af1965  (R&D Systems)

Journal: Science immunology

Article Title: Protein kinase R is an innate immune sensor of proteotoxic stress via accumulation of cytoplasmic IL-24

doi: 10.1126/sciimmunol.abi6763

Figure Lengend Snippet: (A) CRISPR-Cas9 gene editing was used to delete IL24, PRKRA, and TARBP2 genes in THP-1 cell lines to generate cells deficient in IL-24, PACT, and TRBP (respectively). Cells were then treated for 16 hours with delanzomib (200 nM), poly(dA:dT) (1 μg/ml), or vehicle control (Ctrl) as indicated, and induction of IFNB1 mRNA was assessed by qPCR. (B and C) IL24 was CRISPR- Cas9– deleted from iβ5−/− or iβ1−/− cells, and (B) induction of IFNB1 mRNA was assessed by qPCR in iβ5−/−, IL-24−/−, iβ5−/−IL-24−/−, and parental THP-1 cell lines. (C) Immunoblotting was performed on parental, iβ5−/−, and iβ1−/− THP-1 cells sufficient or deficient in IL-24 to assess phosphorylation (p) of STAT1, PKR, and eIF2α. (D) IL-24 tagged with HA or empty vector control was stably expressed in IL-24−/− or IL-24−/−PKR−/− THP-1 cells by lentiviral transfection. Cells were treated with delanzomib (200 nM) for 16 hours, and induction of IFNB1 was assessed by qPCR. (E) The IL-24 receptor subunit, IL20RB was CRISPR-Cas9–deleted from THP-1 cells, cells were then treated with delanzomib (200 nM) for 16 hours and induction of IFNB1 was assessed by qPCR. (F) PKR-IP and WCLs from THP-1, iβ5−/−, iβ5−/−PKR−/−, and iβ5−/− IL-24−/− cell lines were immunoblotted for PKR, IL-24, and actin (loading control). (G) IL-24–Halo was stably expressed in U2OS cells. Super-resolution microscopy was then performed to monitor IL-24–Halo cellular localization during proteasome inhibitor treatment (delanzomib, 200 nM). Egress of IL-24–Halo from the ER to the cytosol could be inhibited by kifunesine. Representative images of specified timepoints are displayed, IL-24–Halo in red, ER-Tracker in green and DAPI in blue. White scale bars, 10 μM. (H) Three to seven representative cells from each time point from (G) were analyzed using Imaris software to determine percentage IL-24–Halo outside the ER (cytoplasmic IL-24). (I) THP-1 cells were treated with delanzomib (200 nM) and/or kifunesine (50 nM) for 14 hours and induction of IFNB1 and IL6 was assessed by qPCR. (J) Expression of IL-24 splice isoforms, and β-tubulin mRNA expression was analyzed by PCR in PRAAS patient and HC fibroblast lines, under normal conditions (lanes 1 to 5) and serum starvation (lanes 6 to 10). (K) THP-1 cells were stably transfected with HA-tagged full length IL-24 or IL-24 ΔEx5 (as indicated). Differentiation into macrophages was performed using PMA (72 hours), and IF for IL-24 (HA tag) in red and DAPI in blue was assessed at baseline and 14 hours after treatment with delanzomib (200 nM). Data are pooled from three to four experiments, and statistical significance was assessed by ratio paired t test, where * indicates P < 0.05, ** indicates P < 0.01, and ns indicates not significant, with the exception of (H) where statistical significance was assessed by two-way ANOVA with Bonferrioni post tests for individual time points, where ** denotes P < 0.01, and * indicates P < 0.05.

Article Snippet: Staining for IL-24 (1:200; R&D Systems, AF1965, donkey anti-goat AF647 secondary antibody (Abcam, ab150131) was performed using the same method; however, culture with brefeldin A was not performed.

