Fig. 8

Modulations of the RIP activity in both SIN γ- and α-retrovectors tightly correlate with those affecting endogenous INSULINgene expression. a EndoC-βΗ2 cells or their derivatives stably expressing fluorescent reporters were transfected with the indicated siRNA. 6 days later, cells were harvested and changes in INSULIN and IAPP mRNA levels were determined through qRT-PCR analyses, and represented as fold change over control siRNA only (siRNA NT, Non Targeting) transfected cells. Each co-transfection (either siRNA NT + siRNA SV40T or siRNA RB + siRNA P130) was done with an equimolar mix (final concentration of 40 nM for each siRNA), while single transfection (either siRNA NT, siRNA SV40T only) was done at a final concentration of 80 nM. The results represent the mean and standard deviation (SD) of at least 3 experiments for each mRNA level analyzed, each point made in duplicate b EndoC-βΗ2 cells or their derivatives expressing the TVA receptor were stably transduced with the indicated γ − or α − retrovector and then transiently transfected with the indicated siRNA at a final concentration of 80 nM (80 nM for the sIRNA in single transfections, 40 nM for each siRNA in co-transfections). GFP fluorescence level was analyzed by flow cytometry 6 days after transfection. Results were expressed as fold variation in MFI (mean of fluorescence intensity) with the value in cells transfected with the siRNA arbitrarily taken as 1. The histograms show the means and SD of three (upper panels) of four (bottom panels) experiments