THC increases p53 protein expression in a time-dependent manner. A, cultured cortical neurons were treated with THC (5 μM) for 5–180 min, cell protein was harvested and analysed for the expression levels of phospho-p53 using western immunoblot. THC significantly increased phospho-p53^ser15 expression at 5 min but not at subsequent time-points (30–180 min). Results are expressed as mean ± S.E.M for 5 observations, ^⁎P < 0.05. Inset; a sample western immunoblot demonstrating the increase in phospho-p53^ser15 expression at 5 min of THC treatment (lane 2) compared to untreated neurons (lane 1), neurons treated with THC for 30 min (lane 3), 60 min (lane 4), 120 min (lane 5) and 180 min (lane 6). T-p53 expression was monitored to ensure equal protein loading. B, a significant increase in phospho-p53^ser15 protein expression was found following treatment with THC at concentrations of 5 μM and 50 μM. Exposure to THC at a concentration of 0.5 μM had no effect on the expression levels of phospho-p53^ser15. Results are expressed as mean ± S.E.M for 5 observations, ^⁎P < 0.05. inset; a sample western immunoblot demonstrating that a 5 min treatment with THC at doses of 5 μM (lane 3) and 50 μM (lane 4) significantly increased phospho-p53^ser15 expression as compared to vehicle-treated neurons (lane 1). Data were normalised to t-p53 and actin expression was monitored to ensure equal protein loading. C, pre-incubation of cortical neurons with AM 251 (1 μM) prior to THC exposure abolished the THC-induced increase in phospho-p53^ser15 expression observed at 5 min. Results are expressed as mean ± S.E.M for 6 observations, ^⁎P < 0.05. D, confocal images of phospho-p53^ser15 immunostaining in (i) vehicle-treated neurons, (ii) THC-treated neurons (5 μM, 5 min), (iii) D-JNK1 (1 μM)-treated cultures and (iv) cultures treated with THC in the presence of D-JNKI (1 μM). Scale bar is 10 μm. E, pre-incubation of cortical neurons with D-JNKI (1 μM). prior to THC (5 μM) exposure abolished the THC-induced increase in phospho-p53^ser15 expression observed at 5 min. Results are expressed as mean ± S.E.M for 6 observations, ^⁎⁎P < 0.01.

THC-induced increase in Bax is reliant on p53. A, cultured cortical neurons were treated with THC (5 μM) for 120 min, cell protein was harvested and analysed for the expression levels of Bax using western immunoblot. THC evoked a significant increase in Bax protein expression, and this was abolished by pre-treatment with the p53 inhibitor, pifithrin-α (100 nM). Results are expressed as mean ± S.E.M for 5 observations, ^⁎P < 0.05. Inset; a sample western immunoblot demonstrating the increase in Bax protein expression following THC treatment (lane 2). Lane 1 represents vehicle-treated neurons, pifithrin-α-treated neurons (lane 3), neurons treated with THC in the presence of pifithrin-α (lane 4). An actin immunoblot confirms equal protein loading. B, images of Bax immunostaining in (i) vehicle-treated neurons, (ii) THC-treated neurons (5 μM, 2 h), (iii) pifithrin-α-treated neurons and (iv) neurons treated with THC in the presence of pifithrin-α. Arrows indicate cells with intense Bax immunoreactivity. Scale bar is 25 μm. C, THC significantly increased phospho-Bcl (p-Bcl) expression at 120 min but not at earlier time-points (5–60 min). Results are expressed as mean ± SEM for 6 observations, normalised to total-Bcl expression, ^⁎⁎P < 0.01. Inset; a sample western immunoblot demonstrating the increase in phospho-Bcl expression at 120 min of THC treatment (lane 5) compared to vehicle-treated neurons (lane 1), neurons treated with THC for 5 min (lane 2), 30 min (lane 3), 60 min (lane 4). D, neurons were pre-incubated with pifithrin-α (100 nM), treated with THC (5 μM) for 120 min and analysed for the expression levels of phospho-Bcl protein using western immunoblotting. Exposure to pifithrin-α abolished the THC-induced increase in Bax expression. Results are expressed as mean ± S.E.M for 5 observations, normalised to total-Bcl expression, ^⁎P < 0.05. Inset: a sample western immunoblot demonstrating phospho-Bcl (p-Bcl) and total-Bcl protein expression in cells treated with vehicle control (lane 1), THC (lane 2), pifithrin-α (lane 3) and THC + pifithrin-α (lane 4).

THC-induced apoptosis is mediated via p53. A, treatment of primary cortical neurons with THC (5 μM; 120 min) significantly increased caspase-3 activity as assessed by the cleavage of the fluorogenic DEVD substrate. The stimulatory effect of THC on caspase-3 activity was prevented by pifithrin-α (100 nM). Exposure of cells to pifithrin-α had no effect on caspase-3 activity. Results are expressed as the mean ± S.E.M for 4 observations, ^⁎P < 0.05. B, cultured cortical neurons were treated with THC (5 μM; 120 min) in the presence or absence of pifithrin-α (100 nM) and cell viability was assessed by TUNEL staining. THC significantly increased DNA fragmentation and this was prevented by pifithrin-α. Results are expressed as mean ± SEM for 4 observations, ^⁎⁎⁎P < 0.001. C, images of TUNEL staining in (i) vehicle-treated neurons, (ii) THC-treated neurons, (iii) pifithrin-α-treated cultures and (iv) cultures treated with THC in the presence of pifithrin-α. Arrows indicate apoptotic neurons displaying DNA fragmentation following exposure to THC. Scale bar is 25 μm.

siRNA knockdown of p53 prevents THC-induced DNA fragmentation. A, exposure of neurons to THC (5 μM) for 2 h significantly increased the percentage of cells with DNA fragmentation. Treatment with p53 siRNA (100 nM; 48 h) prior to THC treatment prevented the THC-induced increase in DNA fragmentation. Results are expressed as mean ± S.E.M for 6 observations, ^⁎⁎⁎P < 0.001 versus vCon; ^⁎⁎ versus p53 siRNA transfected cells exposed to THC. B, Representative confocal images of TUNEL stained neurons in (i) cells exposed to vehicle control, (ii) cells exposed to THC (5 μM, 2 h), (iii) cells pre-treated with p53 siRNA and (iv) cells exposed to THC (5 μM, 2 h) following p53 siRNA pre-treatment. Arrows indicate TUNEL-positive apoptotic cells.