Fig. 2: GSK3β is necessary for the necrotic response elicited by DMNQ.
From: GSK3β is a key regulator of the ROS-dependent necrotic death induced by the quinone DMNQ
a Cytofluorimetric analysis measuring cell death percentages (PI positivity) in the different U87MG clones treated with the indicated concentrations of DMNQ for 24 h. Data are presented as mean ± SD. n = 3. b Cytofluorimetric analysis measuring cell death percentages (PI positivity). U87MG cells were transfected with siRNAs against GSK3β or control. After 48 h they were treated with 30 µM of DMNQ for further 24 h. Data are presented as mean ± SD. n = 3. c Immunoblot analysis of GSK3β levels in U87MG cells transfected with siRNAs against GSK3β or control. Actin was used as loading control. d Immunoblot analysis of GSK3β levels in U87MG GSK3β+/+ and GSK3β−/− cells retrovirally infected with the GSK3β-GFP fusions WT or its catalytically inactive mutant K85A (KM). Actin was used as loading control. e Cytofluorimetric analysis measuring cell death percentages (PI positivity) in the indicated U87MG cell lines treated with 30 µM of DMNQ for 24 h. Data are presented as mean ± SD. n = 3. f Immunoblot analysis of Caspase-3, Caspase-2, and HDAC4 caspase-dependent processing in the indicated U87MG cell lines treated with 30 µM of DMNQ. Incubation with the combination TRAIL (2.5 ng/ml) and bortezomib (0.1 µM) for 20 h was used to trigger apoptosis. Actin was used as loading control. g Cytofluorimetric analysis measuring cell death percentages (PI positivity) in the indicated U87MG cell lines treated or not with the combination TRAIL (2.5 ng/ml) and bortezomib (0.1 µM) for 24 h. Data are presented as mean ± SD. n = 3. h Microscopic images of the indicated U87MG cell lines treated for 24 h with DMNQ (30 µM). Arrows point to membrane blistering and arrowheads to examples of necrotic cells. Phase contrast images were obtained with Leica DMi1 microscope with a 10x objective. i qRT-PCR analysis of GSK3β mRNA levels in the silenced IMR90-E1A//BCL2/C9DN cells. Data are from three experiments; +SD. j Cytofluorimetric analysis measuring cell death percentages (PI positivity). IMR90-E1A/BCL2/C9DN cells were transfected with siRNAs against GSK3β or control and after 48 h treated with 100 µM of DMNQ for further 24 h. Data are presented as mean ± SD. n = 3.
