT#12 and WT#3 that are spread throughout the WT. B) Table summarizing the distribution of SIX2+CITED1+ spots per cluster and the percentage of total SIX2+CITED1+ spots compared to the total number of spots per sample (right side column) C) List of top gene ontology (GO) biological process of hFK nephrogenic zone (cluster 5, SIX2+CITED1+ spots) compared to WT#3 blastema (cluster 4) and WT#12 blastema (cluster 6) SIX2+CITED1+ spots (left panels) and in WT#3 blastema (cluster 4) or WT#12 blastema (cluster 6) SIX2+CITED1+ spots compared to hFK nephrogenic zone (cluster 5, SIX2+CITED1+ spots, right panels). Fold enrichment of each gene set is shown, P < 0.05; upregulated DE genes were used for each comparison. D) Pearson correlation between the expression of CITED1 and SIX2 was performed on SIX2+CITED1+ spots found in clusters of WT#3 (cluster 4), hFK (cluster 5), and WT#12 (cluster 6), with dots representing spots; dots color intensity indicating the number of spots superimposed with a similar expression of SIX2 (x-axis) and CITED1 (y-axis).
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Advanced science (Weinheim, Baden-Wurttemberg, Germany)
July 01 2023

Fig 3: SIX2+CITED1+ cell self-renewal: the role of the extracellular matrix niche. A,B) Representative immunofluorescence staining showing the distribution of ITGß1 (green) and SIX2 (red) in hFK (10 WGA) and WT (WT#12, favorable stage III) A) and for ITGß1 (red) and CITED1 (green) in hFK (10 WGA) and WT (WT#8 favorable stage II. B) Nuclei stained with DAPI (blue); scale bars 50 and 75 µm, respectively. C) Heatmap showing gene expression profile for integrins in SIX2+CITED1+ cells from hFK (17, 17.2, and 17.5 WGA) and WT (WT#3 anaplastic stage I, WT#4: non-anaplastic, stage III, and WT#5: non-anaplastic chemo-treated, stage IV). D) Densitometric analysis by western blot (WB) of ITGß1 expression in freshly isolated SIX2+CITED1+ cells from WT (WT#8,11,12, favorable stage II, favorable stage III, and favorable stage II) versus SIX2+CITED1+ cells from hFK (15,16,18 WGA) showing higher expression of ITGß1 in hFK cells; ß-actin was used as housekeeping protein for normalization. WB bands are presented below the graph, *p < 0.05. E) Representative immunofluorescence staining of SIX2 (red) and CITED1 (green) in SIX2+CITED1+ cells from hFK (17 WGA) cultured for 72 h with/without 1 µg mL-1 anti-ITGß1 or 0.5 µg mL-1 anti-ITGß4 neutralizing antibody showing increased expression of CITED1 in cells treated with anti-ITGß1. Nuclei stained with DAPI (blue). Scale bar = 50 µm. F,G) Percentage of SIX2+CITED1+ cells and total SIX2+ cells from hFK (17 WGA) by flow cytometry analysis after F) 5 d or G) 28 d of culture with/without anti-ITGß1 or anti-ITGß4 neutralizing antibody or a combination of both. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; mean ± SEM. H) Densitometric analysis by WB of LEF1 protein expression in hFK SIX2+CITED1+ cells (17 WGA) cultured for 28 d with/without anti-ITGß1 or anti-ITGß4 neutralizing antibody ß-actin was used as housekeeping control. WB bands are presented below the graph. *p < 0.05. I) Kaplan-Meier survival analysis of mice injected with WT SIX2+CITED1+ cells, without treatment (control, n = 4) or with the treatment of anti-ITGß1 (n = 5) or anti-ITGß4 (n = 4), endpoint tumor size 1.5 cm. J) Schematic representation showing the proposed role of ITGß1 and ITGß4 in WT SIX2+CITED1+ cells.
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Advanced science (Weinheim, Baden-Wurttemberg, Germany)
July 01 2023

Fig 4: SIX2+CITED1+ cells in hFK and WT have different transcriptional signatures. A,B) Periodic acid Schiff (PAS, left, whole image) and H&E (right, close-up images) staining of hFK (A,10 WGA) and WT#4 (B, favorable stage III) show the nephrogenic zone (white dotted line) and differentiating structures (second panel: ureteric bud, UB; cap mesenchyme CM; tubule, glomerulus, and stroma) of hFK, and unorganized WT histology with triphasic components (second panel, stroma, blastema, and epithelial structures including abortive glomeruli and tubules). 10× images acquired and composed using Photoshop DC (Adobe) for whole images, right panels of 20X images. C,D) SIX2 (red) and CITED1 (green) immunofluorescence staining of C) hFK 10 WGA and D) WT#4. SIX2+CITED1+ co-expression in hFK (C, second panel) in the nephrogenic niche (uninduced cap mesenchyme, UCM) but absent within developing (renal vesicle, C-shape, S-shape) and mature (glomerulus and tubule) structures. SIX2+CITED1+ expression is dispersed throughout the WT (D, second panels) in blastema but not in stroma or abortive structures (glomerulus and tubule). Nuclei stained with DAPI (blue), 10× images acquired and composed using Photoshop DC (Adobe) for whole images, right panels of 20× images. E) SmartFlare technique validation by flow cytometry. SIX2-Cy5 and CITED1-Cy3 probes (top left and right panel respectively) were individually used to isolate cells from hFK (17.4 WGA). Flow cytometry confirmed that 99.7% of SIX2+ cells and 94.3% of CITED1+ cells co-express both mRNA and protein (bottom left and right panels). F) FACS sorting (by Smartflares): 5.96% of cells from hFK 16.4 WGA are SIX2+CITED1+ cells, 0.46% from WT#3 (unfavorable stage I), 8.56% from WT#4 (favorable stage III) and 28.2% from WT#5 (favorable chemotherapy-treated stage IV). G,H) Bulk RNA-seq analysis of hFK (17, 17.2, and 17.5 WGA) and WT (n = 3, as in F). PCA (principal component analysis, G) describes 49.43% and 18.14% of the variability, along PC1 and PC2 respectively, within the expression data set. SIX2+CITED1+ cells from WT cluster independently of SIX2+CITED1+ cells from hFK. H) Hierarchical clustering of total gene expression in SIX2+CITED1+ cells from hFK and WT highlights higher similarity among SIX2+CITED1+ cells from different hFK versus higher divergence of SIX2+CITED1+ cells from different WT.
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Advanced science (Weinheim, Baden-Wurttemberg, Germany)
July 01 2023

Fig 5: WT-derived SIX2+CITED1+ cells can be expanded in vitro and generate in vivo xenografts. A flow cytometry analysis of SIX2+CITED1+ cells (%) from hFK (17 WGA) cultured for 5 days on matrigel, collagen I (COL1), fibronectin (FN1), collagen16 (COL16), or laminin511 (LAM511). *p < 0.05; ***p < 0.001; mean ± SEM. B) Flow cytometry analysis of SIX2+CITED1+ cells (%) from hFK (17 WGA) cultured for 28 d on matrigel or LAM511. C) Schematic representation of in vivo limiting dilution and serial xenografts experiments (created with Biorender). D) FACS sorting of SIX2+CITED1+ cells using Smartflare probes from different WT subtypes with favorable histology, stage II (WT#8: 14.7%, WT#11: 30%, and WT#13: 10.7%). E) Representative H&E staining (left panel), SIX2 (red) and CITED1 (green, center) and human mitochondria (red) immunofluorescence staining (right panel) of xenograft generated from freshly isolated WT#8 (favorable stage II)-SIX2+CITED1+ cells (4 months after injection) and cultured WT#8-SIX2+CITED1+ cells (passage 6, 4 months after injection). Classical triphasic WT structures are shown (b: blastema, e: epithelium, and s: stroma; white lines). Nuclei stained with DAPI (blue), scale bar = 50 µm. F) Survival analysis of mice after injection of cultured SIX2+CITED1+ cells from WT#8 (favorable stage II) either without treatment (control, n = 12) or after vincristine treatment (450 µg kg-1 vincristine sulfate IP every 4 days, n = 4).

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CITED1 monoclonal antibody (M03), clone 5H6

The CITED1 monoclonal antibody (M03), clone 5H6 from Abnova Corporation is a Mouse Monoclonal antibody to CITED1. This antibody recognizes Human antigen. The CITED1 monoclonal antibody (M03), clone 5H6 has been validated for the following applications: EIA, Immunoassay, ELISA, Immunohistochemistry, Immunohistochemistry - fixed, and Western Blot.

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Mouse monoclonal antibody raised against a partial recombinant CITED1

PloS one

Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Advanced science (Weinheim, Baden-Wurttemberg, Germany)

CITED1 monoclonal antibody (M03), clone 5H6 from Abnova Corporation

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Sequence

Abnova Corporation

Citations (2)

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CITED1 monoclonal antibody (M03), clone 5H6 from Abnova Corporation

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