Fig.5.

Sirtinol treatment increased caspase activity in MDC1A cells in a p53-dependent process and also induced nuclear accumulation of the ER stress marker DDIT3 (CHOP). A. Treatment with sirtinol at 5μM for 24 h caused a 3–4X increase in caspase 3/7 (DEVDase) activity in differentiated cultures of 38/03 and 50/04 MDC1A myogenic cells, but had little effect on caspase activity in cultures of 09Ubic and 15Vbic healthy control myogenic cells. ^**P < 0.01 by ANOVA with n as indicated. B. As in panel A, treatment with sirtinol at 5μM for 24 h was accompanied by a 3–4X increase of caspase (DEVDase) activity. This sirtinol-induced caspase activation was prevented when 25μM pifithrin-alpha was added at the same time as sirtinol, indicating that the sirtinol-induced caspase activation was p53 dependent. For healthy controls, 15Vbic cells were used. For MDC1A, 50/04 cells were used. Error bars = SE. ^**P < 0.01 by ANOVA with n = 3. C-E. Differentiated MDC1A 50/04 cultures were treated with sirtinol (5μM for 24 h) or staurosporine (1μM for 4.5 h), i.e., conditions we had previously shown to increase caspase (DEVDase) activity. Under these conditions, DDIT3 (also known as CHOP; red), a marker for ER stress, accumulated in the nuclei (blue) of cells treated with sirtinol but not in the nuclei of untreated or staurosporine-treated cells. Though not shown, CHOP also accumulated in the nuclei of sirtinol-treated healthy control cells 15Vbic. Bar in panel C = 20μm.