Fig 3. Effects of PITWT, PIT271 and/or shRNA for PIT-1 on the proliferation and survival of GH4C1 cells.

A. Expression of mRNA's for PIT-1 or PIT271 (upper panel), as well as for PRL (lower panel), in the different cell lines in the absence or presence of 1 μg/ml doxycycline for 5 days. The cells used were from the same experiment as those grown on parallel plates to measure proliferation rate (Fig. 3B). 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 can thus be compared. Values correspond to mean ± SEM (n = 3). The significance of the effect of docycycline-induction on the expression of endogenous PIT-1, of the transgene or PRL was evaluated by the Student's t-test. +: p<0,05; ++: p<0,01; +++: p<0,001. B. Effect of the expression of PITWT, PIT271 and/or shRNA for PIT-1 on the proliferation of GH4C1 or GH4C1-sh1 cells as well as on CV1 cells. Cells were counted on triplicate wells. Induction of the expression of the shRNA (GH4C1-sh1 cells) and of the transcription factors was started on day 0 and continued throughout the experiment. 'Ctrl' represent GH4C1, GH4C1-sh1 or CV1 cells not infected with pInducer-20 vector expressing a transcription factor. Counting of CV1 cells was discontinued after 5 days, as cells became too confluent. Values correspond to mean ± SEM (n = 3). C. Effect of the expression of PITWT, PIT271 and/or shRNA for PIT-1 on the proliferation of GH4C1 or GH4C1-sh1 cells. Data represent the pooled results of the four experiments (with triplicate wells in each) of which one is illustrated on Fig. 3B, and correspond to the cell numbers found in the absence or the presence of doxycycline five day (D5) after the start of the induction. Mean ±SEM, +: p<0,05, ++: p<0,01, relative to the same population without doxycycline, *: p<0,05. D. Effect of the expression of PITWT, PIT271 and/or shRNA for PIT-1 on DNA fragmentation as evaluated by Tunel assay following 4 days exposure to doxycycline. Labeled and unlabeled cells were counted from 6 micrographs taken at random from duplicate wells and the experiment was repeated twice. Mean ±SEM, +: p<0,05, relative to the same population without doxycycline. E. Effect of the expression of PITWT, PIT271 and/or shRNA for PIT-1 on the division of GH4C1 or GH4C1-sh1 cells using EDU as cell division marker. EDU was present for 15–17h between day 3 and 4 of the induction by doxycycline. Labeled and unlabeled cells were counted from 6 micrographs taken at random from duplicate wells and experiments were repeated three times. Numbers given within the columns correspond to the population doubling time in hours as calculated from the labeling percentages. Mean ±SEM, +: p<0,05, ++: p<0,01, relative to the same population without doxycycline, **: p<0,01.