Effect of <i>Hras</i>^<i>G12V</i> on function of cultured SSCs.

A. Derivation of cell lines and in vitro induction of HrasG12V. Two different adult cell line pairs (WT & FR) each from the different reporter systems were established. Dissociated testicular cells were plated and colonies were picked, enriched, and serially passaged on feeders. B. Hras transcript levels (RT-qPCR) in different cell lines. Primers were designed to recognize a common region shared by WT and HrasG12V. N = 3 experiments. C. Western blot for HRAS in SSCs from iWT and iFR. Activated HRAS proteins were pulled down with RAF1 RAS-binding domain and probed by an anti-HRAS antibody (left). In iFR, activated HRAS was observed (black arrow). Open triangle denotes non-specific band. Total lysates (right) from iWT, iFR, and control Hras were probed concurrently. Lysate from Hras-overexpressing 293T cells served as a positive control. D. Growth ratio of different SSC cell lines. Shown are average weekly growth ratios (4 passages over a month). In all cases, the growth ratios of induced lines were not different from their parental cell lines. E. In vitro competition assay. FR2 (tdTomato+) and iFR2 (GFP+) cells were mixed at 1:1 ratio, allocated to low (0.1 ng/ml) and high (10 ng/ml) FGF2 media, and passaged 5 times. At each passage, GFP+ cell ratios were measured by FACS. F. Phosphorylation of ERK after FGF2 stimulation. Top: Representative western blot (top) from iWT and iFR for pERK, ERK, and loading control, cyclophilin B. Bottom: Quantification based on average densitometric values of western blots from three different sets of clones (bottom). At 0’, iFR exhibited more activation of pErk than iWT (t-test; P = 0.08; n = 3), whereas no significant difference was seen at 5’, 15’ and 20’ between iWT and iFR.