Fig 2: Migration and invasion assay of CD133-/A2B5+ GICs and matched non-initiating tumor cells in vitro. Cells passing the matrigel or polycarbonate filters were determined. The number of (A) invading and (C) migrating GICs populations was significantly increased compared with non-initiating tumor cells populations (×100); scale bar=50 µm. Quantified data of (B) invading and (D) migrating GICs compared with non-initiating tumor cells demonstrated a statistical increase. Both migration and invasion of GICs-RFP or non-initiating tumor cells-RFP, which were transfected with RFP, were not significantly different from the corresponding GICs or non-initiating tumor cells. RFP, red fluorescence protein; GICs, glioma-initiating cells.

Fig 3: Multiple FVIII (red), CD133 (blue) and pStat3 (green) confocal immunofluoresce reactionPCNSL tumor vessels lined by FVIII positive endothelial cells (A, B, C red signal) and expressing both pStat3 ( A, B, D green signal) and CD133 ( A, C, D blue signal). An orange signal of FVIII and pStat3 colocalization (A, B orange arrow) is present. Note a CD133 blue cells (A, C, D, arrowhead) expressing FVIII red (A, C arrowhead) and pStat3 (D arrowhead) green fluorescence in the vessel and a CD133 blue cells (C, arrow) expressing pStat3 (A, B, D arrow) near the vessel. Scale bar: A–D 8 µm.

Fig 4: Long-term adaptation to PA phenocopy obesity-induced stem-cell features.a Distributions of relative barcode frequency for three tumors in each group. b Apoptotic rate of parental and adapted HCC1806 cells which were treated with 400 µM PA and vehicle (Ctrl) for 48 h. Both early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptotic cells were included for the apoptotic rate % calculation. Data are represented as mean ± SEM of 3 replicates. c Time-dependent proliferation assay of parental and adapted HCC1806 following 48 h. Cells were exposed to 400 µM PA and vehicle (Ctrl). Cell growth was determined by high content imaging and represented as % confluence normalized to t = 0. For each time point, data are represented as mean ± SEM of 6 replicates. d Representative contour plots of mass cytometry data colored by density of cells showing the changes between parental and adapted HCC1806 cells. Color code represents the cell density from low (blue) to high (red). e Representative tSNE plots of single parental and adapted HCC1806 cells colored by expression of CD133, Axl, and CD44. f Tumor incidence following orthotopic implantation of the indicated number of parental and adapted E0771 cells into chow and HFD-fed mice. The frequency of cancer stem-like cells was calculated by the extreme limiting dilution analysis. The default Chisquare test in ELDA was performed to evaluate the differences between parental and adapted cells (NS, P value >0.05). g The distribution of genes induced by obesity (obese and overweight compared to non-obese patients) in PM hormone negative breast cancers patients among the gene expression changes observed in PA-adapted cell lines. We have included the mutual information value (MI) and its associated z-score reported by iPAGE. For visualization, the enrichment/depletion of the query gene-set was determined using the hyper-geometric test and the resulting p-value was used to define an enrichment score that is shown as a heatmap across the expression bins. The obesity-induced genes were significantly enriched in the top-most bin. The red and blue borders in the heatmap denoted statistical significance for enrichment and depletion respectively. Source data are provided as a Source data file.

Fig 5: Pericentrosomal CD133 captures GABARAP at the centrosome, thereby inhibiting GABARAP-mediated ULK1 activation. (A) Endogenous CD133 interacts with GABARAP in SK-N-DZ cells. Five hundred micrograms of the cell lysate from SK-N-DZ cells was immunoprecipitated with an anti-GABARAP antibody. As a control, immunoprecipitates with preimmune mouse immunoglobulin-G (IgG) were used. Immunoprecipitates were then subjected to immunoblotting using an anti-CD133 antibody. (B) GABARAP interacts with CD133 (WT), but not CD133 (EE). Five hundred micrograms of the cell lysate from HEK293T cells transfected with the indicated vectors was immunoprecipitated with an anti-Flag antibody. Immunoprecipitates were then subjected to immunoblotting using an anti-CD133 antibody. The 5% input was also subjected to immunoblotting using anti-CD133 and anti-Flag antibodies. (C) The interaction between GABARAP and ULK1 was inhibited by CD133. Five hundred micrograms of the cell lysate from HEK293T cells transfected with the indicated expression vectors was immunoprecipitated with an anti-Flag antibody (left). Immunoprecipitates were then subjected to immunoblotting using an anti-Myc antibody (left). The 5% input was also subjected to immunoblotting using anti-CD133, anti-Myc, and anti-Flag antibodies (right). (D) CD133 inhibits the ULK1-mediated phosphorylation of ATG13. Immunoblot of CD133, Myc-ULK1, ULK1S555-p, HA-ATG13, and ATG13-S318-p in RPE1 cells transfected with the expression vector indicated above each lane. An immunoblot of ?-tubulin served as a loading control. (E) The knockdown of CD133 enhances the ULK1-mediated phosphorylation of ATG13 in SK-N-DZ cells. Immunoblot of CD133, ATG13, and ATG13-S318-p in SK-N-DZ cells transfected with CD133 siRNA or its control. An immunoblot of ?-tubulin served as a loading control.