Fig 2: Comparative CD44-promoter activities of HepG2 and HepG2_Rego_R cells. Promoter activity of the CD44 gene in HepG2_Rego_R cells was significantly enhanced, and on using shRNA against FOXM1, it decreased by approximately 65%. The CD44 promoter activity was not changed in the off-target shRNA-treated HepG2_Rego_R cells compared with that in the control HepG2_Rego_R cells. Data are presented as mean ± SEM (n = 4) and were analyzed using one-way ANOVA and the Tukey post hoc test (* p < 0.05 and *** p < 0.001).

Fig 3: SENP2 is involved in cell migration and sphere formation. (A,B) SENP2 promotes the migration of HeLa cell. SENP2-knockdown HeLa cells were transfected with the indicated plasmid and subjected to measure the cell migratory ability using a Boyden chamber analysis with 10% FBS in the low compartment. Migrating cells were analyzed by a crystal violet assay (A) and counted with microscopy (B). Error bars show standard deviations from 10 microscopic fields from three independent experiments. Statistical significance was ascertained with Student’s t-test. (C,D) SENP2 promotes the migration of MDA-MB-231 cell. MDA-MB-231-2B cells were transfected with the indicated plasmid and subjected to measure the cell migratory ability using a Boyden chamber analysis. The migratory ability was determined as above. (E) The SENP2 enhances the expression of MMP9 and CD44. MDA-MB-231-2B cells were re-introduced HA-SENP2 or SENP2-DM plasmid. The resulted cell lysates were used to analyze the expression level of MMP9 and CD44 by immunoblotting with indicated antibodies. (F,G) SENP2 involves in sphere cell formation. MDA-MB-231-2B cells were transfected with the indicated plasmid and used MEGM medium to culture sphere cell (F). The diameters of sphere cell were measured with ImageJ software (G). Error bars show standard deviations from =100 sphere cells in three experiments. Statistical significance was ascertained with Student’s t-test.

Fig 4: ITGß3 is necessary for OPN-a-induced growth inhibitionA. The expression of various integrin and CD44 isoform mRNAs was assessed by RT real-time PCR. Integrin a4, a5, and ß3 expression levels were lower in A549 cells than in CL1-5 cells. The difference in ITGß3 expression between CL1-5 and A549 cells was the most significant. *, p < 0.05; **, p < 0.01, ***, p < 0.001. B. Growth curves for OPN-a stably-expressing CL1-5 cells treated with si-ITGß3 or si-NC. The OPN-a stable clone (OPN-a/si-NC) grew more slowly than VC/si-NC cells. ITGß3 knockdown reversed OPN-a-induced growth inhibition in CL1-5 cells (OPN-a/si-ITGß3). ITGß3 also increased growth in CL1-5 cells (comparison between VC/si-NC and VC/si-ITGß3). C. The efficacy of si-ITGß3 in the CL1-5/OPN-a stable clone was measured by RT real-time PCR in cells in (B). **, p < 0.01, ***, p < 0.001. D. CL1-5 cells transfected with si-ITGß3 or si-NC were treated with purified OPN-a. Similar to ITGß3 knockdown in the CL1-5/OPN-a stable clone (B), ITGß3 knockdown in CL1-5 cells reversed OPN-a-induced growth inhibition. *, p < 0.05. E. A549 cells exogenously expressing ITGß3 were treated with purified OPN-a. Overexpression of ITGß3 in greatly reduced A549 cell growth, suggesting that ITGß3 is required for OPN-a-induced growth inhibition. **, p < 0.01. F. The expression of ITGß3 protein in CL1-5/si-ITGß3 and A549/ITGß3 cells in (D & E). G. Purified OPN-a inhibited growth in H460/ITGß3 and H1299/ITGß3 cells, but did not affect growth in H460 and H1299 cells.

Fig 5: SDCBP depletion is associated with stemness. (A) HNSCC cell lines (parental, P, and CDDP-resistant, R, cells) were depleted of SDCBP and analyzed by qRT-PCR to determine the mRNA expression of the indicated stem cell genes. The most significant differences were found in CD44 for all the cell lines, followed by ABCB1 in HTB-43 and JHU029 cells (parental and resistant cells) and Notch1 in all the cell lines except the HTB-43 cells; (B) Western blot of cells analyzed in Figure 3A. Quantification of SDCBP/Vinculin, p-Src/Src ratio, CD44/Vinculin, and Sox2/Vinculin expression is indicated. Note that the p-Src/Src ratio decreases when SDCBP is inhibited, followed by CD44 and Sox2; (C) Analysis of ABCB1 expression by flow cytometry (plots are shown in Figure S5A); (D) The indicated HNSCC cell lines were depleted of SDCBP, and apoptosis induction was analyzed. The table shows the percentage of apoptosis, including both early apoptosis and late apoptosis (corresponding plots are shown in Figure S6). * p < 0.05, ** p < 0.01, *** p < 0.001.