Journal: BMC Biology

Article Title: 20-Hydroxyecdysone counteracts insulin to promote programmed cell death by modifying phosphoglycerate kinase 1

doi: 10.1186/s12915-023-01621-2

Figure Lengend Snippet: PGK1 knockdown at the wandering stage delays metamorphosis. A Morphology, HE staining, and caspase-3 location in the fat body after dsGFP and dsPGK1 knockdown. PGK1 knockdown (500 ng/larva administered in the sixth-instar at 72 h, three times over 24 h intervals). Images were obtained after the first RNAi exposure for 60 h. dsGFP was used as a negative control. Rabbit polyclonal antibodies against caspase-3 were used as the primary antibody. Green fluorescence indicates caspase-3 presence. Nuclei were stained with DAPI (blue). A’ Quantification of fluorescent caspase-3 in A . ** p < 0.01, n = 3. B TEM analysis of the fat body from A . LD indicates lipid droplets. Red arrows indicate autolysosomes or autophagosomes. Blue arrows represent the apoptotic nuclei. B’ Quantification of autophagosomes, autolysosomes, and apoptotic nucleus in B . * p < 0.05, n = 3. C and C’ The levels of ATG8-II and cleaved-caspase-3 in the fat body based on A . Western blot detection using anti-ATG8 and anti-caspase-3 antibodies. * p < 0.05, n = 3. D and D’ PGK1 acetylation levels in dsGFP and dsPGK1 fat body from A . PGK1 proteins from the fat body were purified by CNBr-activated Sepharose 4B with anti-PGK1 monoclonal antibody. * p < 0.05, n = 3. E Interference efficiency analysis of PGK1 at protein levels from the input of D . * p < 0.05, n = 3. F The Vmax of 3-PG production was determined from A in vitro. PGK1 proteins from the fat body were purified by CNBr-activated Sepharose 4B with the anti-PGK1 monoclonal antibody. n = 3. G and H Concentrations of glucose ( G ) and lactate ( H ) in H. armigera hemolymph from A . n = 3. I Phenotypes after PGK1 knockdown as A (500 ng/larva administered in sixth-instar at 72 h, three times over 24 h intervals). Images were obtained after the first RNAi exposure for 80 h. I’ Percentages of phenotypes in I based on three repeats (thirty larvae for each repeat). * p < 0.05, ** p < 0.01, n = 3. J The pupation time from 6th-6 h to pupa 0 d after RNAi exposure in I . * p < 0.05, n = 3. K The average body weight of pupa after RNAi exposure in I . n = 3. Bars indicate means ± SD for three independent experiments with five larvae per replicate. Statistical analyses were performed using two-tailed Student’s t tests (*: p < 0.05 and **: p < 0.01)

Article Snippet: Antibodies were commercially purchased including mouse monoclonal antibody anti-PGK1 (Santa Cruz Biotechnology, Cat. sc-130335; RRID: AB_627677), mouse monoclonal antibody anti-phosphate-tyrosine (anti-pY; Santa Cruz Biotechnology, Cat. sc-7020; RRID: AB_628123), rabbit polyclonal antibodies anti-acetylated lysine (anti-Ac; Cell Signaling Technology, Cat. 9441), rabbit monoclonal antibody anti-β-actin (ABclonal, Cat. AC026), mouse anti-GFP-Tag mAb (ABclonal, Cat. AE012), mouse anti-His-Tag mAb (ABclonal, Cat. AE003), mouse control IgG (ABclonal, Cat. AC011), and alkaline phosphatase-labeled goat anti-rabbit or horse anti-mouse IgG secondary antibodies (ZSGB-BIO, Cat. ZB-2308; ZB-2310).

Techniques: Staining, Negative Control, Fluorescence, Western Blot, Purification, In Vitro, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: SIRT3 alleviates imiquimod-induced psoriatic dermatitis through deacetylation of XBP1s and modulation of TLR7/8 inducing IL-23 production in macrophages

doi: 10.3389/fimmu.2023.1128543

Figure Lengend Snippet: SIRT3 interacted with XBP1s and regulated XBP1s level through deacetylation activity. (A) Fluorescence analysis of SIRT3 and XBP1s in BMDMs stimulated by IMQ for 12 hours. (B) HEK293T cells were transfected with indicated plasmids, and the interactions between SIRT3 and XBP1s were analyzed. (C) The deacetylation effect of SIRT3 on XBP1s was analyzed in HEK293T. (D) BMDMs pretreated with or without 50μM 3-TYP for 1 hour were stimulated with 20μM IMQ for 0, 3, 6, and 9 hours and total cell lysates were immunoprecipitated with anti-XBP1s antibodies and immunoblotted with anti-Ac-k antibodies. Values are expressed as mean ± SEM. **: P<0.01. IMQ, imiquimod; Ac-K, Acetylated-Lysine; HEK293T, Human embryonic kidney cell line HEK293T; IP, immunoprecipitation.

Article Snippet: Antibodies against XBP1s and Ac-K(Acetylated-Lysine) were obtained from Cell Signaling Technology (Danvers, MA, USA), and anti-SIRT3 antibodies were obtained from Proteintech (Wuhan, China) and Everest Biotech (Oxfordshire, UK).

