Journal: Cardiovascular Research

Article Title: Ex vivo Ikkβ ablation rescues the immunopotency of mesenchymal stromal cells from diabetics with advanced atherosclerosis

doi: 10.1093/cvr/cvaa118

Figure Lengend Snippet: Advanced glycolytic end (AGE) products activates IKKβ signalling cascade and reduce Mesenchymal stromal cells (MSCs) immunopotency. (A) Western-blot analysis representative of two independent experiments with similar results showing the activation of the IRAK4–TAK1–IKKβ–p65/NF-κB signalling cascade on healthy MSCs that are exposed to 5, 10, and 15 µg/mL AGE products for 24 h. (B–D) AGE induced an increase in the production of IKKβ–NF-κB-regulated pro-inflammatory cytokine IL-6 (***P = 0.0002, n = 8), and chemokines IL-8/CXCL8 (***P = 0.0006, n = 8), MCP-1/CCL2 (***P = 0.0002, n = 8). (E) Reduced immunosuppressive function of MSCs after AGE products (***P = 0.0002, n = 8). Each dot represents different biological replicates (i.e. single healthy control MSCs). Mann–Whitney U test was used to compare two independent groups. Abbreviations: MSCs, mesenchymal stromal cells; TAK-1, transforming growth factor‐β‐activating kinase1; IKKβ, inhibitor of nuclear factor kappa-B kinase subunit beta; p65, nuclear factor kappa-light-chain-enhancer of activated B cells RelA subunit; IRAK4, interleukin-1 receptor-associated kinase 4; IL-6, interleukin-6; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; PBMCs, peripheric blood mononuclear cells.

Article Snippet: Six different human IKKβ guide RNAs (gRNAs) (see Supplementary material online , Table S1 ) and non-targeting gRNAs were purchased from GenScript (NJ, USA) and sub-cloned in pLentiCRISPR v2 using BsmBI restriction site (Addgene Plasmid No. 52961).

Techniques: Western Blot, Activation Assay, Control, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Ex vivo Ikkβ ablation rescues the immunopotency of mesenchymal stromal cells from diabetics with advanced atherosclerosis

doi: 10.1093/cvr/cvaa118

Figure Lengend Snippet: Constitutively activated forms of inflammation-activated protein kinases and increased pro-inflammatory cytokine secretion in atherosclerosis+T2DM MSCs. Augmented active T-loop phosphorylated forms of (A) IRAK 4 (*P = 0.05, atherosclerosis MSCs n = 5, atherosclerosis+T2DM MSCs n = 6), (B) TAK1 (*P = 0.02, atherosclerosis MSCs n = 10, atherosclerosis+T2DM MSCs n = 12) and (C) IKKβ (****P < 0.000, atherosclerosis MSCs n = 10, atherosclerosis+T2DM MSCs n = 11); (D) Increased phosphorylation of p65 NF-κB transcription factor on Ser536 located in its transactivation domain (*P = 0.05, atherosclerosis MSCs n = 10, atherosclerosis+T2DM MSCs n = 12) and (E–G) Increased production of IKKβ–NF-κB-dependent pro-inflammatory cytokine IL-6 (***P = 0.0002, atherosclerosis MSCs n = 12, atherosclerosis+T2DM MSCs n = 11), and chemokines IL-8/CXCL8 (***P = 0.0006, atherosclerosis MSCs n = 11, atherosclerosis+T2DM MSCs n = 10), MCP-1/CCL2 (***P = 0.0002, atherosclerosis MSCs n = 11, atherosclerosis+T2DM MSCs n = 11). Each dot represents different biological samples (i.e. MSC from different donors). Mann–Whitney U test was used to compare two independent groups. Abbreviations: T2DM, type 2 diabetes mellitus; MSCs, mesenchymal stromal cells; TAK-1, transforming growth factor‐β‐activating kinase1; IKKβ, inhibitor of nuclear factor kappa-B kinase subunit beta; p65, nuclear factor kappa-light-chain-enhancer of activated B cells RelA subunit; IRAK4, interleukin-1 receptor-associated kinase 4; IL-6, interleukin-6; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1.

Article Snippet: Six different human IKKβ guide RNAs (gRNAs) (see Supplementary material online , Table S1 ) and non-targeting gRNAs were purchased from GenScript (NJ, USA) and sub-cloned in pLentiCRISPR v2 using BsmBI restriction site (Addgene Plasmid No. 52961).

