CD44 Antibody - #DF6392

Fig. 2 RBA-NPs display targeting capability in vitro. a, b Confocal micrographs showed that DiD-NPs (red) enjoyed significantly improved cell uptake compared with free DiD in LPS + IFN-γ activated RAW264.7 cells. Scale bar = 10 μm. c Flow cytometry analysis showed that the fluorescence intensity of DiD-NPs in LPS + IFN-γ activated RAW264.7 cells was higher than that of free DiD in RAW264.7. d The expression of CD44 and folate receptors increased significantly on the surface of LPS + IFN-γ activated RAW264.7 cells (d). Scale bar = 10 μm. e LPS + IFN-γ activated macrophages were pretreated with HA or FA or both HA and FA to compete for the overexpressed CD44 or folate receptors, and cellular uptake of DiD-NPs was reduced. f Confocal micrographs showed co-localization of DiD-NPs (orange) with CD44 receptor (red) and folate receptor (green). Scale bar = 20 μm. Cell nuclei was stained with DAPI (blue). All results are shown as mean ± SD. *P 

Figure 5 Tumor spheres isolated from hepatocellular carcinoma HepG2 cells obtained the characteristics of cancer stem cells. (A) Comparison of the protein levels of CD133 and CD44 in HCCLM3 and HepG2 cells. The protein expression of (B) CD133 and (C) CD44 was dramatically increased in HepG2 cells compared with those in HCCLM3 cells. ∗P < 0.05, compared with the HCCLM3 cells. (D) The images of 3D tumor spheroids were captured using the fluorescence microscope on Day 0 in attachment surface plates and Days 0, 3, 5, 7, and 10 in ultra-low attachment surface plates with CSC medium (magnification 200 ×); scale bar: 20 μm. (E) The protein expression of CD133 and CD44 was analyzed by Western blot in HepG2 and CSC-like cells. The protein expression of (F) CD133 and (G) CD44 was dramatically increased in CSC-like cells compared with HepG2 cells. Data are presented as mean ± SEM; ∗P < 0.05, ∗∗P < 0.01. (H) The expression of CD133 and CD44 were assessed by immunofluorescence staining in HepG2 and tumor sphere cells. Blue represents DAPI; green represents CD44; red represents CD133. Magnification: 400 ×. (I) CSC augments tumor-initiating in vivo. Representative xenograft tumors derived from CSC-like cells compared to HepG2 cells after two weeks subcutaneous injection in nude mice (n = 3). Left panel: HepG2 cells; right: CSCs. (J) The protein levels of CD133 and CD44 were analyzed by Western blot in the tumor tissues isolated from xenograft models. GAPDH served as a sample loading control. The protein levels of CSC markers (K) CD133 and (L) CD44 were dramatically upregulated in right tissues compared with left tissues. Data are presented as mean ± SEM; ∗P < 0.05, ∗∗∗P < 0.001.

Fig. 3. Targeting ability of HTC@DOX. (A) Boxplot of CD44 expression difference between OC tumour and normal tissues using GEPIA (http://gepia.cancer-pku.cn/). The red and gray box respectively represent tumour and normal tissues. (B) CD44 protein expression level of ID8, NRK-49F, AML-12 and H9c2 cells, *compared to the ID8 group (n = 3). (C) Fluorescent inverted microscope images and quantitative of ID8 cells incubated with HTC@DOX and HA + HTC@DOX for 6 h (scale = 20 μm), *compared to the HA + HTC@DOX group (n = 3). (D) Fluorescent images of tumour-bearing mice at different time points and various organs and tumours at 24 h after tail vein injection of fluorescent dye DIR and DIR-loaded nanoparticle HT@DIR (n = 5). (E) Quantitative graph of fluorescence intensity of tumour from tumour-bearing mice at different time points (n = 5), *compared to the DIR group.