Figure 2

AN01 and AN03 enrich for self-renewing CRC CICs, AN01 is a GBM and muscle stem cell marker. (A) Schematic of FACS approach. POP92 spheres were dissociated, stained with indicated antibody and viability dye, and top and bottom 10% of expressing cells were sorted into sphere formation assays (SFAs). Primary sphere formation was assessed by sphere counting. Primary spheres were dissociated, cells were counted and equal numbers were seeded into secondary SFAs. (B) Bar charts showing the mean sphere forming efficiency (SFE) as % of cells giving rise to a sphere, for FACS sorted high and low staining AN01 (integrin α7) and (C) AN02 (HLA-A1) and (D) AN03 (integrin β6), with individual biological replicates indicated as unfilled circles. The primary SFA is shown to the left of the dotted vertical line, and the secondary SFA is shown to the right. Error bars represent SEM, and significance was calculated by paired, two tailed t-test. (E) Human muscle stem cells are readily detected by FACS. Biopsy sample is initially gated for live (7−AAD^negative left), CD45, CD31, Cd11b, Glycophorin A, CD31, and CD34 all negative (top). True human muscle stem cells are detected by selecting CD56 and integrin α7 double positive cells with both AN01 (right) and the commercial antibody (left). (F) Histograms of cells stained with AN01 and commercial antibody reveal the same percentage of integrin α7 positive cells after gating for live and lineage negative cells. (G,H) Box and whisker plots showing relative sphere formation for (G) human, patient-derived GBM cell lines BT799 and (H) GBM87, FACS sorted into four quadrants based on AN01 (integrin α7) and CD133 expression. Sorted populations were: α7+/CD133^high, α7+/CD133^low, α7−/CD133^high and α7−/CD133^low. Sphere formation is represented for each sorted quadrant as % sphere formation relative to the mean α7−/AC133^low (double negative) control. Individual replicates are illustrated as filled circles, the box represents standard deviation of the mean. Results shown are from one representative experiment. N = 2 biological replicates. (I) Presto blue measure of cell viability from BT799 SFA (see (G)) and (J) GBM87 SFA (see(H)). Error bars represent standard deviation from 6 replicates from a single representative experiment. (N = 2). *p < 0.05, **p < 0.01, ***p < 0.001.