Techniques: CRISPR, Western Blot, Plasmid Preparation, Stable Transfection, Transfection, Microscopy, Software, Expressing

Journal: Science immunology

Article Title: Protein kinase R is an innate immune sensor of proteotoxic stress via accumulation of cytoplasmic IL-24

doi: 10.1126/sciimmunol.abi6763

Figure Lengend Snippet: (A) Lysates from patient fibroblasts were immunoblotted for (p)PKR and (p)eIF2α, as well as IL-24 and actin. (B) Patient PBMCs were treated with C16 (0.5 μM) for 8 hours, and protein lysates were then immunoblotted for (p)PKR, (p)STAT1, and (p)eIF2α, as well as IL-24 and actin. (C) PBMC samples from PRAAS and SAVI patients as well as HC samples were cultured with C16 for 8 to 14 hours. Expression of IFNB1 and IFNA1 was assessed by qPCR. IFN-αβ induction of ISGs was assessed by NanoString Technologies to generate an ISG score. Significance was assessed by Wilcoxon matched-pair signed-rank test, where * indicates P < 0.05 and ** denotes P < 0.01. (D) iPSC-derived macrophages from PRAAS patient #3 and a HC were assessed for expression of IFNB1, IFNA1, and IFNA2 at baseline and after 8 hours of treatment with C16 (1 μM). Significance was assessed by Student’s t test, * indicates P < 0.05.

Article Snippet: Staining for IL-24 (1:200; R&D Systems, AF1965, donkey anti-goat AF647 secondary antibody (Abcam, ab150131) was performed using the same method; however, culture with brefeldin A was not performed.

Techniques: Cell Culture, Expressing, Derivative Assay

Journal: Science immunology

Article Title: Protein kinase R is an innate immune sensor of proteotoxic stress via accumulation of cytoplasmic IL-24

doi: 10.1126/sciimmunol.abi6763

Figure Lengend Snippet: (A) CRISPR-Cas9 gene editing was used to delete IL24, PRKRA, and TARBP2 genes in THP-1 cell lines to generate cells deficient in IL-24, PACT, and TRBP (respectively). Cells were then treated for 16 hours with delanzomib (200 nM), poly(dA:dT) (1 μg/ml), or vehicle control (Ctrl) as indicated, and induction of IFNB1 mRNA was assessed by qPCR. (B and C) IL24 was CRISPR- Cas9– deleted from iβ5−/− or iβ1−/− cells, and (B) induction of IFNB1 mRNA was assessed by qPCR in iβ5−/−, IL-24−/−, iβ5−/−IL-24−/−, and parental THP-1 cell lines. (C) Immunoblotting was performed on parental, iβ5−/−, and iβ1−/− THP-1 cells sufficient or deficient in IL-24 to assess phosphorylation (p) of STAT1, PKR, and eIF2α. (D) IL-24 tagged with HA or empty vector control was stably expressed in IL-24−/− or IL-24−/−PKR−/− THP-1 cells by lentiviral transfection. Cells were treated with delanzomib (200 nM) for 16 hours, and induction of IFNB1 was assessed by qPCR. (E) The IL-24 receptor subunit, IL20RB was CRISPR-Cas9–deleted from THP-1 cells, cells were then treated with delanzomib (200 nM) for 16 hours and induction of IFNB1 was assessed by qPCR. (F) PKR-IP and WCLs from THP-1, iβ5−/−, iβ5−/−PKR−/−, and iβ5−/− IL-24−/− cell lines were immunoblotted for PKR, IL-24, and actin (loading control). (G) IL-24–Halo was stably expressed in U2OS cells. Super-resolution microscopy was then performed to monitor IL-24–Halo cellular localization during proteasome inhibitor treatment (delanzomib, 200 nM). Egress of IL-24–Halo from the ER to the cytosol could be inhibited by kifunesine. Representative images of specified timepoints are displayed, IL-24–Halo in red, ER-Tracker in green and DAPI in blue. White scale bars, 10 μM. (H) Three to seven representative cells from each time point from (G) were analyzed using Imaris software to determine percentage IL-24–Halo outside the ER (cytoplasmic IL-24). (I) THP-1 cells were treated with delanzomib (200 nM) and/or kifunesine (50 nM) for 14 hours and induction of IFNB1 and IL6 was assessed by qPCR. (J) Expression of IL-24 splice isoforms, and β-tubulin mRNA expression was analyzed by PCR in PRAAS patient and HC fibroblast lines, under normal conditions (lanes 1 to 5) and serum starvation (lanes 6 to 10). (K) THP-1 cells were stably transfected with HA-tagged full length IL-24 or IL-24 ΔEx5 (as indicated). Differentiation into macrophages was performed using PMA (72 hours), and IF for IL-24 (HA tag) in red and DAPI in blue was assessed at baseline and 14 hours after treatment with delanzomib (200 nM). Data are pooled from three to four experiments, and statistical significance was assessed by ratio paired t test, where * indicates P < 0.05, ** indicates P < 0.01, and ns indicates not significant, with the exception of (H) where statistical significance was assessed by two-way ANOVA with Bonferrioni post tests for individual time points, where ** denotes P < 0.01, and * indicates P < 0.05.

