Fig 2: Testing the effect of doxorubicin (DOX) on p53 level in osteosarcoma cells. (A) DOX inhibits topoisomerase II (TOP2) leading to DNA damage in the form of double-strand breaks (DSBs). As a consequence, the DNA damage response and p53 pathways are activated, which leads to cell cycle arrest and cell death. (B,C) DOX increases levels of active p53 in Cd44-negative osteosarcoma cells. The cells were treated as in Figure 4. The histogram in (B) shows mean value of p53 protein level ±SD from three independent experiments. The values were normalized to loading control. Student’s t-test values: **: p ≤ 0.01. Representative immunoblots for detection of total and phosphorylated p53 are shown in (C). Please notice that phosphorylated p53 was detected by reprobing lower membrane part from the experiment in Figure 4F, therefore the β-actin is the same.

Fig 3: Dexamethasone decreased the expression of cancer stem cell markers. (A) GR-Control/Knockdown/Knockout treated with ethanol (mock), 100 nmol/L dexamethasone, and 1 mmol/L RU486 for 48 h were subjected to RNA extraction, RT and real-time PCR. Expression of each specific gene was normalized to that of GAPDH. Transcription amount is presented relative to that of mock treatment in each cell line. Each value represents the mean ± SD from at least 3 independent experiments. *P < 0.05; #P > 0.05. (B) GR-Control/Knockdown/Knockout cultured for 48 h in the presence of ethanol (mock), 100 nmol/L dexamethasone, and 1 mmol/L RU486 were analyzed on western blot analysis, using an antibody to CD44, GR, C-MYC, β-catenin, Snail, Oct-4. GAPDH was used as a reference control. Data were obtained from at least three independent experiments. The data are presented as mean ± SD. (C) GR bands of GR- Control/Knockdown/Knockout cell lines were analyzed by Image J. *P < 0.05.

Fig 4: Cellular uptake analysis of CUR and HZ-CUR and Western blot analysis of CD44 expression in CRC cells. Cells were treated with the indicated doses of the agents for 24 h. (A) Fluorescence images (400×) of CUR (green). Nuclei were stained with DAPI (blue). Scale bar, 200 μm. (B–D) CUR contents in HCT116, HCT8, and HT29 cells, respectively. (E) Western blot analysis of CD44 expression in CRC cells treated with OXA for 12, 24, and 48 h. α-Tubulin was used as loading control. (F) Western blot analysis of CD44 expression and (G) relative CUR content in HCT116 and HT29 cells (CD44+/CD44−). Knockdown of CD44 in HCT116 and HT29 cells was conducted with specific siRNAs. Values are means ± SD, n = 3. Groups marked with different letters (a, b, c or A, B, C) in the same cell line have significant differences (p < 0.05).

Fig 5: USP38 restrains cancer stem cell population.a Typical oncosphere formed by HCT116 or SW620 cells transfected with control shRNA or shRNA targeting USP38. Right panel is quantification of oncospheres. b Typical oncosphere formed by HCT116 cells transfected with control overexpression vector or USP38 overexpression vector. Lower panel is quantification of oncospheres. c Flow cytometry analysis of CD133 and CD44 double positive cells in HCT116 or SW620 cells transfected with control shRNA or shRNA targeting USP38. d Flow cytometry analysis of CD133 and CD44 double positive cells in HCT116 cells transfected with control overexpression vector or USP38 overexpression vector. e Flow cytometry analysis of apoptotic HCT116 cells transfected with control shRNA, shRNA targeting USP38, control overexpression vector or USP38 overexpression vector and treated with indicated chemotherapeutics. f Protein levels of cancer stem cell-related genes in HCT116 or SW620 cells transfected with control shRNA or shRNA targeting USP38 and HCT116 cells transfected with control overexpression vector or USP38 overexpression vector. Right panels are quantification results. g mRNA levels of cancer stem cell related genes in HCT116 or SW620 cells transfected with control shRNA or shRNA targeting USP38 and HCT116 cells transfected with control overexpression vector or USP38 overexpression vector.