Fig 2: WNT inhibition during pellet culture enhanced homogeneity of hiPSC chondrogenesis.A Experimental scheme of WNT inhibition. B C59 treatment during pellet culture enhanced Saf-O staining and decreased off-target cells (yellow arrowheads) as compared to other WNT inhibition culture regiments. Top row scale bar = 400 µm. Bottom row scale bar = 200 µm. The experiment was performed twice with similar results. C Pellets treated with C59 in only pellet culture exhibited an increased GAG/DNA ratio compared to pellets treated with other culture regiments. *p = 0.00001 at d28. #p = 0.0228 at d42. Mean ± SEM. n = 4 pellets per group. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test at a specific time point. D C59 significantly decreased, but WNT3A significantly increased, CD146+/CD166+/CD45- progenitors at the Cp stage. Different letters are significantly different (a vs. b, p = 0.0005; a vs. c, p = 0.0021; b vs. c, p = 0.0001). Mean ± SEM. n = 3 per group (independent experiment). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. E RNA-FISH of d28 pellets showing C59-treated pellets had decreased WNT3A and WNT4 labeling (green) but more homogenous COL2A1 distribution (red) in the pellets. Scale bar = 200 µm. The experiment was performed twice with similar results.

Fig 3: SLC25A1 expression correlates with metastatic disease and promotes stemness in NSCLC. a Representative SLC25A1 immunohistochemical staining of tissue microarrays of lung adenocarcinomas. b Representative images of an immunoreactive tumor, normal adjacent tissue (NAT) and metastatic lymph node. c Quantitative analysis of SLC25A1 staining based on percentage of reactive cells. d The cell cycle profile of H1299 cells grown as monolayers or spheres is shown. e Quantitative real-time PCR (normalized values) on the indicated gene products performed in either monolayer (red bars) or spheres of H1299 cells and analyzed in triplicate samples. f Real-time PCR of the indicated genes performed in naive H1299 or SLC25A1-expressing cells. g–i FACS analysis of CD166, CD133 expression and of the CD44 high, CD24 low population in naive H1299 and SLC25A1-expressing cells. j Quantification of the indicated markers from three independent experiments each with duplicate values. k To study self-renewal, cells from SLC25A1-spheres were sorted for CD44/CD24 high or low populations and re-plated independently as single cells.

Fig 4: a Growth curves of H1299 or SLC25A1-expressing cells cultured as spheres, dissociated and counted at each of the indicated time points. The arrows indicate the loss of viability that occurs in the first days of sphere formation, likely reflective of differentiated cells dying by anoikis. b Control or SLC25A1-expressing cells were dissociated and plated as single spheres in semisolid media, methylcellulose. After 1 week, spheres were isolated, dissociated again and re-plated in the same conditions, producing second-generation spheres. c Quantification of the experiments shown in b. d, e FACS analysis of CD133 and CD166 in the indicated cell lines either mock transfected (gray bars) or transfected with the vector expressing SLC25A1 (black bars). f Growth curves of H1299 cells infected with lentivirus control shRNA, or with either of two SLC25A1 shRNAs (825 and 350). g, h Sphere-forming ability of H1299 cells infected with lentivirus control or with the indicated SLC25A1-shRNAs, dissociated and plated as single cells in semisolid media. i CD166 and CD44 expression in cells infected with PLKO control lentivirus or with lentivirus harboring the specific SLC25A1 shRNA. j, k Sphere-forming ability of cells expressing SLC25A1 wild-type or mutant SLC25A1R282G/R285C j, or treated with the SLC25A1 inhibitors BTA (2 mM) and CTPI (1 mM) k. Bars represent the standard deviation, asterisks refer to *P = 0.05; **P = 0.01; ***P = 0.001; ****P = 0.0001 by unpaired T-test

Fig 5: Proteomic analysis of cellular proteins targeted by US12 family members in PM samples.(A-B) Workflows of proteomic series 1 and 2 respectively. (C-E) Quantitation of PM proteins in enriched KEGG pathways identified using DAVID. Relative abundance of each protein is expressed relative to the sample with the highest abundance (set to 1). NQ – not quantified. A number of the US12-21 mutant targets were also regulated by the US18 and US20 mutants (B7-H6, ULBP2, IL6ST, KIT, KITLG JAM3, ACVR1, ACVRL1, IFNGR1, JAM3, MPZL1, CXADR, ALCAM, SDC4, CD99, SDC1). A subset of these proteins were also regulated by the US14 and/or US16 mutants (KIT, KITLG, ACVR1, IFNGR1, JAM3, MPZL1, CD99, SDC1). We estimated p values for the ratios of each mutant compared to HCMV Merlin using Benjamini-Hochberg corrected Significance B values (Cox and Mann, 2008): *p<0.05, **p<0.0001. For proteomic series 1, ratios were calculated as US12 family deletion mutant / average HCMV and for proteomic series 2, US12 family deletion mutant / HCMV. All proteins quantified by 2 or more peptides were included in this calculation. SDC1 was quantified by a single peptide in proteomic series 2.DOI: http://dx.doi.org/10.7554/eLife.22206.007