Journal: Inflammation Research

Article Title: NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype

doi: 10.1007/s00011-024-01892-7

Figure Lengend Snippet: NLRP1 expression is associated with senescence. A Human fibroblasts were exposed to 20 Gy ionizing irradiation (IR). On day 1, 5 and 7, NLRP1 and senescence protein expression were analyzed by immunoblotting. B Representative images of Ki67 and NLRP1 immunofluorescence. Scale bar = 50 μm. C , D IL-1β, IL18, IL-6 and IL-8 were quantified by ELISA. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, *** P < 0.001 differences between time points after irradiation and day 0. E , F Human fibroblasts were treated with Valboropro (Vbpro) to induce NLRP1 expression. After 24 h, NLRP1 and IL-6 protein expression were analyzed by immunoblotting and cytokines were analyzed by ELISA. Human fibroblasts were irradiated ( G ) or stimulated with palbociclib (Palbo) ( H ) to induce two different senescence models. Then, cells were transfected with a non-targeting control siRNA (Control) or with siRNAs against NLRP1 (siNLRP1). Expression of NLRP1 and senescence-associated proteins p16, p21, p53 and IL-6 were assessed by immunoblotting and IL6, IL-8 or IL-18 were quantified by ELISA. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, *** P < 0.001 irradiated vs. control. aaa P < 0.001, IR + siRNA vs. IR cells (color figure online)

Article Snippet: Monoclonal antibodies specific for NLRP1 (NBP1-54899), NLRP3 (NBP2-12446) and p21/CIP1/CDKN1A (NBP2-29463) were purchased from Novus Biologicals (Colorado, USA).

Techniques: Expressing, Irradiation, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Inflammation Research

Article Title: NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype

doi: 10.1007/s00011-024-01892-7

Figure Lengend Snippet: NLRP1 contributes to cellular senescence in vivo. A Nlrp1 protein expression in liver from WT mice at 1 month after IR. B Serum levels of IL-18 after IR. C Effect of IR on the bodyweight of WT and Nlrp1 knockout (KO) mice. D Protein expression in liver from IR and non-IR WT and Nlrp1 KO mice of senescent markers (IL-6, p16 and p21). Densitometry in Supplementary Fig. 6. E Serum levels of IL-6 in serum from IR and non-IR WT and Nlrp1 KO mice. F Heat map depicting expression of 44 mouse cytokines in serum at 5 weeks after IR of WT and Nlrp1 KO mice. n = 6 mice per group. G IL-6 releases from healthy fibroblasts was assessed after 24 and 48 h of incubation with media containing serum from IR and non-IR WT and Nlrp1 KO mice. H Representative liver section stainings of hematoxylin and eosin (H&E). I Representative liver section immunostainings of NLRP1 and p16. All data are presented as means ± SEM, n = 6–8 mice per group; * P < 0.05, ** P < 0.005, *** P < 0.001 irradiated vs. control. aa P < 0.005 IR WT vs. IR KO mice; bb P < 0.005 IR KO vs. IR KO mice (color figure online)

Article Snippet: Monoclonal antibodies specific for NLRP1 (NBP1-54899), NLRP3 (NBP2-12446) and p21/CIP1/CDKN1A (NBP2-29463) were purchased from Novus Biologicals (Colorado, USA).

Techniques: In Vivo, Expressing, Knock-Out, Incubation, Irradiation, Control

Journal: Inflammation Research

Article Title: NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype

doi: 10.1007/s00011-024-01892-7

Figure Lengend Snippet: NLRP1 senses DNA damage dependent of cGAS activation. A Protein expression levels of NLRP1 and cGAS after 24 h exposition to gDNA from non-irradiated and irradiated cells. B IL-6 and IL-18 release to the medium after the same experimental condition. Levels were determined by ELISA assay. C , D Protein expression levels of NLRP1 and cGAS and IL-6 and IL-18 release after 24 h exposition to a non-irradiated or irradiated synthetic double-stranded DNA sequence, poly(dA-dT), Cytokine levels were determined by ELISA assay. Cytokine levels were determined by ELISA assay. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, *** P < 0.001 gDNA vs. control cells. a P < 0.05, aa P < 0.005, aaa P < 0.001, IR gDNA vs. control cells. E Human fibroblasts were irradiated to induces senescence. Then, cells were transfected with a non-targeting control siRNA (Control) or with siRNAs against cGAS (sicGAS). Expression of NLRP1, NLRP3, IL-1β, cGAS and senescent protein p16, p21 was assessed by immunoblotting. F IL-18 release of non-irradiated, irradiated and irradiated and transfected with siRNAs against cGAS. Cytokine levels were determined by ELISA assay. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, IR vs. control cells. aa P < 0.005, IR vs. sicGAS (color figure online)