Techniques: Phospho-proteomics, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Ex vivo Ikkβ ablation rescues the immunopotency of mesenchymal stromal cells from diabetics with advanced atherosclerosis

doi: 10.1093/cvr/cvaa118

Figure Lengend Snippet: Pharmacological inhibition of IKKβ reduces pro-inflammatory cytokine and chemokine secretion and rescues in vitro immunopotency of atherosclerosis+T2DM MSCs. (A–C) MLN120B treatment reduces the secretion of IL-6 (***P = 0.001, n = 11), IL-8/CXCL8 (**P = 0.003, n = 9), and MCP-1/CCL2(**P = 0.005, n = 10) in atherosclerosis+T2DM MSCs. (D) Representative example of flow cytometry proliferation. Histograms show CFSE dilution following in vitro proliferation of monocyte-depleted peripheral blood mononuclear cells (PBMCs) in co-culture with atherosclerosis+T2DM MSCs with or without the use of the IKKβ inhibitor MLN120B. (E) MLN120B improved the ability of atherosclerosis+T2DM MSCs to suppress the proliferation of activated T‐cells (***P = 0.0005, n = 12) while not affecting (F) CD4+ T cell viability (n = 12). (G and H) Similar immunopotency and CD4+ T cell viability in both cell–cell contact (n = 4) and trans-well conditions (n = 4) in MLN120B-treated atherosclerosis+T2DM MSCs. Each dot represents different biological samples (i.e. MSC from different donors). Mann–Whitney U test was used to compare two independent groups and Wilcoxon test was used for paired comparisons. Abbreviations: T2DM, type 2 diabetes mellitus; MSCs, mesenchymal stromal cells; IL 6, interleukin-6; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; FSC‐A, forward scatter area; SSC‐A, side scatter area; SSC‐H, side scatter height; SSC‐W, side scatter width; 7-AAD, 7-aminoactinomycin D; EI, expansion index; CFSE, carboxyfluorescein succinimidyl ester.

Article Snippet: Six different human IKKβ guide RNAs (gRNAs) (see Supplementary material online , Table S1 ) and non-targeting gRNAs were purchased from GenScript (NJ, USA) and sub-cloned in pLentiCRISPR v2 using BsmBI restriction site (Addgene Plasmid No. 52961).

Techniques: Inhibition, In Vitro, Flow Cytometry, Co-Culture Assay, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Ex vivo Ikkβ ablation rescues the immunopotency of mesenchymal stromal cells from diabetics with advanced atherosclerosis

doi: 10.1093/cvr/cvaa118

Figure Lengend Snippet: IKKβ knockdown in atherosclerosis+T2DM-MSCs enhances their immunoprotective effects in the permanent LAD ligation model. (A) Representative western blots showing the efficacy of two different gRNA molecules targeting the IKBKB locus in one control MSCs left either untreated or exposed to 10 ng/mL TNF-α for 10 min. Note that the KD efficiency was evaluated in all the MSCs samples used in vivo (see Supplementary material online, Figure S11). The effect of reducing the expression level of IKKβ in atherosclerosis+T2DM MSCs was evaluated on their (B) immunopotency in vitro (***P = 0.001, n = 11) and (C) ability to attract PBMCs (*P = 0.02, n = 4). (D) Haematoxylin and Eosin (H&E) staining of the injured (i.e. purple: infiltration of leucocytes) and viable myocardium (i.e. pink) (**P = 0.007, n = 8), (E) CD4+ (*P = 0.01, n = 7), (F) Monocyte/macrophage (MOMA-2 staining) (*P = 0.01, n = 7), and (G) FOXP3 (**P = 0.007, n = 8) content in heart sections and (H) IL-6 plasma levels (*P = 0.01, n = 7) were assessed. Each dot represents different animals injected with six different biological samples (i.e. MSC from different donors). Wilcoxon test used for paired groups Abbreviations: T2DM, type 2 diabetes mellitus; PBMCs, peripheric blood mononuclear cells; MOMA, monoclonal anti-macrophage antibody; IL 6, interleukin-6.

