PCI-24781 modulates the expression of HIF-1α.

<p>(<b>A</b>) All cell lines as well as normal lymphocytes were subjected to lysis followed by Western blotting using HIF-1α antibody. (<b>B</b>) All cell lines were incubated with indicated concentration of PCI-24781 (PCI) under normoxia for 24 hr followed by cell lysis and western blotting using HIF-1α antibody. (<b>C</b>) SUDHL4 and (<b>D</b>) OCI-LY3 cells were incubated with or without PCI for 0-24 hr followed by western blot analysis for HIF-1α. Bands were quantified by densitometry and change in HIF1α protein over time is shown by the line graph on the right side of each blot. (* = P<0.05).</p

PI3K/AKT pathway in HIF-1α regulation.

<p>(<b>A</b>) To check the expression of HIF-1α in SUDHL6 and OCI-LY3 cells expressing Myr-Akt and wt-Akt, cells were subjected to lysis followed by western blotting using HIF-1α antibody. (<b>B</b>) SUDHL4-and OCI-LY3 cells were incubated with indicated concentration of LY294002 (LY) for 24 hr followed by cell lysis and Western blotting using HIF-1α antibody. (<b>C</b>) Malignant cells obtained from blood sample of CLL patient were incubated with indicated concentration of PCI or LY alone or in combination for 16 hr followed by cell lysis and western blotting using specific antibodies as indicated. (<b>D</b>) SUDHL4 and OCI-LY3 cells were treated with PCI and LY either alone or in combination for 24 hr followed by cell lysis and western blotting using specific antibodies for pAkt and Akt. Actin is used as an internal control.</p

PCI-24781 induces AMPK activation.

<p>SUDHL4 and OCI-LY3 cells were treated with indicated concentration of PCI or LY alone and in combination for 24hr followed by western blotting using specific antibodies. Actin is used as an internal control. </p

PCI-24781-induced suppression in HIF-1α protein does not require proteasomal degradation.

<p>OCI-LY3 cells were incubated with indicated concentrations of PCI-24781 or MG132 alone or in combination for 24 hr followed by western blotting using specific antibodies to HIF-1α and HIF-1α-OH. </p

Knock down of HIF-1α enhances PCI induced apoptosis.

<p>(<b>A</b>) HIF-1α was knocked down in SUDHL4 and OCI-LY3 cell lines. Cells were transduced with scrambled or HIF-1α shRNA by spin infection using GIPZ lentivirus system.  After selection in puromycin for 2 weeks, cells were subjected to Western blotting to check the protein level and after positive selection cells were treated with indicated concentration of PCI-24781 for 48 hours, followed by annexin V/PI staining and analyzed by flow cytometry. Western blot next to each bar graph shows knock down of HIF-1α (<b>B</b>) SUDHL4, SUDHL6 and OCI-LY3 cells were incubated with indicated concentration of PCI-24781 and PX-478 either alone or in combination for 24 hr followed by cell lysis and western blotting. Actin is used as an internal control for all western blots. (<b>C</b>) SUDHL4, SUDHL6 and OCI-LY3 cells were treated with indicated concentration of PCI or PX-478 alone or in combination for 48 hr followed by annexin V/PI staining and analyzed by flow cytometry. (<b>D</b>) OCI-LY3 cells were transfected with wt-HIF-1α or mutant (P/A) HIF-1α plasmids using Amaxa Kit L and selected in neomycin for one week. After selection cells were treated with indicated concentration of PCI for 48 hr followed by AnnexinV/PI staining and analyzed by flow cytometry. (ns= not significant,. * = P<0.05; **= p<0.01; *** = P<0.001).</p

PCI-24781- Induced autophagy is mediated by HIF-1α.

<p>(<b>A</b>) SUDH4, SUDHL6 and OCI-LY3 cells were incubated with increasing concentration of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for LC3B and p62 protein using specific antibodies. (<b>B</b>) SUDHL4 and OCI-LY3 cells were incubated with 0.5 or 1.0 µM of PCI respectively for 0-16 hr followed by cell lysis and western blotting for LC3B protein using specific antibody. (<b>C</b>) SUDHL4 and OCI-LY3 cells expressing scrambled or HIF-1α shRNA were treated with PCI for 24 hr followed by further incubation with acridine orange for 15 minute. Cells were washed twice with PBS and analyzed by flow cytomerty for the quantification of cells with high AVO population. (<b>D</b>) OCI-LY3 cells with or without HIF-1α shRNA were treated with PCI-24781 for 6 hr followed by cell lysis and western blotting for autophagy markers p62 and LC3B proteins. Actin is used as an internal control for western blotting. (* = P<0.05; **= p<0.01; *** = P<0.001).</p

PCI-24781 induces prosurvival autophagy.

<p>(<b>A</b>) SUDHL4 and OCI-LY3 cells were treated with 0.5 µM PCI and 5 mM 3-MA or 10 nM Bafilomycin (Baf) either alone or in combination for 6 hr followed by cell lysis and western blotting for LC3B protein. Actin is used as an internal control. (<b>B</b>) SUDHL4 cells were treated with 3 mM 3-MA and indicated concentration of PCI-24781 either alone or in combination for 16 hr followed by additional incubation with 2 µg/mL acridine orange for 15 minutes. Percentage of cells representing formation of Acidic vesicular organelles was quantified by flow cytometry. (<b>C</b>) SUDHL4 cells were treated with PCI or 3 –MA either alone or in combination for 48 hr followed by further incubation with 2 µg/mL acridine orange and analyzed by flow cytometry. DAPI was added to cells prior to acquisition. Cells were gated on AVO low and AVO high population and apoptosis was measured on each population by gating on Annexin V–APC (FL8) and DAPI (FL6) positive cells. (<b>D</b>) Bar graph representation of % of apoptosis on low AVO population obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081333#pone-0081333-g005" target="_blank">Figure 5 C</a>. (<b>E</b>) SUDHL4 and OCI-LY3 cells were incubated with PCI-24781 and Chloroquine (CQ) either alone or in combination for 72 hr followed by annexin V/ PI staining and flow cytometry. (* = P<0.05; **= p<0.01).</p

PCI-24781 induced Apoptosis is mediated by PI3K/Akt pathway.

<p>(<b>A</b>) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. (<b>B</b>) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. (<b>C</b>) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. (<b>D</b>) SUDHL6 and OCI-LY3 cells were transfected with Myr-Akt or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. (<b>E</b>) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).</p