Article Snippet: Monoclonal antibodies specific for NLRP1 (NBP1-54899), NLRP3 (NBP2-12446) and p21/CIP1/CDKN1A (NBP2-29463) were purchased from Novus Biologicals (Colorado, USA).

Techniques: Activation Assay, Expressing, Irradiation, Enzyme-linked Immunosorbent Assay, Sequencing, Control, Transfection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The p53/Retinoblastoma-mediated Repression of Testicular Orphan Receptor-2 in the Rhesus Monkey with Cryptorchidism

doi: 10.1074/jbc.m910158199

Figure Lengend Snippet: FIG. 2. Induction of p53 and p21 and hypophosphorylation of Rb in the context of surgery-induced cryptorchidism in the rhesus monkey. Testis total RNA was prepared as described in Fig. 1. A, the total RNA samples obtained from the indicated rhesus monkey testes were subjected to Northern blot analysis using a-32P-labeled human p53 or p21 cDNA fragments. The same hybridization membrane was stripped and rehybridized with a b-actin gene cDNA probe to serve as an internal control for the normalization of RNA. Molecular weight markers shown on the gel are 18 and 28 S rRNAs. B, protein extracts obtained from the indicated rhesus monkey testes were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to Immobilon P membrane. The membrane represented in the upper panel was first incubated with blocking buffer and then incubated with an anti-human p53 polycolonal antibody followed by an alkaline phosphatase-conjugated secondary antibody. The protein extracts were also immunoprecipitated with either 5 ml of anti-human p21 (middle panel) monoclonal antibody or 5 ml of anti-human Rb (lower panel) monoclonal antibody. Immunoprecipitates were separated and transferred to membranes, and Western blot analysis was performed using the same method described for p53. pp-Rb, hyperphosphorylated (inactive) form of Rb. p-Rb, hypophosphorylated (active) form of Rb.

Article Snippet: The supernatant containing 500 mg of protein was incubated with primary antibody, either 65951A (Pharmingin) for p21 or 14001A (Pharmingin) for Rb, at 4 °C for 2 h before the addition of protein A/G-Sepharose beads (Santa Cruz).

Techniques: Northern Blot, Labeling, Hybridization, Membrane, Control, Molecular Weight, Polyacrylamide Gel Electrophoresis, Incubation, Blocking Assay, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The p53/Retinoblastoma-mediated Repression of Testicular Orphan Receptor-2 in the Rhesus Monkey with Cryptorchidism

doi: 10.1074/jbc.m910158199

Figure Lengend Snippet: FIG. 7. Our model linking cryp- torchidism and the repression of TR2 involves the p53-Rb signaling path- way. Due to increased testis temperature via cryptorchidism, expression of p53 is increased. This increase in p53 then up- regulates p21, which in turn reduces CDK activity. Rb, existing in either its hyper- phosphorylated (inactive) or hypophos- phorylated (active) form, is pushed to- ward an increase in activity due to the decrease in CDK function. Active Rb pro- tein then binds transcription factor E2F, effectively reducing expression of TR2. It has been demonstrated that TR2 has the ability to regulate expression of several target genes, thereby modulating other signaling pathways. Thus, the repression of TR2 through up-regulation of p53 caused by cryptorchidism may have wide- spread effects. pp-Rb, hyperphosphory- lated (inactive) form of Rb; RAR, retinoic acid receptor; RXR, retinoid receptor X; HBV, human hepatitis B virus.

Article Snippet: The supernatant containing 500 mg of protein was incubated with primary antibody, either 65951A (Pharmingin) for p21 or 14001A (Pharmingin) for Rb, at 4 °C for 2 h before the addition of protein A/G-Sepharose beads (Santa Cruz).

Techniques: Expressing, Activity Assay, Protein-Protein interactions, Virus