This observation recommended that overexpression of FHL1C induced cell development arrest and or cell death in Jurkat cells. We to start with examined the cell cycle progression of Jurkat cells transfected with pEGFP or pEGFP FHL1C. The outcomes showed no exceptional variation within the cell cycle distribution between the two groups, even though the num ber of cells overexpressing FHL1C exhibited a slight enhance in G2 M phase. We upcoming determined cell viability after transfection. We discovered the percentage of viable cells decreased continu ously amid Jurkat cells after transfection with pEGFP FHL1C, suggesting that overexpression of FHL1C may lead to cell death. Next, we straight estimated apoptosis right after overexpres sion of FHL1C. Jurkat cells were transfected as described over, and apoptosis was determined by flow cytometric analysis with annexin V and PI staining.

In the GFP cell population, there was a substantial raise of annexin V cells between the pEGFP FHL1C transfected Jurkat cells compared with that amid the pEGFP transfected Jurkat cells, suggesting that overexpression of FHL1C induced apoptosis in Jurkat moreover cells. Annexin V and PI staining distin guishes early apoptotic and late apop totic cells. As Figure 3C and D had been proven, overexpression of FHL1C resulted in an in crease of both early and late apoptotic cells among Jurkat cells. We also examined the morphology of Jurkat cells transfected with pEGFP or pEGFP FHL1C by Hoechst staining and TEM. The outcomes confirmed that there were additional apoptotic cells with condensed nuclei amid Jurkat cells overexpress ing FHL1C.

At the molecular degree, overexpression of FHL1C in Jurkat cells diminished the expression of anti apoptosis molecules, which includes Bcl two and Bcl x1, and greater expression from the apoptosis related molecule caspase three. These results strongly recommend that overexpression of FHL1C induces apoptosis of T ALL cells. FHL1C induces apoptosis of Jurkat likewise cells by way of suppression of RBP J mediated transactivation Similar to its murine homolog KyoT2, FHL1C also possesses a C terminal RBPmotif, suggesting that FHL1C interacts with RBP J and suppresses RBP J mediated transactivation. To confirm an interaction amongst FHL1C and RBP J, we performed co immunoprecipitation. HeLa cells had been co transfected with expression vectors for Myc tagged RBP J and EGFP tagged FHL1C, and immunoprecipitation was per formed with an anti Myc antibody.

Co precipitated proteins were detected employing an anti FHL1 antibody by western blotting examination. The results showed that GFP FHL1C was very well co precipitated with RBP J, suggesting that FHL1C interacts with RBP J. Furthermore, we carried out reporter assays utilizing HeLa and Cos7 cells by transfection with pEGFP FHL1C in addition to a NIC expression vector. As being a result, more than expression of FHL1C suppressed transactivation in the reporter harboring RBP J binding web sites by NIC within a dose dependent method. This result demonstrated that FHL1C suppresses RBP J mediated transactivation by competing with NIC. We subsequent determined no matter whether FHL1C induced apop tosis of Jurkat cells as a result of suppression of RBP J mediated transactivation by overexpressing RBP J VP16, a constitutively activated RBP J.

Jurkat cells have been transfected with pEGFP FHL1C alone or co transfected with pEGFP FHL1C and pCMX VP16 RBP J, followed by evaluation of apoptosis. The outcomes showed that Jurkat cells did not undergo apoptosis following transfection with pCMX VP16 RBP J alone, and overexpression of FHL1C alone induced apoptosis, which was constant together with the benefits shown above. Co transfection of cells with vec tors carrying FHL1C and RBP J VP16 resulted in effi cient attenuation of the FHL1C induced apoptosis. This effect was proportional to the volume of RBP J VP16.