DMOG inhibits lysine demethylases and increases levels of the Suv39h1 methyltransferase.

<p>(<b>A</b>) MEFs were incubated in either solvent (Control: DMSO) or DMOG (1 mM) for 24 hr. H3K9me3 levels were detected by immunofluorescent staining with antibody to H3K9me3 and co-stained with DAPI to locate nuclear DNA. (<b>B</b>) HEK293T cells were transiently transfected with vector or KDM4A and allowed to recover for 30 hrs. Cells were either untreated or incubated with DMOG (1 mM) for 24 hrs. H3K9me3 levels were detected by immunofluorescent staining with antibody to H3K9me3. (<b>C</b>) MCF-7 cells were incubated in increasing concentrations of DMOG or CoCl₂ (200 µM) for 24 hrs. Cell extracts were prepared and examined by western blot (WB) analysis to monitor levels of Hif1α and Suv39h1. β-actin levels are shown to demonstrate equal loading. (<b>D</b>) MCF-7 cells were incubated in DMOG (1 mM) or CoCl₂ (200 µM) for the indicated time. Cell extracts were prepared and examined by western blot (WB) analysis to monitor levels of Hif1α and Suv39h1. β-actin levels are shown to demonstrate equal loading. (<b>E</b>) MCF-7 cells expressing either a non-specific shRNA (shRNA^Con) or shRNA targeting Hif1α (shRNA^Hif1α) were incubated with DMOG or CoCl₂ (200 µM) for 24 hrs. Cell extracts were prepared and examined by western blot (WB) analysis to monitor levels of Hif1α and Suv39h1. β-actin levels are shown to demonstrate equal loading.</p

Hif1α increases transcription of the CHD4 and MTA3 genes.

<p>(<b>A</b>) MCF-7 cells stably expressing either a non-specific shRNA (shRNA^Con) or shRNA targeting Hif1α (shRNA^Hif1α) were exposed to DMOG (1 mM) or CoCl₂ (200 µM) for 24 hrs. Cell extracts were prepared and then examined by western blot (WB) analysis to monitor levels of Hif1α. β-actin levels are shown to demonstrate equal loading. (<b>B–E</b>) MCF-7 cells stably expressing either a non-specific shRNA (shRNA^Con) or shRNA targeting Hif1α (shRNA^Hif1α) were exposed to solvent (open bars) or CoCl₂ (200 µM; filled bars) for 8 hr. mRNA levels for the indicated genes were measured by RT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026064#s4" target="_blank">methods</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026064#s2" target="_blank">Results</a> ± SE (n = 3). (<b>F</b>) MCF7 cells stably expressing either a non-specific shRNA (shRNA^Con) or shRNA targeting Hif1α (shRNA^Hif1α) were exposed to CoCl₂ (200 µM) for indicated number of hours. Cell extracts were prepared and then examined by western blot (WB) analysis to monitor levels of CHD4, MTA3 and Hif1α. β-actin levels are shown to demonstrate equal loading.</p

Upregulation of Hif1α promotes radioprotection.

<p>(<b>A</b>) MCF-7 cells were incubated in DMOG (1 mM) or CoCl₂ (200 µM) for 10 hr or exposed to 10Gy of ionizing radiation (IR) for the indicated times. Cell extracts were prepared and examined by western blot (WB) analysis to monitor levels of Hif1α. β-actin levels are shown to demonstrate equal loading. (<b>B</b>) MCF-7 cells were incubated in DMOG (1 mM) or CoCl₂ (200 µM) for the indicated times. Cells were irradiated (10Gy) at time zero where indicated. Cell extracts were prepared and examined by western blot (WB) analysis to monitor levels of Hif1α. β-actin levels are shown to demonstrate equal loading. (<b>C</b>) MEFs were preincubated in DMOG (1 mM) for 1 hr, followed by exposure to IR as indicated. 24 hours later, DMOG was removed and clonogenic cell survival assays carried out. • = DMOG, ○ = solvent (PBS).</p

DMOG improves survival of mice following total body irradiation.

<p>Mice were irradiated (8Gy TBI) and survival monitored over 30 days. DMOG (100 mg/kg) or saline was administered ip 4 hrs before TBI, and 12 hrs and 36 hrs after TBI. Kaplan-Meier survival plots of (<b>A</b>) C57BL/6J (**p = 0.037) and (<b>B</b>) Balb/c mice (**p = 0.019) are shown.</p

DMOG protects cells from radiation by stabilizing the Hif1α protein.

<p>(<b>A</b>) MEFs expressing a non-specific shRNA (○, •; sh^Con) or shRNA targeting Hif1α (□, ▪; sh^Hif1α) were untreated (○, □) or preincubated for 1 hr in DMOG (•, ▪; 1 mM), irradiated at the indicated dose and allowed to recover for 24 hr. DMOG was removed by medium exchange and clonogenic cell survival assays carried out following 10–12 days growth in culture. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026064#s2" target="_blank">Results</a> ± SE (n = 6). (<b>B</b>) Wild type MEFs or MEFs derived from mice with a double knockout of Suv39h1 and Suv39h2 (MEF-Suv^DKO) were incubated with DMOG (1 mM) or CoCl₂ (200 µM) for 24 hrs. Cell extracts were prepared and examined by western blot (WB) analysis to monitor levels of Hif1α. β-actin levels are shown to demonstrate equal loading. (<b>C</b>) MEFs derived from mice with a double knockout of Suv39h1 and Suv39h2 (MEF-Suv^DKO) were incubated with solvent (○) or DMOG (•; 1 mM) for 1 hr, irradiated at the indicated dose and allowed to recover for 24 hrs. DMOG was removed by medium exchange, cells allowed to recover for 10–12 days, and clonogenic cell survival assays carried out. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026064#s2" target="_blank">Results</a> ± SE (n = 3).</p

Knockdown of individual proteasome subunits results in NSCLC cytotoxicity.

<p>Diagram of the 26 S proteasome showing multiple whole genome shRNA screen hits with the following color code: top hit (red), strong hit (>1 shRNA sequence per gene in both cell lines, dark orange), minor hit (1 shRNA sequence per gene in both cell lines, light orange), chymotrypsin-like proteolytic catalytic site (not a hit but highlighted for illustrative purposes, green). Each hit is labeled using the last two alphanumeric characters of the gene’s HUGO nomenclature; e.g., A1 = PSMA1, B5 = PSMB5, M1 = SHFM1.</p

Proteasome inhibition delays DNA repair <i>in vivo</i>.

<p>(A) FANCD2 immunofluorescence in 10 Gy irradiated vs. unirradiated NCI-H460 xenografts recovered from mice with or without doxycycline-induced <i>PSMA1</i> shRNA expression in tumor cells. Bar = 10 µm. (B) γ-H2AX immunohistochemistry in NCI-H460 xenograft tumors with or without doxycycline-induced <i>PSMA1</i> shRNA knockdown, recovered from mice 1, 6, and 24 hours after 10 Gy irradiation. Bar = 10 µm. (C) Quantification of immunohistochemistry for γ-H2AX in (B). Cells with ≥5 foci were scored as positive (n>400 cells). All results are mean ± SEM. P values were calculated using a two-tailed Student’s t test.</p

Proteasome inhibition blocks expression of Fanconi Anemia/Homologous Recombination genes.

<p>(A) Diagram of NF-kB promoters on <i>FANCD2</i> and <i>BRCA1</i> genes. (B) Expression by qPCR of <i>FANCD2</i> following proteasome inhibition ±30 nM (A549) or 50 nM (NCI-H460) bortezomib or ± inducible <i>PSMA1</i> shRNA, and ±10 Gy IR. All values are normalized to ACTB and all results are mean ± SEM. (C) As in (B) but with BRCA1. (D) NSCLC cell survival following bortezomib and IR is partially rescued by <i>IkBα</i> siRNA knockdown when performed in advance. Cell viability was assayed using an ATP luminescence-based assay measuring relative luminescent units (RLU). All results are mean ± SEM.</p

Role of proteasome inhibition in modulating NF-κB pathway-mediated expression of Fanconi Anemia (FA)/homologous recombination (HR) genes.

<p>Proteasome inhibition by bortezomib or <i>PSMA1</i> knockdown results in an increase in IκBα, which in turn decreases NF-κB binding to the promoters of FA/HR genes including <i>FANCD2</i> and <i>BRCA1</i>. This reduces the availability of these DNA repair proteins for recruitment to DNA damage sites, resulting in decreased RAD51 focus formation and HR following induction of DNA double strand breaks by ionizing radiation.</p

Proteasome inhibition sensitizes NSCLC cells to radiation.

<p>(A) Western blot showing protein levels of PSMA1 and PSMB5 in A549 and NCI-H460 NSCLC cells after <i>PSMA1</i> shRNA knockdown compared to non-silencing shRNA control. (B) Chymotrypsin-like (CTL) proteasome activity assay in A549 (left) and NCI-H460 (right) NSCLC cells after treatment with bortezomib, or <i>PSMA1</i> siRNA knockdown. All results are mean ± SEM and normalized to DMSO vehicle control. (C) Clonogenic survival assay of A549 (left) and NCI-H460 (right) following IR and bortezomib. Marked bars show the percent kill of bortezomib-treated samples compared to DMSO vehicle control at each IR dose. All results are mean ± SEM and normalized to DMSO vehicle control. (D) Apoptosis detection assay of NCI-H460 following 2 and 4 Gy IR and 50 nM bortezomib. Bars show percentage of cells in early apoptosis (left) or late apoptosis (right) via Annexin V and propidum iodide staining, respectively. All results are mean ± SD and P values were calculated using a two-tailed Student’s <i>t</i> test.</p