# CD44 Depletion in Glioblastoma Cells Suppresses Growth and Stemness and Induces Senescence
## 3 Results  
CD44 Depletion Diminishes the Proliferative Capacity and Induces Cellular Senescence in U251 MG Cells  
In an attempt to explore the role of CD44 in GBM progression, we utilized the CRISPR/Cas9 gene editing to generate stable CD44 knock-out (KO) clones of U251 MG cells, a well-established, highly proliferative, and invasive GBM cell line. We designed two different single-guide RNAs (sgRNAs) targeting exon 2, one of the conserved exons encoding part of N-terminus of CD44 (Figure S1). Upon lentiviral delivery and puromycin selection, potential CD44 KO clones were screened by immunoblotting and Clones #1, #2, and #3, derived by using sgRNA #1, and Clones #20 and #28, derived by using sgRNA #2, were selected for further experiments (Figure 1a). Since CD44 has been connected with cell cycle progression and proliferation of cancer cells of various origins, we analysed the proliferation of CD44 KO cells using a cell counting assay; we observed a significant reduction in cell numbers of KO clones compared to the parental cells (Figure 1b). To elucidate whether the decreased number of cells was due to impaired proliferative capacity or induction of apoptosis, immunostaining against the nuclear proliferation marker Ki67 was performed. We observed a reduced number of Ki67-positive nuclei in CD44 KO clones, such as Clones #3 and #28, compared to control cells, when cells were grown in a complete medium. In addition, parental cells deprived of serum also exhibited fewer Ki67-positive nuclei (Figure 1c,d). Furthermore, immunoblotting against cleaved caspase-3 was negative in CD44 KO clones, as well as in parental cells under these conditions ([PERSON], unpublished data [3]), consistent with the notionthat the reduction in the number of CD44 KO cells was due to impaired proliferation and not to induction of apoptosis. Figure 1: CD44 depletion reduces proliferation of U251 MG cells and induces cellular senescence. (a) Immunoblot analysis of full length (FL) and cleavage products (TM-ICD) of CD44 in either parental  
or selected clones of CD44 KO clones of U251 MG cells after utilizing two different single-guide RNA (sgRNA) constructs (1 and 2). Tubulin was used as the loading control. Uncropped immunoblots are shown in Figure S6. **(b)** Proliferation assay was performed by counting cells of parental U251 MG and selected CD44 KO clones after 6 days in culture. (**c**,**d**) Immunofluorescence analysis of Ki67 expression in parental or CD44 KO U251 MG cells cultured in 10% FBS containing medium for 24 h; culturing of parental U251 MG cells in serum-free medium was used as the negative control (**c**). Quantification of Ki67-positive nuclei was carried out by using ImageJ software (**d**). Zeiss Axivision was used to take the micrographs; bars, 20 \(\upmu\)m. (**e**) Immunoblotting for phospho-(p)CREB after stimulation for 24 h in parental U251 MG cells and CD44 KO clones cultured in media containing 10% or 0% FBS. GAPDH served as the loading control; the results are shown as the fold difference after normalization to GAPDH when cells were cultured in 10% FBS-containing medium. Uncropped immunoblots are shown in Figure S7. **(f)** A senescence-associated-\(\beta\)-gal assay was carried out in parental U251 MG and selected CD44 KO clones after the cells had been cultured for 24 h in medium with 0 or 5% FBS. Zeiss Axivision was utilized to take the micrographs. Bars represent 100 \(\upmu\)m. **(g)** Immunoblotting analysis for p16 was performed following stimulation of cells for 24 h in medium containing 0% or 10% FBS; GAPDH was used as the loading control. Uncropped immunoblots are shown in Figure S8. Quantification of p16 after normalization to GAPDH is depicted above the immunoblots. All graph bars are illustrated as the average \(\pm\) SEM based on at least three independent experiments. Asterisks depict significant differences compared to the control: *_p_ < 0.05, **_p_ < 0.01.