Fig 2: Gly-tRF is involved in the maintenance of LCSC-like properties. A, C Flow cytometric analysis to detect the percentage of representative LCSC surface markers (CD133, CD13, EpCAM, CD44) in HCCLM3 and Huh7 cells stably transfected with Gly-tRF NC-mimic and Gly-tRF mimic. The statistical graph shows result of three independent experiments. B, D Sphere formation assay to reflect the sphere formation ability of those cells with LCSC-like properties in HCCLM3 and Huh7 transfected with Gly-tRF NC-mimic and Gly-tRF mimic. Scale bar = 100 µm. The statistical graph shows the diameter of spheres in three independent experiments. Data are shown as mean ± SD. ns means no significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 3: X-ray irradiation enriched the stemness of surviving cancer cells in vitro and in vivo. (a) Established pancreatic cancer cell lines (SW1990, Panc1, and AsPC1) were exposed to various doses (0, 4, 8, and 10 Gy) X-ray irradiation and analyzed for CD133+ cancer cells by flow cytometry at 0, 24, 48, 72 h post treatment. (b) Western blot analysis of the protein expression levels of stem cell related markers (CD133, Oct4, Sox2, and Nanog) in pancreatic cancer cell lines (SW1990, Panc1, and AsPC1) at 48 h post-treated with various doses (0, 4, 8, or 10 Gy) X-ray irradiation. GAPDH expression was detected as a loading control. (c) qRT-PCR analysis of stem cell-related markers (CD133, Oct4, Sox2, and Nanog) in pancreatic cancer cell lines (SW1990, Panc1, and AsPC1) at 48 h post-treated with various doses (0, 4, 8, or 10 Gy) X-ray irradiation. GAPDH was detected as a loading control. (d) Schematic representation of the in vivo irradiation protocol. SW1990 cancer cells were used to establish subcutaneous tumors. When the tumors reached a volume of 1mm3, the nude mice received 4 Gy X-ray irradiation every 2 d for 6 d. Fresh tumor tissues were harvested 5 d after the last irradiation. (e) Fresh tumor tissues were digested into single cell suspension and the percentages of CD133+ were accessed by flow cytometry. Experiments were repeated three times, and the data are expressed as the mean ± SEM. *P < .05, **P < .01, ***P < .001. Statistical analysis was performed using Student's t-test (c, e right).

Fig 4: Compound 1 preferentially targets CD133+ CSCs. (A,B) Representative flow cytometry plots of the percentage of early apoptosis (Annexin-V+), late apoptosis (Annexin-V+/DAPI+) and dead (DAPI+) cells in the total PDAC2 cell population (top) or within the CD133-positive population (bottom) treated with Cpd. 1 at 2.5 µM. Cells were analyzed (A) 24 h and (B) 48 h post treatment initiation. (C) Fold-change in total apoptosis (early and late apoptosis) for each group and treatment shown in panels A and B, (Student’s t-test). * p < 0.05; *** p <0.001; ns, not significant.

Fig 5: Expression of ALDH1, CD133 and CD44 prior to and following induction of differentiation in SP cells. SP or non-SP cells were incubated with anti-ALDH1, anti-CD133 and anti-CD44 antibodies together, and subjected to flow cytometry analysis. (A) Percentage of ALDH1+, CD133+ or CD44+ cells in non-SP and SP cells prior to (day 0) and following differentiation (day 14). During flow cytometry analysis, Left, cells were analyzed for the expression of ALDH1 and CD133; Middle, cells were analyzed for the expression of ALDH1 and CD44; Right, cells were analyzed for the expression of CD133 and CD44. All these were conducted in the same population of SP or non-SP cells. (B) Percentage of only ALDH1+ cells in the same population of non-SP and SP cells in A prior to (day 0) and following differentiation (day 14). (C) Percentage of ALDH1+ cells in non-SP and SP cells prior to (day 0) and following differentiation (day 14) after three independent experiments. **P<0.01. ALDH1, aldehyde dehydrogenase-1; CD44, CD44 antigen; CD133, CD133 antigen; PE, phycoerythrin; APC, allophycocyanin; SSC, side scatter; SP, side population.