Article Snippet: Membranes were blocked in 5% skim milk in tris-buffered saline (TBS) containing 0.1% Tween 20 (Sigma-Aldrich) (TBST) before overnight incubation with specific primary antibodies in 5% bovine serum albumin (BSA) (Sigma-Aldrich) or 5% skim milk in TBST at 4°C: anti-ubiquitin (Santa Cruz Biotechnology, clone P4D1, sc-8017), anti–phosphor-p65 Ser 536 [Cell Signaling Technology (CST), clone 93H1, 3033], anti-iβ5 (Abcam, ab3329), anti-iβ1 (Thermo Fisher Scientific, PA11960), anti–phosphor-PKR Thr 446 (Abcam, clone E120, ab32036), anti–phosphor-eIF2α Ser 51 (CST, clone D9G8, 3398), anti-TFAM (CST, clone D5C8, 8076), anti–phosphor-STAT1 Tyr 701 (CST, clone 58D6, 9167), anti–IL-24 (R&D Systems, AF1965, or Abcam, ab115207), anti-PACT (Santa Cruz Biotechnology, clone D-4, sc-377103), anti-TRBP (Santa Cruz Biotechnology, clone D-5, sc-514124), or anti–actin–horseradish peroxidase (HRP) (1:10,000; Santa Cruz Biotechnology, clone C4, sc-47778).

Techniques: CRISPR, Western Blot, Plasmid Preparation, Stable Transfection, Transfection, Microscopy, Software, Expressing

Journal: Science immunology

Article Title: Protein kinase R is an innate immune sensor of proteotoxic stress via accumulation of cytoplasmic IL-24

doi: 10.1126/sciimmunol.abi6763

Figure Lengend Snippet: (A) Lysates from patient fibroblasts were immunoblotted for (p)PKR and (p)eIF2α, as well as IL-24 and actin. (B) Patient PBMCs were treated with C16 (0.5 μM) for 8 hours, and protein lysates were then immunoblotted for (p)PKR, (p)STAT1, and (p)eIF2α, as well as IL-24 and actin. (C) PBMC samples from PRAAS and SAVI patients as well as HC samples were cultured with C16 for 8 to 14 hours. Expression of IFNB1 and IFNA1 was assessed by qPCR. IFN-αβ induction of ISGs was assessed by NanoString Technologies to generate an ISG score. Significance was assessed by Wilcoxon matched-pair signed-rank test, where * indicates P < 0.05 and ** denotes P < 0.01. (D) iPSC-derived macrophages from PRAAS patient #3 and a HC were assessed for expression of IFNB1, IFNA1, and IFNA2 at baseline and after 8 hours of treatment with C16 (1 μM). Significance was assessed by Student’s t test, * indicates P < 0.05.

Article Snippet: Membranes were blocked in 5% skim milk in tris-buffered saline (TBS) containing 0.1% Tween 20 (Sigma-Aldrich) (TBST) before overnight incubation with specific primary antibodies in 5% bovine serum albumin (BSA) (Sigma-Aldrich) or 5% skim milk in TBST at 4°C: anti-ubiquitin (Santa Cruz Biotechnology, clone P4D1, sc-8017), anti–phosphor-p65 Ser 536 [Cell Signaling Technology (CST), clone 93H1, 3033], anti-iβ5 (Abcam, ab3329), anti-iβ1 (Thermo Fisher Scientific, PA11960), anti–phosphor-PKR Thr 446 (Abcam, clone E120, ab32036), anti–phosphor-eIF2α Ser 51 (CST, clone D9G8, 3398), anti-TFAM (CST, clone D5C8, 8076), anti–phosphor-STAT1 Tyr 701 (CST, clone 58D6, 9167), anti–IL-24 (R&D Systems, AF1965, or Abcam, ab115207), anti-PACT (Santa Cruz Biotechnology, clone D-4, sc-377103), anti-TRBP (Santa Cruz Biotechnology, clone D-5, sc-514124), or anti–actin–horseradish peroxidase (HRP) (1:10,000; Santa Cruz Biotechnology, clone C4, sc-47778).

Techniques: Cell Culture, Expressing, Derivative Assay