Article Snippet: Six different human IKKβ guide RNAs (gRNAs) (see Supplementary material online , Table S1 ) and non-targeting gRNAs were purchased from GenScript (NJ, USA) and sub-cloned in pLentiCRISPR v2 using BsmBI restriction site (Addgene Plasmid No. 52961).

Techniques: Knockdown, Ligation, Western Blot, Control, In Vivo, Expressing, In Vitro, Staining, Clinical Proteomics, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Papaverine and its derivatives radiosensitize solid tumors by inhibiting mitochondrial metabolism

doi: 10.1073/pnas.1808945115

Figure Lengend Snippet: Papaverine radiosensitizes through complex I inhibition. (A) Western blot of NDI1 expression in mitochondrial fractions of parent A549 and NDUFV1 KO cells. (B) Representative trypan blue viability (n = 3) of cells grown in galactose-only media (T = 96 h). Values represent mean viable cells ± SD. (C) Seahorse analysis of the response of parent A549 and NDUFV1 KO ± NDI1 cells to 10 μM papaverine or 1 μM rotenone. Values are mean ± SD. (D) Quantification of pimonidazole staining in NDUFV1 KO NDI1 flank tumors treated with 2 mg/kg PPV or vehicle (n = 3). Value is mean pimonidazole-positive cells from 20 images per tumor ± SEM. P value was calculated with t test. (E) Quantification of tumor growth delay of heterotopic NDUFV1 KO NDI1 flank xenografts receiving either 8 Gy XRT (magenta) or 2 mg/kg PPV 35 min before 8 Gy XRT (blue) (n = 4). Curves represent mean tumor volumes ± SEM. P values were calculated against XRT with t test. n.s., not significant.

Article Snippet: Three separate human NDUFV1 guide RNAs (gRNAs) were obtained from GenScript (catalog no. SC1678, item no. U5053CH250_1-3).

Techniques: Inhibition, Western Blot, Expressing, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Neuronal-specific TNFAIP1 ablation attenuates postoperative cognitive dysfunction via targeting SNAP25 for K48-linked ubiquitination

doi: 10.1186/s12964-023-01390-z

Figure Lengend Snippet: Identification of TNFAIP1 as an interacting protein with SNAP25. A Network view of E3–SNAP25 interactions (left panel) and the E3 hierarchical tree for SNAP25 (right panel). The predicted E3 ligases surrounding SNAP25 are represented different colors and characters according to their respective ubiquitin ligase domains. The size of the nodes, width of the edges, and shade of the edges indicate the confidence score. The position of the predicted ubiquitin ligases in the E3 family hierarchical tree is displayed. The abbreviations in each circle, such as "BO", "C" and "H", denote the E3 family, and the numbers in parentheses following each ubiquitin ligase family denote the number of corresponding predicted E3-SNAP25 interactions. B Western blot analysis of TNFAIP1 protein levels in the hippocampus of mice undergoing anesthesia and surgery (AS). Quantification results normalized to GAPDH are presented as mean ± SEM ( n = 6). ** P < 0.01, unpaired t -test. C, D Co-IP using anti-Flag or anti-Myc antibody confirmed the interaction between exogenous TNFAIP1 and SNAP25 in HEK293T cells transfected with plasmids expressing Flag-TNFAIP1 and Myc-SNAP25. E Co-IP with an anti-TNFAIP1 antibody in SH-SY5Y cells confirmed the interaction between endogenous TNFAIP1 and SNAP25. F Co-IP with an anti-SNAP25 antibody in HT22 cells confirmed the interaction between endogenous TNFAIP1 and SNAP25. G TNFAIP1 directly interacted with SNAP25 in vitro. Purified GST (control) or GST-TNFAIP1 were mixed with His-SNAP25 and subjected to GST pull-down followed by IB for His. H Diagram illustrating the full-length and different truncations of TNFAIP1. Plus indicates binding, minus indicates no binding, and aa indicates amino acids. I Mapping of interacting domains of TNFAIP1 and SNAP25 by Co-IP with an anti-Flag antibody, followed by IB with anti-Flag or anti-Myc antibody

Article Snippet: Small interfering RNAs (siRNAs) targeting human and mouse TNFAIP1 were synthesized by RiboBio (China) with the sequences 5'-GGUUGGGCAACAAGUAUGUTT-3' and 5'-CGTCGCATTCATGTCAAGCGCTATA-3', respectively.

