Journal: International Journal of Molecular Sciences

Article Title: E2f2 Attenuates Apoptosis of Activated T Lymphocytes and Protects from Immune-Mediated Injury through Repression of Fas and FasL

doi: 10.3390/ijms23010311

Figure Lengend Snippet: Apoptosis in TCR-stimulated E2f2 −/− lymphocytes is linked to upregulation of Fas and FasL and activation of the pathway: ( A ) Reverse transcription qPCR analysis of Fas and FasL expression in freshly purified WT and E2f2 −/− T cells unstimulated or after stimulation with anti-CD3 during the indicated time. Eef1a1 was used as normalization control. Results are expressed as fold-over WT at 0 h (mean ± SD) from 3 to 5 independent experiments. *** p < 0.0001; * p < 0.05. ( B ) Representative Western blot analysis of Fas and Fas L in extracts prepared from freshly purified WT and E2F2 −/− T cells unstimulated or after stimulation with anti-CD3 for the indicated time. Expression of Hsp90α/β was used as loading control. Numbers below the bands correspond to the relative densitometric values, expressed as fold-over WT at 0 h. Similar results were obtained in at least 4 independent experiments. ( C ) Representative results of Annexin V-FITC and PI staining followed by FACS analysis performed with WT and E2f2 −/− T cells after stimulation with anti-CD3 for 48 h and incubation with vehicle or anti-FasL antagonistic antibody for the last 24 h. ( D ) Results of early apoptosis observed under the indicated conditions, expressed as the percentage of cells (mean ± SD) from 4 independent experiments. *** p < 0.0001, ** p < 0.01. ( E ) Representative Western blot analysis of Cyclin A2 (CycA2) and Cleaved Caspase 3 (Casp3) in extracts prepared from WT and E2f2 −/− T cells 48 h after stimulation with anti-CD3 and incubation with anti-FasL antagonistic antibody or vehicle (vehic) for the last 24 h. Numbers below the bands correspond to the relative densitometric values, expressed as fold-over WT vehicle.

Article Snippet: Western blots were performed with 20 μg of total protein extract, using antibodies against Cleaved Caspase 3 (9664S), phospho-Chk1 (Ser345, 133D3) (Cell Signaling, Danvers, MA, USA), phospho-Rpa2 (Ser4/Ser8, A300-245A-M, Thermo Fisher Scientific), phospho-histone H2AX (Ser139, 07-164, Millipore, Billerica, MA, USA), β-actin (A5441) (Sigma), E2F1 (sc-251), Parp-1 (sc-53643), Fas (sc-1023), FasL (sc-6237), Mcm2 (sc-9839), Cyclin A2 (sc-596), Chk1 (sc-7898), p53 (sc-126), and Hsp 90α/β (sc-13119) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA).

Techniques: Activation Assay, Reverse Transcription, Expressing, Purification, Control, Western Blot, Staining, Incubation