Journal: Autophagy

Article Title: N-3 PUFAs induce inflammatory tolerance by formation of KEAP1-containing SQSTM1/p62-bodies and activation of NFE2L2

doi: 10.1080/15548627.2017.1345411

Figure Lengend Snippet: The n-3 PUFA DHA strongly reduced expression of CXCL10 by affecting several signaling pathways. (A) MDMs were treated with DHA (70 µM) for 16 h before stimulation with LPS (100 ng/ml) for 3 h. Transcript levels of 579 genes (nCounter GX Human Immunology kit assay) were determined. Transcripts that were more than 1.5-times increased by LPS in both donors were selected and the fold change between vehicle- and DHA-treated cells calculated, mean ± SD, n = 2. (B) QRT-PCR analysis of CXCL10 and (C) TNF in MDM treated with DHA, OA or AA for 16 h and LPS for 3 h, n = 5 (Friedman test with Dunn's). (D) IB analysis and quantification of IRF3 and RELA phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 h, n = 5. (Friedman test with Dunn's), of (E) TBK1, IKBKB, STAT1 and STAT3 phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test, one-tailed) and of (F) IRF1 and RELA in cytosolic (Cyt) and nuclear (Nuc) fractions in MDMs treated with DHA, OA or AA for 16 h and LPS for 2 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test). (G) IFNB1 mRNA levels after incubation with DHA for 16 h and LPS for 2 h, n = 6 (Wilcoxon matched-pairs signed rank test).

Article Snippet: The following were from Cell Signaling Technology: IKBKB/IKKβ (8943); IRF1 (4302); IRF3 (4302S), MAP1LC3B (3868); MAPK1/3/p44/42 (9107); RELA/p65 (3034); phospho-CHUK/IKBKB/IKKα/β (S176/180; 2697); phospho-IRF3 (S396; 4302); phospho-NFKB2/p105 (S933; 4806); phospho-RELA/p65 (S536; 3033); phospho-STAT1 (Y701; 7649); phospho-STAT3 (Y705; 9145); phospho-TBK1 (S172; 5483); STAT1 (14994); STAT3 (9139); TAX1BP1 (5105); TBK1 (3013).

Techniques: Expressing, Quantitative RT-PCR, One-tailed Test, Incubation

Journal: Oncoimmunology

Article Title: Bufalin Suppresses Migration and Invasion of Hepatocellular Carcinoma Cells Elicited by Poly (I:C) Therapy

doi: 10.1080/2162402X.2018.1426434

Figure Lengend Snippet: Bufalin inhibits poly (I:C)-triggered activation of TBK1-IRF3 pathway in HCC cells. (A to E) MHCC97 H cells were treated with poly (I:C) (5 μg/ml), bufalin (5 nM) or vehicles (DMSO, 2hrs) as indicated. The amounts of indicated proteins contained in 100 μg whole cell extracts (WCL) were determined by ELISA assays. In (E), equal amounts of WCL or nuclear extracts were examined for indicated proteins by using Western blotting. (F and G) MHCC97 H cells were transiently transfected with Ifnb (F) or NF-κB (G) reporters for 24 H, and treated with poly (I:C) (5 μg/ml), bufalin (5 nM) or vehicles for 6 H. Reporter activation was measured by dual-luciferase activity assays. In (A to D), (F) and (G), data are means ± SD of triplicates. Data are representative of three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA followed by Bonferroni multiple comparisons).

Article Snippet: Abs specific for IRF3 (#4302), Myc-tag, p65 (L8F6, #6956), phospho-IkBa (Ser32/Ser36) (5A5, #9246), TBK1 (#3013), phospho-IRF3 (Ser396) (4D4G, #4947), and phospho-TBK1 (Ser172) (#5483) were from Cell Signaling Technology (Beverly, MA). β-actin antibody, poly (I:C), bufalin and other non-specified reagents were purchased from Sigma (St. Louis, MO).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Luciferase, Activity Assay

