Fig 2: Flow cytometric analysis. Expression levels of (A) CD44+ and (B) CD133+ in cells with different ALDH activity are shown. The percentages in (A) indicate CD44+ cells in the groups of cells with different ALDH activity. The percentages in (B) indicate CD133+ cells in the groups of cells with different ALDH activity. (C) Statistical analysis of the expression of CD133+. ALDH, aldehyde dehydrogenase.

Fig 3: NAC maintains circulating EPC population in mice with hyperlipidemia following PM exposure. Circulating cells were isolated from LDLR KO mice fed HFD with or without PM exposure. Following red blood cell lysis, murine circulating CD34+/CD133+ cell population was analyzed by flow cytometry. Circulating EPC levels were significantly decreased in both 1-week and 6-month LDLR KO PM + HFD mice compared with HFD mice. NAC effectively maintained the EPC population. One week PM + ND exposure noticeably decreased EPC population while 1 week HFD had no effect on EPC compared with ND. n=5-8. *P<0.01 vs. ND; **P<0.01 vs. HFD; #P<0.01 vs. PM + ND; ##P<0.01 vs. PM + HFD. ND, normal diet; HFD, high-fat diet; NAC, N-acetyl cysteine; PM, ambient fine particulate matter; LDLR, low-density lipoprotein receptor; KO, knockout; EPC, endothelial progenitor cell.

Fig 4: Elevated FUBP1 enhances the tumorigenicity of CRC cells in vivo. (A, C, E) Representative images of the tumors were shown. (A) Tumors formed by FUBP1-transfected SW48 cells and vector control. (C) Tumors formed by FUBP1-silenced LoVo cells and control LoVo cells. (E) Tumors formed by FUBP1-silenced cells which were CD133+ALDH1+ cells sorted from LoVo cells and its control cells. Cells (1 × 106, 1 × 105, 1 × 104, 1 × 103, or 1 × 102) were implanted into BALB/c nude mice (n = 6, per group). Tumor formation growth curves following implantation of the 1 × 106 (B) (D)or 1 × 105 (F) indicated cells. P values were determined by two-tailed Student’s t-test. (G) Representative images of IHC staining of CD133, ALDH1, and FUBP1 in tumor tissues of mice originated from the indicated cells. Scale bar, 50 µm. (H) Representative images of FUBP1, CD133, and ALDH1 IHC staining of CRC Tissue Microarrays (HCol-A150CS-02-M-013, 75 cases). Scale bar, 100 µm. (I) FUBP1 expression with the expression of CD133 and ALDH1 in 75 CRC patient specimens was determined by Pearson’s correlation analysis. All bars represented the mean ± SD of three independent experiments, and *, **, and *** denotes P < 0.05, P < 0.01, and P < 0.001, respectively.

Fig 5: (A) FACS analysis for CD44 or Sca-1-positive subpopulations in primary #1 cells after Wnt small-molecule inhibitors for 24 h. CWP291, 1 µM CWP232291; BC21, 10 µM BC21; CCT, 20 µM CCT031374; PKF, 2 µM PKF 118–774; Cisplatin, 1 µM cisplatin. Right, representative FACS images showing reduced CD44 or Sca-1-positive cells in primary #1 cells after CWP232291 treatment. (B) Sphere-forming assay showing reduced tumorsphere formation from primary #1 cells after the treatment for 7 days with Wnt inhibitors. The spheres greater than 100 mm of diameter were enumerated. Right, representative images of tumor spheres after the treatment with Wnt inhibitors. (C) CWP232291 inhibited primary (with CWP232291) and second sphere formation (without CWP232291) in primary #1 and SW620. (D) Representative images of tumorspheres from primary #1 cells of (C). (E) Representative IF images for CD133, Lgr-5, and Sca-1, putative mouse intestinal cancer stem cell markers, in vehicle- or CWP232291-treated allografts of SCID mice. SCID mice injected with primary #1 cell were treated with 100 mg/kg CWP232291 twice a week for 8 weeks. Bottom, IF scoring for CD133, Lgr-5, and Sca-1-positive tumor cells in vehicle- (n = 3) or CWP232291-treated (n = 3) allografts. Bar = 100 µm. (F) FACS analysis for CD133 in CWP232291- or vehicle-treated allografts. Single cell suspensions were obtained from allografts after dispase digestion. FACS, fluorescence-activated cell sorting; IF, immunofluorescence. *p < 0.05