The combined loss of direct activator proteins does not protect cells from nitric oxide-induced cell death.

<p>Wild type and <i>Bim^−/−/Puma^−/−</i> MEFs were treated with 0 and 400 µM DETA-NO for 24 and 48 hours and cell death was measured by percent LDH release (A). BID protein expression was assessed in <i>Bim^−/−/Puma^−/−</i> MEFs stably expressing scrambled shRNA or shRNA targeted against <i>Bid</i> (B). <i>Bim^−/−/Puma^−/−</i> MEFs stably expressing scrambled shRNA or shRNA targeted against <i>Bid</i> were treated with 0, 100, 200 and 400 µM DETA-NO at the indicated concentrations for 48 hours and cell death was measured by percent LDH release (C).</p

p38 and ERK are activated by NO, but do not mediate NO-induced cell death.

<p>Phospho-p38 was measured to assess p38 activity in <i>Bax^−/−/Bak^−/−</i> MEFs treated with 400 µM DETA-NO for 8, 16 and 24 hours (A). Percent LDH release was measured in wild type and <i>p38^−/−</i> MEFs treated with 0, 100, 200 and 400 µM DETA-NO for 24 hours (B). Phospho-ERK was measured to assess ERK activity in <i>Bax^−/−/Bak^−/−</i> MEFs treated with 400 µM DETA-NO for 8, 16 and 24 hours (C). Phospho-ERK was measured in <i>Bax^−/−/Bak^−/−</i> MEFs pretreated with 10 µM of the ERK inhibitor UO126 followed by 400 µM DETA-NO (D). Percent LDH release was measured in wild type MEFs pretreated with UO126 (10 µM) followed by 0, 100, 200 (E).</p

MCL-1 is degraded in response to nitric oxide treatment and is dependent on the ASK1/JNK1 pathway.

<p>MCL-1 protein expression was measured in <i>Bax^−/−/Bak^−/−</i> MEFs treated with 400 µM DETA-NO for 0, 8, 16 and 24 hours (A). MCL-1 degradation was measured in <i>Bax^−/−/Bak^−/−</i> MEFs pre-treated with or without 20 µM MG132 followed by 0 or 400 µM DETA-NO for 24 hours (B). MCL-1 protein expression was measured in <i>Jnk1^−/−</i> (C), <i>Ask1^−/−</i> (D) MEFs treated with 400 µM DETA-NO for the indicated time points.</p

BAX and BAK mediate nitric oxide-indeced cell death.

<p><i>Bax^−/−/Bak^−/−</i> MEFs were infected with either Bax, Bak or GFP as a control. BAX and BAK expression was verified by Western analysis (A,B). <i>Bax^−/−/Bak^−/−</i> MEFs expressing GFP, BAK or BAX were treated with 0, 100, 200 and 400 µM DETA-NO for 48 hours and cell death was measured by LDH release (C).</p

NO-induced apoptosis is not due to ROS or peroxynitrite generation.

<p><i>Bax^−/−/Bak^−/−</i> MEFs adapted to glucose or galactose were treated with 400 µM DETA-NO or 10 µM rotenone for 48 hours and cell death was measured by percent LDH release (A). Wild type MEFs were pre-treated with the ROS inhibitor EUK-134 (20 µM) followed by 0, 100, 200 and 400 µM DETA-NO for 24 hours and cell death was measured by LDH release (B). PEITC is known to generate endogenous ROS and was used as a positive control. Wild type MEFs were pre-treated with the peroxynitrite scavengers, uric acid (1 mM) and ebselen (10 µM) followed by 0, 100, 200 and 400 µM DETA-NO for 24 hours and cell death was measured by percent LDH release (C and D). Wild type MEFs were pre-treated with the nitric oxide scavenger PTIO (1 mM) followed by 0, 100, 200 and 400 µM DETA-NO for 24 hours and cell death was measured by percent LDH release (E).</p

<i>Ask1</i> is required for NO-induced cell death.

<p>Phospho-JNK was measured to assess JNK activity in <i>Ask1^−/−</i> and <i>Bax^−/−/Bak^−/−</i> MEFs treated with 0 or 400 µM DETA-NO (A). Percent LDH release was measured in wild type and Ask<i>1^−/−</i> MEFs treated with 0, 100, 200 and 400 µM DETA-NO (B).</p

The individual loss of the BH3-only proteins BID, BIM, PUMA, BAD and NOXA does not protect against nitric oxide-induced cell death.

<p>Wild type, <i>Bid^−/−</i> (A), <i>Bim^−/−</i> (B), <i>Puma^−/−</i> (C), <i>Bad^−/−</i> (D) and <i>Noxa^−/−</i> (E) MEFs were treated with 0 and 400 µM DETA-NO for 24 and 48 hours. Percent cell death was measured by LDH release.</p