Techniques: Activity Assay, Fluorescence, Transfection, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: SIRT3 alleviates imiquimod-induced psoriatic dermatitis through deacetylation of XBP1s and modulation of TLR7/8 inducing IL-23 production in macrophages

doi: 10.3389/fimmu.2023.1128543

Figure Lengend Snippet: Deletion of Sirt3 exacerbated increased XBP1s acetylation and transcription of downstream cytokines. (A) BMDMs derived from WT and Sirt3 -/- mice were stimulated with 20μM IMQ for 0, 3, 6, and 9 hours and total cell lysates were immunoprecipitated with anti-XBP1s antibodies; the overall XBP1s levels and its acetylation levels were examined by western blots. (B) mRNA levels of inflammatory cytokines were determined by qRT-PCR. Values are expressed as mean ± SEM. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001. IMQ, imiquimod; IP, immunoprecipitation; Ac-K, Acetylated-Lysine. WT, wild type; KO, Sirt3 knockout.

Article Snippet: Antibodies against XBP1s and Ac-K(Acetylated-Lysine) were obtained from Cell Signaling Technology (Danvers, MA, USA), and anti-SIRT3 antibodies were obtained from Proteintech (Wuhan, China) and Everest Biotech (Oxfordshire, UK).

Techniques: Derivative Assay, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Knock-Out

Journal: Frontiers in Immunology

Article Title: SIRT3 alleviates imiquimod-induced psoriatic dermatitis through deacetylation of XBP1s and modulation of TLR7/8 inducing IL-23 production in macrophages

doi: 10.3389/fimmu.2023.1128543

Figure Lengend Snippet: SIRT3 modulated TLR7/8 response through XBP1s. (A) WT and Sirt3 -/- BMDMs pretreated with or without 50μM 3-TYP for 1 hour and 20μM MKC8866 for 24 hours were stimulated with 20μM IMQ for 9 hours and total cell lysates were immunoprecipitated with anti-XBP1s antibodies; the overall XBP1s levels and its acetylation levels were examined by western blots. (B) mRNA levels of inflammatory cytokines were determined by qRT-PCR. Values are expressed as mean ± SEM. ns: P>=0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001. IMQ, imiquimod; IP, immunoprecipitation; Ac-K, Acetylated-Lysine; WT, wild type; KO, Sirt3 knockout.

Article Snippet: Antibodies against XBP1s and Ac-K(Acetylated-Lysine) were obtained from Cell Signaling Technology (Danvers, MA, USA), and anti-SIRT3 antibodies were obtained from Proteintech (Wuhan, China) and Everest Biotech (Oxfordshire, UK).

Techniques: Immunoprecipitation, Western Blot, Quantitative RT-PCR, Knock-Out

Journal: Frontiers in Immunology

Article Title: SIRT3 alleviates imiquimod-induced psoriatic dermatitis through deacetylation of XBP1s and modulation of TLR7/8 inducing IL-23 production in macrophages

doi: 10.3389/fimmu.2023.1128543

Figure Lengend Snippet: Decreased SIRT3 and increased XBP1s acetylation in macrophages from psoriasis patients. (A) CD3+ T cells and CD14+ monocytes from PBMCs of patients with psoriasis and age- and sex-matched healthy volunteers were isolated and SIRT3 levels were examined by western blots. (B) PBMCs of patients with psoriasis and age- and sex-matched healthy volunteers were differentiated to DCs and macrophages, and SIRT3 levels were examined by western blots. (C) mRNA levels of SIRT3 in macrophages from patients with psoriasis and age- and sex-matched healthy volunteers were determined by qRT-PCR. (D) Macrophages derived from patients with psoriasis and age- and sex-matched healthy volunteers were stimulated by IMQ for 12 hours; XBP1s, and its acetylation level were examined by western blots. Values are expressed as mean ± SEM. *: P<0.05, **: P<0.01. CD3, CD3+ T cells. CD14, CD14+ monocytes. DC, dendritic cell; mø, macrophages; IMQ, imiquimod; Ac-K, Acetylated-Lysine; IP, immunoprecipitation.

Article Snippet: Antibodies against XBP1s and Ac-K(Acetylated-Lysine) were obtained from Cell Signaling Technology (Danvers, MA, USA), and anti-SIRT3 antibodies were obtained from Proteintech (Wuhan, China) and Everest Biotech (Oxfordshire, UK).

Techniques: Isolation, Western Blot, Quantitative RT-PCR, Derivative Assay, Immunoprecipitation

Journal: Cell reports

Article Title: Downregulation of hepatic ceruloplasmin ameliorates NAFLD via SCO1-AMPK-LKB1 complex

doi: 10.1016/j.celrep.2022.111498

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-acetylated-Lysine antibody, WB, dil: 1/1,000 , Cell Signaling Technology , Cat#9441; RRID: AB_331805.

Techniques: Magnetic Beads, shRNA, Recombinant, Protease Inhibitor, Lysis, Modification, Transfection, BIA-KA, Mutagenesis, Purification, Reporter Assay, TUNEL Assay, RNA Sequencing Assay, Transgenic Assay, Plasmid Preparation, Software