Journal: Oncoimmunology

Article Title: Bufalin Suppresses Migration and Invasion of Hepatocellular Carcinoma Cells Elicited by Poly (I:C) Therapy

doi: 10.1080/2162402X.2018.1426434

Figure Lengend Snippet: Roles of TBK1 in poly (I:C)– and bufalin–regulated migration and invasion of HCC cells. (A to D) MHCC97 H (A and B) or HepG2-TLR3 (C and D) cells were pretreated with or without TPCA-1 (100 nM) or BX-795 (100 nM) for 30 min, and then treated with poly (I:C) (5 μg/ml), bufalin (5 nM) or vehicles as indicated for 48 H. Migrated cells (A and C) and invaded cells (B and D) were counted (cells of one filed out of 5 fields of membrane under 200 x magnification). (E to J) Tbk1 +/+ or Tbk1 –/– MHCC97 H cells (E to G) or Tbk1 –/– MHCC97 H cells rescued by transfection with empty (Mock) or TBK1 expression vectors for 24 H (H to J) were treated with poly (I:C) (5 μg/ml), bufalin (5 nM) or vehicles as indicated for 48 H, and were examined for migration and invasion as in (A to D). Expression of TBK1 was examined by Western blotting using anti-TBK1 antibody (E and H). In (A to D), (F), (G), (I) and (J), data are means ± SD of triplicates. Data are representative of three independent experiments. ns, not significant; ***, P < 0.001 (one-way ANOVA followed by Bonferroni multiple comparisons).

Article Snippet: Abs specific for IRF3 (#4302), Myc-tag, p65 (L8F6, #6956), phospho-IkBa (Ser32/Ser36) (5A5, #9246), TBK1 (#3013), phospho-IRF3 (Ser396) (4D4G, #4947), and phospho-TBK1 (Ser172) (#5483) were from Cell Signaling Technology (Beverly, MA). β-actin antibody, poly (I:C), bufalin and other non-specified reagents were purchased from Sigma (St. Louis, MO).

Techniques: Migration, Transfection, Expressing, Western Blot

Journal: Oncoimmunology

Article Title: Bufalin Suppresses Migration and Invasion of Hepatocellular Carcinoma Cells Elicited by Poly (I:C) Therapy

doi: 10.1080/2162402X.2018.1426434

Figure Lengend Snippet: Bufalin inhibits the activation of TBK1 and suppresses migration and invasion of HCC cells triggered by poly (I:C) transfection. (A to E) MHCC97 H (A to C) or HepG2 (D and E) cells were transfected with poly (I:C) (5 μg/ml), or treated with bufalin (5 nM) or vehicles as indicated for 1–2 H (A) or 48 H (B to E). The amounts of indicated proteins in equal amounts of whole cell extracts or nuclear extracts were examined by Western blotting (A). Migrated cells (B and D) and invaded cells (C and E) were counted (cells of one filed out of 5 fields of membrane under 200 x magnification). (F and G) Tbk1 +/+ or Tbk1 –/– MHCC97 H cells were were transfected with poly (I:C) (5 μg/ml), or treated with bufalin (5 nM) or vehicles as indicated for 48 H. Migration and invasion were determined as in (B to E). In (B to G), data are means ± SD of triplicates. Data are representative of three independent experiments. ns, not significant; ***, P < 0.001 (one-way ANOVA followed by Bonferroni multiple comparisons).

Article Snippet: Abs specific for IRF3 (#4302), Myc-tag, p65 (L8F6, #6956), phospho-IkBa (Ser32/Ser36) (5A5, #9246), TBK1 (#3013), phospho-IRF3 (Ser396) (4D4G, #4947), and phospho-TBK1 (Ser172) (#5483) were from Cell Signaling Technology (Beverly, MA). β-actin antibody, poly (I:C), bufalin and other non-specified reagents were purchased from Sigma (St. Louis, MO).

Techniques: Activation Assay, Migration, Transfection, Western Blot

Journal: Oncoimmunology

Article Title: Bufalin Suppresses Migration and Invasion of Hepatocellular Carcinoma Cells Elicited by Poly (I:C) Therapy

doi: 10.1080/2162402X.2018.1426434

Figure Lengend Snippet: Bufalin inhibits poly (I:C)-inspired metastasis of HCC cells in vivo. (A and B) Balb/c nu/nu mice with MHCC97 H orthotopical xenografts were treated by intraperitoneal injection of indicated amounts of poly (I:C) (0.5, 1 or 2 mg/kg), bufalin (0.5, 1 or 2 mg/kg) or vehicles as indicated. For the combination treatments, bufalin was administrated at 0.5 mg/kg. On the third day after the second poly (I:C) treatment, liver were examined for apoptosis by TUNEL assays. The section stained with only the HRP-conjugated secondary antibody was used as negative control. The sections were photographed under 400 x magnification, and TUNEL positive cells under each field of 5 fields were counted. (C to F) After six weeks of treatment (0.5 mg/kg poly (I:C) or 0.5 mg/kg bufalin), the lungs were excised and examined for metastases by H&E staining. Typical metastases were shown in (C; 200 x magnification), and the metastases under each field of 5 fields were counted. The orthotopical xenografts examined were: MHCC97 H (C and D), HepG2-TLR3 (E) and Tbk1 +/+ or Tbk1 –/– MHCC97 H (F). In (B), (D), (E) and (F), data are means ± SD (n = 10 per group). In (A) and (C), bars indicate for 50 μm. Data are representative of three independent experiments. ns, not significant; ***, P < 0.001 (one-way ANOVA followed by Bonferroni multiple comparisons).

