Fig 1: Kynurenine and IGF-I pathways were activated in CEFs. (A) Simplified schematic of the kynurenine pathway. (B) Cell lysates of NF-54, CAF-54, and CEF-54 were subjected for immunoblot with indicated antibodies. (C) Western blots in NF-54 cell lysates after treatment with CAF-CM for indicated period. Right: The bands indicate the protein(s) incorporated KYN. (D) The amount of KYN in each CM was examined by ELISA. *P < 0.05 (n = 4. t-test). The data in bar charts are presented as the mean ± SD. (E) NF-54 cells were incubated with recombinant ASPN (20 ng·mL-1). Cell lysates were prepared and subjected to western blot. The band intensities were quantified and normalized to a-tubulin expression, then, expressed as the relative ratio to the control untreated cells in each panel. (F) CD8+ T cells were incubated with NF-54-CM or CEF-54-CM for 2 days and subjected to FACS analysis. Dot plots of 7-AAD and CD8 (left), and histogram of 7-AAD (right). In dot plots, the numbers indicate the percentage of the cells in each fraction. (G) Biotin-labeled NF-54 cells were cocultured with CAF-54 cells for 3 days. Biotin-labeled NFs were isolated by streptavidin-coupled microbeads and cocultured with CD8+ T cells for 2 days. CD8+ T cells were collected, and dead cells (arrow) were detected by 7-AAD staining. (H–M) ASPN was stably overexpressed in NF-50 (H, I) or HSC-43 cells (J–M). (H, J) Western blot analysis of each molecules in cell lysates of parent (pt) cells or ASPN-expressing cells. The value below the KMO panel indicates the ratio of the band intensity to the control after normalized to a-tubulin expression. (K) The amount of KYN in each CM was examined by ELISA. **P < 0.01 (n = 4. t-test). The data in bar charts are presented as the mean ± SD. (I, L, M) CD8+ T cells were incubated with each CM for 2 days and subjected for FACS analysis without fixation. Representative results of three independent assays are shown. Dead cells were evaluated by PI, 7-AAD, or Annexin-V staining, and indicated by arrows in the histogram. (M) +NLG-919 (pre); CM was prepared from HSC-43 ASPN cells pretreated with an IDO-1 inhibitor (5 µm). As a control, NLG-919 was directly added to CD8+ T cells together with HSC-43 ASPN-CM (+NLG-919, post). Boxed area indicates dead CD8+ T cells. (N) PAPP-A-mediated liberation of IGF-I. (O) Western blot of COS-1 cells transfected with IGFBP-4-HA. (P) In vitro cleavage of IGFBP-4-HA by cell lysates of NFs or CEFs was examined. Cleaved and uncleaved IGFBP-4-HA (arrow and arrowhead, respectively) were detected by immunoblot with anti-HA antibody. Longer exposure of cleaved IGFBP-4-HA is shown at the bottom. (Q) Activation of IGF-IR signaling in cancer cells by IGF-I/IGFBP-4 plus CEF-54-CM was examined as described in Section 2.8. (R) Immunoblot of PAPP-A in CEFs and cancer cells. (S) Activation of IGF-IR in OCUM-12 or Li-7 cells was detected by anti-phospho-IGF-IRß antibody. CEF(-) indicates that the IGF-I/IGFBP-4 mixture was directly added to cancer cells. Right panels (+CEF-CM): Activation of IGF-IR was not induced by CEF-CM alone.