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Fig. 2 | Cell Communication and Signaling

Fig. 2

From: Cell death induced by the ER stressor thapsigargin involves death receptor 5, a non-autophagic function of MAP1LC3B, and distinct contributions from unfolded protein response components

Fig. 2

Tg-induced cell death partially requires LC3B. a LNCaP cells were transfected for 2 d with the indicated siRNAs (siCtrl = non-targeting control siRNA). Subsequently, cells were treated with 100 nM Tg or 0.01% DMSO vehicle control (“DMSO”, also transfected with siCtrl) in the additional presence of 2.5 μg/ml propidium iodide to stain dead cells. Cell death was monitored and quantified with the IncuCyte ZOOM as described in Materials and Methods, and displayed as relative values normalized to those obtained in the siCtrl+Tg condition after 48 h of treatment (mean value set to 1). Mean ± SEM from 6 independent experiments. b LNCaP cells were transfected and treated as in (a). After 30 h of treatment with Tg or DMSO (also transfected with siCtrl), whole cell lysates were prepared and subjected to western blotting with the indicated antibodies; cl-PARP = cleaved PARP. The positions of molecular weight markers are indicated to the left. One representative blot out of 4 independent experiments. c Quantification of cleaved PARP protein levels, normalized to the tubulin loading control and then to the siCtrl+Tg condition (mean value set to 1). Mean ± SEM from 4 independent experiments. d HCT116 cells were transfected and treated as in (a), followed by quantification of cell death by propidium iodide staining after 39 h of treatment. Mean ± SEM from 5 independent experiments. e HCT116 cells were transfected as in (d). After 30 h of treatment with Tg or DMSO (also transfected with siCtrl), whole cell lysates were prepared and subjected to western blotting with the indicated antibodies; p55/53 casp8 = procaspase-8, p43/41 casp8 = cleaved caspase-8, cl-PARP = cleaved PARP. The asterisk indicates a nonspecific band. One representative blot out of 3 independent experiments. f Quantification of cleaved caspase-8 bands (upper panel) and cleaved PARP levels (lower panel), normalized to the tubulin loading control and then to the siCtrl+Tg condition (mean value set to 1). Mean ± SEM from 3 independent experiments. For (a, c, d, and f): Dots represent individual values, with a separate color for each experiment. *P < 0.05, ***P < 0.001, ns; not significant, One-way ANOVA compared to the Tg + siCtrl condition

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Cell Communication and Signaling

ISSN: 1478-811X