Article Snippet: Abs specific for IRF3 (#4302), Myc-tag, p65 (L8F6, #6956), phospho-IkBa (Ser32/Ser36) (5A5, #9246), TBK1 (#3013), phospho-IRF3 (Ser396) (4D4G, #4947), and phospho-TBK1 (Ser172) (#5483) were from Cell Signaling Technology (Beverly, MA). β-actin antibody, poly (I:C), bufalin and other non-specified reagents were purchased from Sigma (St. Louis, MO).

Techniques: In Vivo, Injection, TUNEL Assay, Staining, Negative Control

Journal: Oncoimmunology

Article Title: Bufalin Suppresses Migration and Invasion of Hepatocellular Carcinoma Cells Elicited by Poly (I:C) Therapy

doi: 10.1080/2162402X.2018.1426434

Figure Lengend Snippet: Bufalin inhibits poly (I:C)-induced phosphorylation of TBK1 in vivo. Balb/c nu/nu mice with MHCC97 H (A and B) or HepG2-TLR3 (C and D) orthotopical xenografts were treated by intraperitoneal injection of poly (I:C) (0.5 mg/kg), bufalin (0.5 mg/kg) or vehicles as indicated. On the third day after the second poly (I:C) treatment, liver were examined for phospho-TBK1 expression by immunohistochemistry (IHC) assays. The sections were photographed under 400 x magnification (A and C), and the staining was semiquantitatively scored. The section derived from poly (I:C) group and stained in the presence of blocking peptide was used as negative control. In (B) and (D), data are means ± SD (n = 10 per group). In (A) and (C), bars indicate for 50 μm. Data are representative of three independent experiments. ***, P < 0.001 (one-way ANOVA followed by Bonferroni multiple comparisons).

Article Snippet: Abs specific for IRF3 (#4302), Myc-tag, p65 (L8F6, #6956), phospho-IkBa (Ser32/Ser36) (5A5, #9246), TBK1 (#3013), phospho-IRF3 (Ser396) (4D4G, #4947), and phospho-TBK1 (Ser172) (#5483) were from Cell Signaling Technology (Beverly, MA). β-actin antibody, poly (I:C), bufalin and other non-specified reagents were purchased from Sigma (St. Louis, MO).

Techniques: In Vivo, Injection, Expressing, Immunohistochemistry, Staining, Derivative Assay, Blocking Assay, Negative Control

Journal: Theranostics

Article Title: Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes

doi: 10.7150/thno.54695

Figure Lengend Snippet: cGAS-STING pathway was activated by arginine starvation. (A) Cells overexpressing cGAS-V5 were deprived for 72 h and stained with anti-V5 antibodies. Scale bars, 10 μm. (B) CWR22Rv1 cells were deprived for indicate timepoints, and endogenous IRF3 was immunoprecipitated. IRF3 phosphorylation and TBK1 association were determined by immunoblots. Fold changes are listed below each blot. (C) IRF3 phosphorylation level was quantified and normalized with the IRF3. (n = 3, **p < 0.01). (D) RT-qPCR analysis of type I interferon expression in CWR22Rv1 cells starved for 72 h. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (E) IFNβ secretion by CWR22Rv1 cells after arginine starvation. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) (F) IFNβ promoter activity in CWR22Rv1 cells deprived for 72 h. (n = 3, *p < 0.05)

Article Snippet: Cell lysates were resuspended in 4× sample buffer (200 mM Tris, pH 6.8; 8% SDS; 40% glycerol; 0.4% bromophenol blue and 20% 2-mercaptoethanol) and heated at 95 ℃ for 5 min. Antibodies used were OPA1 (67589), DRP1 (8570), Mitofusin-1 (14793), Mitofusin-2 (11925), H2AX (2595), γH2AX (2577), H3 (9715), H3K4me1 (5326), H3K4me3 (9751), IRF3 (11904), p-IRF3 (S396) (29047), TBK1 (3504), p-TBK1 (S172) (5483), p65 (8242) and p-p65 (S536) (3033) from Cell Signaling; H3K4me2 (39679), H3K9me1 (39887), H3K9me2 (39683), H3K9me3 (39765), H3K27me1 (61015), H3K27me2 (39919), H3K27me3 (39155), H3K36me1 (61351), H3K36me2 (39255), H3K36me3 (61101) from Active Motif; α tubulin (sc-23948) from Santa Cruz; Lamin A/C (GTX101127) from GeneTex. α tubulin was in 1:20000 dilution.