Techniques: Ubiquitin Proteomics, Western Blot, Co-Immunoprecipitation Assay, Transfection, Expressing, In Vitro, Purification, Control, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Neuronal-specific TNFAIP1 ablation attenuates postoperative cognitive dysfunction via targeting SNAP25 for K48-linked ubiquitination

doi: 10.1186/s12964-023-01390-z

Figure Lengend Snippet: TNFAIP1 catalyzes K48-linked polyubiquitination of SNAP25 at K69. A Ubiquitination analysis of SNAP25 in lysates from HEK293T cells transfected with Myc-SNAP25, HA-Ub, and full-length Flag-tagged TNFAIP1 or its truncations. B Co-transfection of Flag-TNFAIP1 with Myc-SNAP25 and WT or mutant HA-tagged ubiquitin in HEK293T cells followed by ubiquitination assays of exogenous SNAP25. C Diagram illustrating the predicted ubiquitination sites of SNAP25 (colored in pink, top), and ubiquitination assays of SNAP25 in lysates from HEK293T cells transfected with Flag-TNFAIP1, HA-Ub, and WT or mutant Myc-tagged SNAP25 (bottom). D Western blot analysis of SNAP25 protein levels in HT22 cells treated with MG132 (50 nM, 4 h) and Flag-TNFAIP1. E Quantification results normalized to GAPDH are presented as mean ± SEM ( n = 4). * P < 0.05, ** P < 0.01, one-way ANOVA test. F Immunoblot analysis of SNAP25 in HT22 cells transfected with Flag-TNFAIP1 in the absence or presence of CHX (10 μg/mL). G The relative SNAP25 level for each time point was quantified as mean ± SEM ( n = 3). ** P < 0.01, two-way ANOVA test. H HT22 cells transiently transfected with full-length Flag-tagged TNFAIP1 or its truncations were treated with 10 μg/mL CHX for 0, 3, and 6 h. (I) Quantification of SNAP25 levels relative to GAPDH was shown as mean ± SEM ( n = 3). ** P < 0.01, 97–216 vs. FL; # P < 0.05, ## P < 0.01, 207–316 vs. FL, two-way ANOVA test

Article Snippet: Small interfering RNAs (siRNAs) targeting human and mouse TNFAIP1 were synthesized by RiboBio (China) with the sequences 5'-GGUUGGGCAACAAGUAUGUTT-3' and 5'-CGTCGCATTCATGTCAAGCGCTATA-3', respectively.

Techniques: Ubiquitin Proteomics, Transfection, Cotransfection, Mutagenesis, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Neuronal-specific TNFAIP1 ablation attenuates postoperative cognitive dysfunction via targeting SNAP25 for K48-linked ubiquitination

doi: 10.1186/s12964-023-01390-z

Figure Lengend Snippet: TNFAIP1 affects mitophagy and pyroptosis in vitro. A SH-SY5Y cells expressing control siRNA or siTNFAIP1 were treated with 3% isoflurane (30 min) and 1 μg/mL LPS (12 h), followed by incubation with 10 mM DCFH-DA for 20 min. The mean fluorescence intensity (MFI) of DCFH-DA was measured using flow cytometry to reflect the intracellular ROS level ( n = 3). ** P < 0.01, one-way ANOVA test. B Flow cytometry analysis of cell death in SH-SY5Y cells using Annexin V-FITC/PI double-staining. C Quantification of pyroptotic cells with annexin V-FITC/PI double-positive staining ( n = 3). * P < 0.05, ** P < 0.01, one-way ANOVA test. D Gel images of mitophagy- and pyroptosis-related proteins in TNFAIP1-silenced SH-SY5Y cells. GAPDH served as a loading control. E HT22 cells expressing empty vector or TNFAIP1 were exposed to 3% isoflurane (30 min) and 1 μg/mL LPS (12 h), followed by incubation with 10 mM DCFH-DA for 20 min. The intracellular ROS level was measured by the MFI of DCFH-DA ( n = 3). ** P < 0.01, one-way ANOVA test. F Flow cytometry analysis of double staining with Annexin V-FITC/PI in HT22 cells overexpressing TNFAIP1 after Iso + LPS treatment. G Quantification of pyroptotic cells with annexin V-FITC/PI double-positive staining ( n = 3). ** P < 0.01, one-way ANOVA test. H Gel images of mitophagy- and pyroptosis-related proteins in TNFAIP1-overexpressed HT22 cells. GAPDH served as a loading control

Article Snippet: Small interfering RNAs (siRNAs) targeting human and mouse TNFAIP1 were synthesized by RiboBio (China) with the sequences 5'-GGUUGGGCAACAAGUAUGUTT-3' and 5'-CGTCGCATTCATGTCAAGCGCTATA-3', respectively.

