Increased colony formation in soft agar is mediated by factors secreted by G2B-10A cells. (A) Paraformaldehyde (PFA) fixation abrogates the ability of G2B-10A cells to stimulate R2-T1AS colony formation. As previously, 1 × 104 (denoted as 1) R2-T1AS cells, were plated in soft agar cultures either alone (red bar), or with 8 × 104 (denoted 8) live G2B-10A cells (G2B-10A, red stippled bar), or mixed with 8 × 104 (denoted as 8) PFA-fixed G2B-10A cells (G2B-10A(PFA), grey stippled bar). Data show mean numbers derived from 4 independent experiments (± standard error), with each experiment performed in 4 replicate wells. (B) Medium conditioned by G2B-10A cells enhances colony formation. To produce conditioned media, G2B-10A cells, or G2B-10A(PFA) cells, or no cells (mock) were plated in low-glucose, 5%-serum medium, on tissue culture plates. Conditioned media harvested after a 3-day incubation were used to set up the soft agar cultures. In all soft agar cultures, 1 × 104 R2-T1AS cells were plated alone (denoted as 1 in the graph labels) in either mock medium (red bar), G2B-10A - conditioned medium (G2B-10A CM, green bar), or in G2B-10A(PFA) - conditioned medium (G2B-10A(PFA) CM, grey bar). Data show mean of 3 independent assays (± standard error), with each assay performed in 4 replicate wells. **p < 0.001, ns - not significant.