Fig. 1

Ru1 negatively affects PaCSCs molecular and functional properties. A Relative toxicity (LU = light units) ± SD in Panc185 and PancA6L adherent (non-CSCs) and sphere (CSCs) cultures treated at the indicated doses of Ru1 for 24, 48 or 72 h. Toxicity was determined using the ToxiLight assay kit at the indicated hours post treatment. B Representative flow cytometric plots of AnnexinV-FITC staining in Panc185 and PancA6L sphere-derived cells treated for 72 h with Ru1 at 100 and 250µM. C Top: Representative images of Panc185 spheres in the absence (untreated) or presence of Ru1. Cells were treated with Ru1 for 24 h at 100µM prior to establishing spheres. Bottom: Mean fold change ± SD in the number (no.) of spheres formed compared to control (set as 1.0). **** p < 0.0001, as determined by unpaired two-sided Student’s t-test. D Top: Representative images of Panc185 colonies in the absence (untreated) or presence of Ru1. Cells were treated with 24 h with the Ru1 at 100µM during day 1 post seeding. Bottom: Mean fold change ± SD in the colony efficiency in untreated and Ru1-treated samples, compared to control (set as 1.0). **** p < 0.0001, as determined by unpaired two-sided Student’s t-test. E Mean percentage of Autofluorescent+ or CD133+ cells ± SD, determined by flow cytometry, in untreated and Ru1-treated Panc185 cells (* p <0.05, ** p < 0.01, **** p < 0.0001, ns = not significant, as determined by unpaired Student’s t test). F Sum total of tumors obtained from untreated or Ru1-treated Panc185 and PancA6L injections, from two independent Limiting Dilution Analysis (LDA) assays. Cells were treated with Ru1 for 24h at 100µM prior to injection. CSC frequencies were calculated using the ELDA software. G Average tumor weights ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by unpaired two-sided Student’s t-test; nd= not determined. H Representative flow cytometric plots of AnnexinV-FITC/DAPI staining in Panc185 and PancA6L cells, treated for 24 h with Ru1 at 100 µM, and subsequently injected in vivo. I Top: Representative images of zebrafish embryo tails at 6 days post injection of 100-150 untreated or Ru1-treated H2b-mCherry-labelled Panc185 cells. Scale bar = 250 μM. Bottom: Mean ± SEM of proliferation ratio observed between untreated (Unt) or Ru1-treated determined on day 6 post injection. Cells were treated with Ru1 for 24 h at 100 µM prior to injection. Proliferation ratios are represented in comparison to 1 dpi (day post injection) (red line). * p < 0.05, as determined by unpaired two-sided Student’s t-test. J Mean number (no.) of spheres ± SEM determined at 7 (1^st generation), 14 (2^nd generation), and 21 (3^rd generation) days post seeding, from CD133-negative, CD133-positive, Autofluorescent (Fluo)-negative, or Fluo-positive cells sorted from Panc185 cells and treated at d0 with Ru1 (100µM) for 24h. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by unpaired two-sided Student’s t-test