Techniques: In Vitro, Expressing, Control, Incubation, Fluorescence, Flow Cytometry, Double Staining, Staining, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Neuronal-specific TNFAIP1 ablation attenuates postoperative cognitive dysfunction via targeting SNAP25 for K48-linked ubiquitination

doi: 10.1186/s12964-023-01390-z

Figure Lengend Snippet: SNAP25 overexpression rescues the defective mitophagy and excessive pyroptosis phenotypes induced by TNFAIP1 overexpression. A HT22 cells were transfected with indicated plasmids and treated with 3% isoflurane (30 min) and 1 μg/mL LPS (12 h), followed by incubation with 10 mM DCFH-DA for 20 min. The intracellular ROS level was reflected by the MFI of DCFH-DA ( n = 3). ** P < 0.01, one-way ANOVA test. B Flow cytometry analysis of double staining with Annexin V-FITC/PI under Iso + LPS conditions in HT22 cells overexpressing indicated plasmids. C Quantification of pyroptotic cells with annexin V-FITC/PI double-positive staining ( n = 3). ** P < 0.01, one-way ANOVA test. D Gel images of mitophagy- and pyroptosis-related proteins in Iso + LPS-treated HT22 cells transfected with indicated plasmids. GAPDH served as a loading control. E Quantification results normalized to GAPDH were presented as mean ± SEM ( n = 4). * P < 0.05, ** P < 0.01, one-way ANOVA test

Article Snippet: Small interfering RNAs (siRNAs) targeting human and mouse TNFAIP1 were synthesized by RiboBio (China) with the sequences 5'-GGUUGGGCAACAAGUAUGUTT-3' and 5'-CGTCGCATTCATGTCAAGCGCTATA-3', respectively.

Techniques: Over Expression, Transfection, Incubation, Flow Cytometry, Double Staining, Staining, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Neuronal-specific TNFAIP1 ablation attenuates postoperative cognitive dysfunction via targeting SNAP25 for K48-linked ubiquitination

doi: 10.1186/s12964-023-01390-z

Figure Lengend Snippet: TNFAIP1 knockdown ameliorates postoperative cognitive impairment, which is abrogated by SNAP25 knockdown. A Experimental design. Two weeks after AAV9 injection, mice were subjected to laparotomy under isoflurane anesthesia. The OFT and FCT were performed from the third to fifth day. The hippocampus of different groups was harvested after behavioral assessment. The total moving distance ( B ) time spent in the center region ( C ) in the OFT. D Freezing response of mice was assessed by contextual fear conditioning as the proportion of immobility ( n = 12). ** P < 0.01, one-way ANOVA test. E H&E (400 × , bar: 50 μm) and TUNEL staining (400 × , bar: 50 μm) of hippocampal CA1 samples following the indicated treatments. TUNEL-positive cells are shown in red, and nuclei are shown in blue. F The Percentage of TUNEL-positive cells in the hippocampi of mice ( n = 3). ** P < 0.01, one-way ANOVA test. G Gel images of mitophagy- and pyroptosis-related proteins in the hippocampi microinjected with the indicated viruses. GAPDH served as a loading control (left panel). Quantification results normalized to GAPDH were represented as mean ± SEM (right panel, n = 4). * P < 0.05, ** P < 0.01, one-way ANOVA test. The concentration of IL-1β and IL-18 was measured by ELISA in the serum ( H-I ) and hippocampi ( J-K ) of mice following the indicated treatments ( n = 4). * P < 0.05, ** P < 0.01, one-way ANOVA test

Article Snippet: Small interfering RNAs (siRNAs) targeting human and mouse TNFAIP1 were synthesized by RiboBio (China) with the sequences 5'-GGUUGGGCAACAAGUAUGUTT-3' and 5'-CGTCGCATTCATGTCAAGCGCTATA-3', respectively.

Techniques: Knockdown, Injection, TUNEL Assay, Staining, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay