Transformed cells are preferentially sensitive to PCTAIRE1 knockdown-effect with TRAIL treatment.

<p>(A-C) Human diploid fibroblasts IMR-90 (A), 267B1 immortalized normal prostate epithelial cells (B), and K-ras transformed 267B1 (267B1/K-ras) cells (C) were transfected with scrambled RNA or three different siRNAs targeting PCTAIRE1 (siRNAs: si-1472, si-1566, si-1656). Lysates of cells 72 hours post-transfection were prepared and analyzed by immunoblotting using rabbit anti-PCTAIRE1 (top) or anti-beta-actin (bottom) antibodies. (D-F) IMR-90, 267B1 and 267B1/K-ras cells were transfected with siRNAs. After 48 hours, cells were stimulated with TRAIL at various concentrations as indicated. After 24 hours, cellular ATP levels were measured, and the data expressed as a ratio between cells cultured with and without TRAIL (mean ± SD; n = 3).</p

PCTAIRE1-Knockdown Sensitizes Cancer Cells to TNF Family Cytokines

<div><p>While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of <i>PCTAIRE1</i> sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells.</p></div

PCTAIRE1 knockdown sensitizes PPC1 cells to anti-Fas antibody and TRAIL.

<p>(A) PPC1 cells were transfected with scrambled RNA or three different siRNAs targeting PCTAIRE1 (siRNAs 1472, 1566, 1656). Lysates from cells at 48 hours post-siRNA transfection were prepared, normalized for total protein content, and aliquots were analyzed by immunoblotting using anti-PCTAIRE1 (top) or anti-alpha-tubulin (bottom) antibodies. (B, C) PPC1 cells were transfected with control RNA (purple “x”) or various siRNAs targeting PCTAIRE1 (blue diamonds, 1472; red squares, 1566, green triangles, 1656). After 48 hours, cells were stimulated with either anti-Fas antibody (CH-11) (B) or TRAIL (C) at various concentrations as indicated. After 24 hours, cellular ATP levels were measured as a surrogate indicator of cell viability using Cell Titer Glo reagents, and the data are expressed as the ratio between cells cultured with and without anti-Fas (B), and TRAIL (C). (D) Apoptosis analyses were performed using an annexinV kit. PPC1 cells were transfected with scramble RNA of siRNAs targeting PCTAIRE1. After 48 hours, cells were treated with anti-Fas antibody (100 ng/ml) or TRAIL (50 ng/ml) for 4 hours. *P < 0.05, ***P < 0.001 by t-test. All data represent mean ± SD (n = 3). (E, F) Clonogenic survival assays. PPC1 cells were seeded in 6 well (35 mm) dishes at 1.0 x 10⁵ cells per well and then reverse-transfected with scramble-control or PCTAIRE1-targeting siRNAs as indicated. After 48 hours, anti-Fas antibody (E, 10 ng/ml) or TRAIL (F, 20 ng/ml) was added and cells were cultured for 3 days before fixing and staining with 0.5% crystal violet dye. (G, H) PPC1 cells were transfected with control RNA or three different siRNAs targeting PCTAIRE1. After 48 hours, the cells were stimulated with Fas (G, 10 ng/ml) or TRAIL (H, 10 ng/ml). After 3 hours, cell lysates were prepared, normalized for total protein content, and analyzed by immunoblotting using an antibody specific for cleaved caspase-8. The partially cleaved 43/41 kDa bands and fully cleaved 18 kDa band are indicated, as are molecular weight markers (kDa). The blot was reprobed with beta-actin antibody as a loading control. (I) PPC1 cells were transfected with scramble-control or siRNA targeting PCTAIRE1 (si-1472). After 48 hours, cells were or were not stimulated with anti-Fas antibody (CH-11, 10 ng/ml). After 3 hours, cell lysates were harvested and immunoprecipitated with anti-caspase 8 antibody, followed by immunoblotting with the indicated antibodies. The red arrowheads indicate non-specific bands.</p

Targeting PCTAIRE1 using an inducible shRNA vector in PPC1 cells.

<p>(A) PPC1 cells stably containing inducible shRNAs targeting different sites on PCTAIRE1 mRNA (shRNA#1, #2) or scramble-control were cultured for 48 hours with doxycycline (Dox, 100 ng/ml). Protein lysates were generated, normalized for total protein concentration, and analyzed by SDS-PAGE/immunoblotting using antibodies for PCTAIRE1 (top) and beta-actin (bottom). (B, C) PPC1 cells were cultured with (ON) or without (OFF) 100 ng/ml Dox for 48 hours, then stimulated with various concentrations of either anti-Fas antibody CH-11 (B) or TRAIL (C). After 24 hours, cellular ATP levels were measured, and the data expressed as a ratio relative to cells cultured without anti-Fas or TRAIL (mean ± SD; n = 3). (D, E) Clonogenic survival assays. PPC1 cells stably containing inducible shRNAs (scramble or shRNA#2) were cultured with (ON) or without (OFF) 100 ng/ml Dox for 48 hours, then stimulated with anti-Fas antibody (CH-11, 10 ng/ml) or TRAIL (20 ng/ml). Cells were cultured for 3 days before fixing and staining with 0.5% crystal violet dye.</p

PCTAIRE1-knockdown modulates RIPK1 expression.

<p>(A) PPC1 cells were transfected with control RNA or three different siRNAs targeting PCTAIRE1. After 48 hours, cell lysates were prepared with RIPA buffer, normalized for total protein content, and analyzed by immunoblotting using the indicated antibodies. (B) PPC1 cells were transfected with indicated siRNAs for 48 hours, whereupon cell lysates were collected in 1x Laemmli buffer and subjected to immunoblotting analysis for RIPK1 (top) and beta-actin (middle). (C, D) PPC1 cells were transfected with the indicated siRNAs. After 48 hours, cells were treated with the proteasome inhibitor MG-132 (10 μM) for 5 hours before preparation of cell lysates (C: 1x Laemmli buffer, D: RIPA buffer). (E) PPC1 cells were transfected with control RNA or siRNA targeting RIPK1. After 48 hours, cell lysates were prepared in RIPA buffer, normalized for total protein content, and analyzed by immunoblotting using the indicated antibodies. (F, G) PPC1 cells were transfected with control RNA or siRNA targeting RIPK1. After 48 hours, cells were stimulated with either anti-Fas antibody (CH-11) (F) or TRAIL (G) at various concentrations as indicated. After 24 hours, cellular ATP levels were measured as a surrogate indicator of cell viability using Cell Titer Glo reagents, and the data expressed as a ratio between cells cultured with and without anti-Fas (F) or TRAIL (G). (H) Clonogenic survival assays. PPC1 cells were seeded in 6 well (35 mm) dishes at 1.0 x 10⁵ cells per well, and then reverse-transfected with scramble-control, PCTAIRE1-targeting siRNA (si-1472), or RIPK1-targeting siRNA as indicated. After 48 hours, anti-Fas antibody (top, 2.5 ng/ml) or TRAIL (bottom, 10 ng/ml) was added and cells were cultured for 3 days before fixing and staining with 0.5% crystal violet dye.</p

Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

<div><p>Perturbation of endoplasmic reticulum (ER) homeostasis triggers the ER stress response (also known as Unfolded Protein Response), a hallmark of many pathological disorders. However the connection between ER stress and inflammation remains largely unexplored. Recent data suggest that ER stress controls the activity of inflammasomes, key signaling platforms that mediate innate immune responses. Here we report that expression of NLRP1, a core inflammasome component, is specifically up-regulated during severe ER stress conditions in human cell lines. Both IRE1α and PERK, but not the ATF6 pathway, modulate <i>NLRP1</i> gene expression. Furthermore, using mutagenesis, chromatin immunoprecipitation and CRISPR-Cas9-mediated genome editing technology, we demonstrate that ATF4 transcription factor directly binds to <i>NLRP1</i> promoter during ER stress. Although involved in different types of inflammatory responses, XBP-1 splicing was not required for <i>NLRP1</i> induction. This study provides further evidence that links ER stress with innate</p></div

NLRP1 mRNA and protein are up-regulated upon ER stress.

<p>(A) Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 levels were measured by quantitative real-time PCR (qPCR) using cyclophillin A as an endogenous control. Semi-quantitative RT-PCR using a different NLRP1 primer set and GAPDH as a control is also shown. (B) HeLa cells were treated either with BFA or TG for the indicated times. NLRP1 mRNA levels were measured by qPCR and RT-PCR. Spliced and un-spliced XBP-1 forms were also evaluated by RT-PCR. (C) HCT116 cells were treated with the indicated stimuli for 24 hours. NLRP1 and NOD1 mRNA levels were measured by qPCR. (D) Cell lysates from wild-type or <i>NLRP1</i>^{<i>−/−</i>} HeLa, THP-1 and K562 cells, untreated or treated with BFA for 20 hours, were normalized for total protein content. Cell extracts were then subjected to SDS-PAGE/immunoblot analysis before and after immunoprecipitation with NLRP1 antibody. Vinculin was detected as loading control. NLRP1 mRNA levels were also measured by RT-PCR. Each panel is representative of at least three independent experiments. (DMSO: dimethyl sulfoxide, TM: tunicamycin, TG: thapsigargin, MSU: monosodium urate crystals, BFA: brefeldin A, PolyI:C: polyinosinic-polycytidylic acid, FLA: flagellin, MDP: muramyl dipeptide, R837: Imiquimod)</p

NLRP1 mRNA up-regulation is dependent on both IRE1α and PERK pathways.

<p>(A) IRE1α, PERK and ATF6 levels were reduced using siRNA. Upon treatment with ER stress, mRNA levels were measured by qPCR and RT-PCR. IRE1α, PERK and ATF6 knock-down was verified by SDS-PAGE/immunoblotting. (B) Stably transduced HeLa cells were cultured in presence or absence of doxycycline (Dox) for 24 hours and then treated overnight with 2μM BFA. mRNA levels were measured by qPCR and RT-PCR. IRE1α, PERK and ATF6 knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.</p

Atf4 but not Xbp-1s stimulates <i>NLRP1</i> gene expression during ER stress.

<p>(A) HeLa cells were infected with increasing concentrations of murine Xbp-1s and Atf4 adenovirus for 24 hours and NLRP1 mRNA was measured by qPCR. (B) IRE1α, PERK, ATF6 and XBP-1s were down-regulated using siRNA in HeLa cells. Cells were treated with BFA for 20 hours and mRNA levels were measured by qPCR. IRE1α, PERK, ATF6 and XBP-1s knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.</p

Cardiac Glycosides Activate the Tumor Suppressor and Viral Restriction Factor Promyelocytic Leukemia Protein (PML)

<div><p>Cardiac glycosides (CGs), inhibitors of Na⁺/K⁺-ATPase (NKA), used clinically to treat heart failure, have garnered recent attention as potential anti-cancer and anti-viral agents. A high-throughput phenotypic screen designed to identify modulators of promyelocytic leukemia protein (PML) nuclear body (NB) formation revealed the CG gitoxigenin as a potent activator of PML. We demonstrate that multiple structurally distinct CGs activate the formation of PML NBs and induce PML protein SUMOylation in an NKA-dependent fashion. CG effects on PML occur at the post-transcriptional level, mechanistically distinct from previously described PML activators and are mediated through signaling events downstream of NKA. Curiously, genomic deletion of PML in human cancer cells failed to abrogate the cytotoxic effects of CGs and other apoptotic stimuli such as ceramide and arsenic trioxide that were previously shown to function through PML in mice. These findings suggest that alternative pathways can compensate for PML loss to mediate apoptosis in response to CGs and other apoptotic stimuli.</p></div