Fig 2. Development of GH4C1 cells clones with inducible expression of shRNA and/or PITWT and PIT271.

A. Pit-1 mRNA levels in GH4C1 cells 48 hours after transfection of siRNA's (Ambion) specific for rat Pit-1. Results are expressed relative to levels found in Control ('Ctrl') cells corresponding to non-transfected cells and correspond to the mean ± SEM of two experiments. 'Mock' corresponds to GH4C1 cells transfected with an unrelated non-specific control siRNA. B. Time-course of PIT-1 mRNA levels following induction of shRNA expression in GH4C1 cells (polyclonal population) infected with shRNA expressing conditional lentiviral vectors. Results are the mean ± SEM of two experiments and are expressed relative to levels found in Control ('Ctrl') cells corresponding to GH4C1 cells infected with the sh1 expressing vector and not exposed to the inducer doxycycline. Induction was achieved by exposure to 1μg/ml doxycycline for 1 to 7 days. C. PIT-1 mRNA levels in individual clones of GH4C1 cells harboring the shRNA expressing conditional lentiviral vectors, 6 days after induction of shRNA expression by 1 μg/ml doxycycline. Results are expressed relative to values found for GH4C1 control cells. D. Western blots of control GH4C1 cells and GH4C1/sh1 A2 clone. Induction by 1 μg/ml doxycycline was maintained for 3 or 7 days. Molecular weight ladder (Thermo Page Ruler) on the right. E. Expression of mRNA's for PIT-1 or PIT271 in the different cell lines in the absence or presence of 1 μg/ml doxycycline for 6 days. 'Pit endo' corresponds to wt endogenous PIT-1, ‘Pit SIR’ (si-resistant) to PITWT or PIT271 expressed from the conditional expression vectors. The results for 'Pit endo' and ‘Pit SIR’ have been obtained by normalizing the values obtained in qPCR to those obtained with known quantities of external controls, and are thus comparable. Results are mean ± SEM of three measures. F. Expression, in the same cells as in Fig. 2E, of PRL mRNA's. Results are expressed relative to values found for GH4C1 control cells.