Both live and PFA-fixed benign G2B-10A cells stimulate R2-T1AS cells to secrete soluble factors. (A-D) Cytokine antibody array analyses of conditioned media derived from: (A) R2-T1AS/G2B-10A co-culture (1.875 × 105 R2-T1AS cells/ml mixed with 1.5 × 106 G2B-10A cells/ml), (B) R2-T1AS/G2B-10A(PFA) co-culture (1.875 × 105 R2-T1AS cells/ml mixed with 1.5 × 106 PFA-fixed G2B-10A cells/ml), (C) R2-T1AS alone (1.875 × 105 R2-T1AS cells/ml). The R2-T1AS cells were plated at 8 times lower density than G2B-10A cells to reflect the 1:8 ratio at which cancer and benign cells were co-cultured in the soft agar assays. (D) G2B-10A alone (1.5 × 106 cells/ml). To produce conditioned media, cells were cultured in suspension, in low-glucose, 5% serum conditions and conditioned media were harvested after 3 days of incubation. All antibody array membranes were incubated with 1 ml of conditioned media from the indicated cultures. All membranes underwent the same treatment and were exposed to the X-ray film for the same time. Membranes shown were generated in a different experiment than the membranes in Figure 6. POS - positive control spots, black solid lines - differentially expressed cytokines, red lines - signals specific to co-cultures, dashed black lines - not reproducible signals. (E) Quantification of the arrays in Figures 9A, 9B, 9C, and 9D. The signals on the R2-T1AS array (red bars), G2B-10A array (green bars), R2-T1AS/G2B-10A array (blue bars) and R2-T1AS/G2B-10A(PFA) array (gray bars) and were quantified by densitometry and normalized to the average of positive control signals (POS). (F) ELISA analysis of IL-6 concentration in the conditioned media from the following cultures: R2-T1AS (red bars), G2B-10A (green bars), R2-T1AS/G2B-10A (blue bars) and R2-T1AS/G2B-10A(PFA) (gray bars).