lef1 c12a5 rabbit mab cell signaling technology  (Cell Signaling Technology Inc)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Identification and Characterization of the Wilms Tumor Cancer Stem Cell.

doi: 10.1002/advs.202206787

Figure Lengend Snippet: Figure 3. SIX2+CITED1+ cell self-renewal: the role of the extracellular matrix niche. A,B) Representative immunofluorescence staining showing the distribution of ITG𝛽1 (green) and SIX2 (red) in hFK (10 WGA) and WT (WT#12, favorable stage III) A) and for ITG𝛽1 (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 ITG𝛽1 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 ITG𝛽1 in hFK cells; 𝛽-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-ITG𝛽1 or 0.5 μg mL−1 anti-ITG𝛽4 neutralizing antibody showing increased expression of CITED1 in cells treated with anti-ITG𝛽1. 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-ITG𝛽1 or anti-ITG𝛽4 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-ITG𝛽1 or anti-ITG𝛽4 neutralizing antibody 𝛽-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-ITG𝛽1 (n = 5) or anti-ITG𝛽4 (n = 4), endpoint tumor size 1.5 cm. J) Schematic representation showing the proposed role of ITG𝛽1 and ITG𝛽4 in WT SIX2+CITED1+ cells.

Article Snippet: #9315 1:1000 WB Cyclin D1 Abcam # 16663 1:1000 WB LEF1 (C12A5) Cell Signaling Tech.

Techniques: Staining, Gene Expression, Western Blot, Expressing, Isolation, Cell Culture, Cytometry, Control, Injection