Figure 7.

CD44 knockdown in MDA-MB-231-BR and its impact on HA metabolism and interaction with the BBB. (A) Representative Western blot (n = 2) result of protein lysates (20 µg) of MDA-MB-231-BR WT, control (scramble), and CD44 KD (shCD44) showing expression of the HA receptor CD44. β-Actin was used as a loading control. (B) Histogram showing the quantified amount of CD44 measured via flow cytometry. One representative experiment is shown (n = 2) (C) Bar graph displaying ELISA results on secreted total HA and LMW-HA levels (ng/10⁶ cells) of MDA-MB-231-BR control (scramble) and CD44 KD (shCD44) cells. Cell supernatant was collected and used for ELISA (representative experiment, n = 3). (D) Histochemical staining of cells in agar for HA using biotinylated HA binding protein, scale corresponds to 50 µm. Fluorescence staining of nuclei (DAPI, blue) and HA (FITC, green) using biotinylated HABP and FITC-conjugated Streptavidin. Scale bars represent 50 µm. (E) Histogram showing the quantified amount of surface HA measured via flow cytometry. One representative experiment is shown (n = 2). (F) The adhesion ability of MDA-MB-231-BR control (scramble) and CD44 knockdown (shCD44) cells to adhere to primary human brain endothelial cells (hBMECs) was analyzed under static conditions. The relative amount (to scramble = 1) of adhesive cells is shown (n = 3). (G) ECIS: normalized resistance at 4 kHz (values were set as 1 prior treatment; representative experiment; n = 3) (H) Bar graphs displaying relative resistance values at 4 kHz under the influence of different tumor cells (n = 3). (I) Invasion potential of MDA-MB-231-BR control (scramble) and CD44 knockdown (shCD44) cells through hBMECs measured by the Transwell assay (n = 3). Statistical significance was determined using unpaired two-tailed Student’s t-tests. The assumption of homogeneity of variance was tested using Levene’s Test of Equality of Variances (p > 0.05). Values are means +/− s.d. * p < 0.05, ** p < 0.005.