Dose dependent response of primary cultures to different drugs.

<p>Primary GBM (G1) cells were treated with lomustine, carmustine, UCN-01, oxaliplatin, temozolomide, and tamoxifen (TAM) for 24 h and cell viability was assessed by MTT assay. The viability in untreated control cells was assumed as 100%. The data represents the mean ± SE (n = 3). * p< 0.01 difference between untreated control vs. TAM treated cells.</p

Tamoxifen induces apoptosis and Par-4 expression in Primary GBM cells.

<p>(A) G1 cells were treated with TAM for 12 h and analyzed for apoptosis by staining cells with Annexin V and PI followed by flow cytometry analysis. The bars in the histogram represent mean ±SE of three independent experiments. * p<0.05- treated vs. untreated control. (B) Cells were exposed to TAM for 24 h and analyzed for PARP by immunoblotting with GAPDH as loading control. (C) Caspase-3 activity analyzed by flow cytometry in control and treated cells with TAM for 24 h in the presence or absence of caspase-3 inhibitor (ZVAD-FMK). The numerical values are percent positive cells for caspase-3 activity. (D) Expression of Par-4 in TAM treated G1 cells depicted by immunoblotting with GAPDH as loading control. (E) Immunofluorescence shows the expression of Par-4 in G1 cells treated with TAM. The cells stained with Cy-3 (red) represents the expression and localization of Par-4 in untreated and treated cells (Scale bar - 20µm).</p

Dose dependent response to different drugs on cell viability.

<p>HNGC-2 cells were treated for 24 h with lomustine, carmustine, UCN-01, oxaliplatin, temozolomide, and tamoxifen (TAM) and cell viability was assessed by MTT assay. The cell viability of vehicle control cells was assumed as 100%. The data represents the mean ± SE (n = 3). * p< 0.01, comparison between vehicle control and treated cells.</p

Effect of Par-4 silencing on apoptosis induced by Tamoxifen.

<p>HNGC-2 cells transfected with control siRNA (Nontarget) or siRNA (100 nM) against Par-4 were treated with Tamoxifen (10µg/mL) for 24 h and flowcytometry analysis was performed to confirm apoptosis by (A)TUNEL assay, (B) Represents the overlay profiles of HNGC-2 cells treated with transfected Si control (pink) and Par-4 siRNA (blue), (C) capase-3 activity. The numerical values in the plots are percent cells positive for apoptosis in (A) and caspase-3 activity in (C). The data is representative of two similar experiments.</p

Role of secretory Par-4 in TAM induced apoptosis.

<p>(A) HNGC-2 cells were treated with TAM for 24 h and extracellular Par-4 was analyzed by western blotting in conditioned medium (neat -upper blot) and after concentrating by 30 KD cut-off filters (lower blot). The adjacent blots show ponceau staining of BSA, used as loading control. Expression of Par-4 in conditioned medium of HeLa cells treated with TRAIL was used as positive control for secretory Par-4. (B)Time dependent induction of GRP78 in HNGC-2 cells treated with TAM (10 µg/mL) was determined by western blotting. GAPDH was used as a loading control. (C) Par-4(red) localization with GRP78 (green) on cell membrane in TAM treated HNGC-2 cells (Scale bar - 20µm). (D) HNGC-2 cells were exposed to GRP78 and Par-4 antibody for 1 h prior to TAM treatment and apoptosis was assessed for caspase-3 activity by flow cytometry analysis. IgG was used as isotype control. Values in plots depicts percent cells positive for caspase-3 activity.</p

Tamoxifen induced Par-4 regulates Akt and ERK signaling in HNGC-2 cells.

<p>HNGC-2 cells treated with TAM (10 µg/mL) for increasing time periods were analyzed for the expression pattern of (A) pAkt (Ser473),total Akt and (B) pERK (42/44) and total ERK by western blotting. GAPDH was used as loading control. (C) HNGC-2 cells were transfected with control siRNA and Par-4 siRNA (100 nM) and analyzed for expression of Par-4 by immunofluorescence. (D) Transfected control and Par-4 silenced cells were analyzed for phosphoAkt (Ser473) total Akt and (E) phosphoERK and total ERK signaling by western blotting. Fold change is shown by bars, mean ± SD of three independent experiments. *p<0.05, control siRNA vs. Par-4 siRNA cells.</p

Expression of Par-4 in HNGC-2 cells treated with a panel of drugs.

<p>(A) Expression of Par-4 was determined by immunoblotting in HNGC-2 cells exposed to lomustine, carmustine, UCN-01, oxaliplatin, tamoxifen (TAM) and temozolomide for 24 h. The bars represent mean ± SE of three independent experiments. *p<0.05, treated vs. vehicle control. (B) Time dependent induction of Par-4 in HNGC-2 cells treated with TAM (10µg/mL). Fold induction of Par-4 mRNA level normalized using 18S rRNA, was assessed by quantitative real-time PCR. The data are expressed as the mean ± SD and are representative of two independent experiments that yielded similar results. (C) Par-4 protein levels relative to GAPDH were analyzed by western blot. (D) Par-4 expression (red) was visualized by probing with Cy3 antibody along with actin as green (Scale bar - 20µm). (E) Cell fraction lysates were used to determine Par-4 upregulation in cytoplasm and nucleus of HNGC-2 cells exposed to TAM (10 µg/mL), Actin and lamin A/C were used as loading controls respectively.</p

Effect of Par-4 silencing on apoptosis in primary GBM cells treated with Tamoxifen.

<p>(A) G1 cells were transfected with control siRNA or Par-4 siRNA (100 nM) and analyzed for its expression by immunoblotting and (B) Immunofluorescence (Scale bar - 20µm). (C) The control and Par-4 transfected cells were exposed to TAM (15µg/ml) for 36 h and assessed for caspase-3 activity. Values in the plot indicate percent cells positive for caspase-3 activity.</p