Techniques: Staining, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Expressing, Activity Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: USP25 Deficiency Exacerbates Acute Pancreatitis via Up-Regulating TBK1–NF-κB Signaling in Macrophages

doi: 10.1016/j.jcmgh.2022.07.013

Figure Lengend Snippet: BMDMs from Usp25 -/- mice show enhanced inflammatory responses. ( A ) Schematic illustration of the experimental model. Acinar cells were isolated from mice and cultured for 0 or 24 hours. ACS was collected and applied to BMDMs harvested from WT and Usp25 -/- mice. BMDM responses then were measured. ( B ) BMDMs from WT and Usp25 -/- mice were exposed to ACS for 60 minutes. Cell lysates were probed for downstream signaling proteins by immunoblotting. ( C–G ) Messenger RNA levels of inflammatory factors in BMDMs from WT and Usp25 -/- mice that were exposed to ACS for 9 hours. ( H ) BMDMs were exposed to ACS for 18 hours, and IFNβ levels in culture medium were determined. ( C–H ) Data are shown as means ± SD; n = 3 independent experiments. ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-IRF3, phosphorylated interferon regulatory factor 3; p-P65, phosphorylated P65 protein; p-TBK1, phosphorylated TANK binding kinase 1; PCR, polymerase chain reaction.

Article Snippet: Antibodies against STING (13647), phosphorylated (p)-TBK1 (Ser172; 5483), TBK1 (3013), p–IRF-3 (Ser396; 4947), IRF-3 (4302), p–NF-κB p65 (Ser536; 3033), NF-κB p65 (8242), NF-kappa-B inhibitor alpha (4812), p-stress-activated protein kinase/JNK (Thr183/Tyr185; 4668), SAPK/JNK (9252), p-Erk1/2 (Thr202/Tyr204; 4370), Erk1/2 (4695), p-p38 (Thr180/Tyr182; 9211), p38 (8690), and glyceraldehyde-3-phosphate dehydrogenase (5174) were obtained from Cell Signaling Technology (Pudong, Shanghai, China).

Techniques: Isolation, Cell Culture, Western Blot, Binding Assay, Polymerase Chain Reaction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: USP25 Deficiency Exacerbates Acute Pancreatitis via Up-Regulating TBK1–NF-κB Signaling in Macrophages

doi: 10.1016/j.jcmgh.2022.07.013

Figure Lengend Snippet: Expression of USP25 in Usp25 -/- BMDMs reverse ACS-induced inflammatory responses in cells and STING agonist activates downstream signaling in both WT and Usp25 -deficient macrophages. Usp25 -/- BMDMs were transfected with control plasmid or plasmid expressing Flag-tagged Usp25 (NM_013918; Genechem). Acinar cells were isolated from WT mice and cultured for 0 or 24 hours. ACS collected from 0 or 24 hours was applied to control- and Usp25 -plasmid–transfected BMDMs. ( A ) ACS exposure of BMDMs was performed for 60 minutes. Lysates then were probed for activation of downstream inflammatory response proteins. Antibody against Flag was used to detect USP25 expression. ( B ) BMDMs were prepared from WT and Usp25 -/- mice. Cells were treated with 40 μg/mL STING agonist DMXAA or vehicle for 1 hour. Lysates then were probed for activation of downstream inflammatory response proteins. Total proteins and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as control. ( C–G ) ACS exposure was called out for 9 hours. Messenger RNA levels of inflammatory factors then were measured by quantitative polymerase chain reaction. ( H–L ) BMDMs were treated with DMXAA for 9 hours. Messenger RNA levels of inflammatory factors then were measured. ( C–L ) Data are shown as means ± SD; n = 3. ∗ P < .05, ∗∗ P < .01 and ∗∗∗ P < .001. Ctrl, control; p-IRF3, phosphorylated interferon regulatory factor 3; p-P65, phosphorylated P65 protein; p-TBK1, phosphorylated TANK binding kinase 1.

Article Snippet: Antibodies against STING (13647), phosphorylated (p)-TBK1 (Ser172; 5483), TBK1 (3013), p–IRF-3 (Ser396; 4947), IRF-3 (4302), p–NF-κB p65 (Ser536; 3033), NF-κB p65 (8242), NF-kappa-B inhibitor alpha (4812), p-stress-activated protein kinase/JNK (Thr183/Tyr185; 4668), SAPK/JNK (9252), p-Erk1/2 (Thr202/Tyr204; 4370), Erk1/2 (4695), p-p38 (Thr180/Tyr182; 9211), p38 (8690), and glyceraldehyde-3-phosphate dehydrogenase (5174) were obtained from Cell Signaling Technology (Pudong, Shanghai, China).

Techniques: Expressing, Transfection, Plasmid Preparation, Isolation, Cell Culture, Activation Assay, Real-time Polymerase Chain Reaction, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: USP25 Deficiency Exacerbates Acute Pancreatitis via Up-Regulating TBK1–NF-κB Signaling in Macrophages

doi: 10.1016/j.jcmgh.2022.07.013

Figure Lengend Snippet: BMDMs deficient in Usp25 show enhanced inflammatory responses to L-arginine. ( A ) Bone marrow of WT mice was irradiated and reconstituted with bone marrow cells from either WT mice (WT→WT) or Usp25 -/- mice (KO→WT). Mice then were challenged with L-arginine to induce pancreatitis. Pancreatic lysates were probed for inflammatory pathway activation by immunoblotting. ( B ) WT and Usp25 -/- mice were injected with saline or L-arginine. Pancreatic lysates were used for immunoblotting. ( C ) Messenger RNA levels of Ccl4 , Ccl5 , Cxcl10 , and Isg15 in the pancreas of mice. ( D ) Serum IFNβ levels were measured by ELISA. ( E ) Messenger RNA levels of Ccl4 , Ccl5 , Cxcl10 , and Isg15 in the pancreas of WT and Usp25 -/- mice. ( F ) Serum IFNβ levels in mice. ( C–F ) Data are shown as means ± SD; n = 5–6. ∗ P < .05 and ∗∗ P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-IRF3, phosphorylated interferon regulatory factor 3; p-P65, phosphorylated P65 protein; p-TBK1, phosphorylated TANK binding kinase 1.

Article Snippet: Antibodies against STING (13647), phosphorylated (p)-TBK1 (Ser172; 5483), TBK1 (3013), p–IRF-3 (Ser396; 4947), IRF-3 (4302), p–NF-κB p65 (Ser536; 3033), NF-κB p65 (8242), NF-kappa-B inhibitor alpha (4812), p-stress-activated protein kinase/JNK (Thr183/Tyr185; 4668), SAPK/JNK (9252), p-Erk1/2 (Thr202/Tyr204; 4370), Erk1/2 (4695), p-p38 (Thr180/Tyr182; 9211), p38 (8690), and glyceraldehyde-3-phosphate dehydrogenase (5174) were obtained from Cell Signaling Technology (Pudong, Shanghai, China).

Techniques: Irradiation, Activation Assay, Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: PLoS ONE

Article Title: SHARPIN Is Essential for Cytokine Production, NF-κB Signaling, and Induction of Th1 Differentiation by Dendritic Cells

doi: 10.1371/journal.pone.0031809

Figure Lengend Snippet: WT and cpdm BMDC (2×10 6 cells in 0.5 mL complete medium) were stimulated with 100 ng/mL LPS (A) or 25 µg/mL poly I:C (B). At 0, 15, 30, and 60 minutes, whole-cell lysates were obtained and subject to immunoblots with antibodies against proteins involved in NF-κB, TBK1/IRF3, ERK1/2, and p38 signaling pathways. Beta-actin was used as loading control. (C) Cellular levels of p-IKK1/2 and p-IκBα in LPS- or poly I:C-stimulated BMDC were quantitated with ImageJ (NIH) and presented as trend lines. are representative of at least two independent experiments.

Article Snippet: Immunoblots were performed with antibodies (Cell Signaling Technology) against p-IKK1/2(#2697), p-IκBα (#9246), IκBα (#4814), p-TBK1(#5483), p-p38 (#9216), p38 (#9212), p-ERK1/2 (#4376), and ERK1/2 (#4695).

Techniques: Western Blot