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: Cancer Research

Article Title: Prophylactic In Vivo Hematopoietic Stem Cell Gene Therapy with an Immune Checkpoint Inhibitor Reverses Tumor Growth in Syngeneic Mouse Tumor Models

doi: 10.1158/0008-5472.can-19-1044

Figure Lengend Snippet: Figure 3. Effect of miR-423-5p target site overexpression on HSPCs. A, Vector structure. HDAd-GFP-miR-423 contains four miR-423-5p target sites in the 30UTR linked to the GFP gene. B, Mouse HSPCs (M; Lin cells from the bone marrow of CD46-transgenic mice) and human HSPCs (Hu; CD34þ cells) were infected with either HDAd-GFP or HDAd-GFP-miR423 at a multiplicity of infection of 500 or 3,000 vp/cell, respectively. Three days later, cell lysates were analyzed by Western blot analysis for CDKN1A. Blots were reprobed with anti-b-actin antibodies to adjust for loading differences. The right panel shows the quantification of CDKN1A signals normalized to b-actin signals. The signals from the corresponding mouse and human HDAd-GFP/mgmt samples were taken as 100%. C, Effect on progenitor colony formation. One day after HDAd infection, mouse Lin cells (2.5 103 cells per 35 mm dish) or human CD34þ cells (3 103 cells/dish) were plated for colony assays. Colonies were counted 12 days later (N ¼ 3; , P < 0.05). Statistical significance was calculated by two-sided Student t test (Microsoft Excel). [In agreement with previous studies (35, 36), infection of HSPCs at relatively high multiplicities of infection slightly reduced the colony forming capacity of HSPCs.] n.s., not significant.

Article Snippet: CDKN1A antibodies (STJ23069, St John's Laboratory) cross-reacted with the mouse and human protein. qRT-PCR for aPD-L1-g 1 mRNA qRT-PCR for aPD-L1-g1 mRNA is described in Supplementary Materials and Methods aPD-L1-g 1 ELISA Recombinant mouse PD-L1 protein (Sino Biological Inc., catalog no. 50010-M08H) at 2 mg/mL were used to coat ELISA plates.

Techniques: Over Expression, Plasmid Preparation, Transgenic Assay, Infection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Pathway Analysis Identifies Amphiregulin as a Key Factor for Cisplatin Resistance of Human Breast Cancer Cells

doi: 10.1074/jbc.m706287200

Figure Lengend Snippet: FIGURE 3. Analysis of AKT kinase, downstream signaling reveals inactiva- tion of the p53 pathway in MCF-7 CisR cells. A, quantification of AKT kinase activity. To measure AKT kinase activity, a solid phase ELISA, which utilizes a specific synthetic peptide as a substrate and a polyclonal antibody that rec- ognizes the phosphorylated form of the substrate, was used. (n 3, ***, p 0.001). B, detection of p53 protein by immunoblotting (IB) using a polyclonal affinity-purified goat Ab specific for p53. MCF-7 cells (lane 1) and MCF-7 CisR cells (lane 2). Lane M, molecular weight marker. p53 is indicated by an arrow. C, quantification of p53 by a sandwich ELISA that measures human total p53 in cell lysates (n 3, ***, p 0.001). D, p21 expression indicates p53 pathway activity. Detection of p21 in whole cell lysates of MCF-7 (lane 1) and MCF-7 CisR cells (lane 2) by immunoblotting using a polyclonal affinity-purified goat Ab specific for p21. Lane M, molecular weight marker. p21 indicated by an arrow. E, to quantify the levels of p21 protein a sandwich ELISA that measures p21 in cell lysates was used. (n 3, ***, p 0.001). F, to quantify BCL-2 expression an ELISA that detects human BCL-2 in cell lysates was used (n 3, ***, p 0.001).

Article Snippet: For immunoblotting we used a polyclonal affinity-purified goat Ab specific for p53 at a concentration of 1 g/ml (AF1355, R & D Systems, Wiesbaden, Germany) and a polyclonal affinity-purified goat Ab specific for p21 at a concentration of 1 g/ml (AF1047, R & D Systems, Wiesbaden, Germany).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Affinity Purification, Molecular Weight, Marker, Sandwich ELISA, Expressing

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The total p21 protein expression (pg/mL) in normal cell line-HUVEC and in tumor cell line-PE/CA-PJ49 cells treated 24 h with CisPt and/or RSV, CRM. The experiments were performed in triplicates. Results are expressed as mean values of three determinations ± standard deviation (SD). (** p < 0.005, *** p < 0.0005; **** p < 0.00005).

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Expressing, Standard Deviation

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The effects of CisPt, RSV, CRM treatment applied alone or in combination for 24 h on the level of p21 protein expression (n-fold p21protein expression) in normal cell line-Huvec and in tumor cell line-PE/CA-PJ49. The n-fold p21expression was calculated using the formula: n-fold p21expression = p21 pg/mL treatment/p21 pg/mL control.

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Expressing, Control

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The effect of treatment with CisPt, RSV, CRM applied independently or in combination on P21 gene expression in tumor cells PE/CA-PJ49 compared to normal cells HUVEC. Each sample was performed in duplicate. The samples were analyzed using the formula 2-ΔΔCt = gene expression. (* p < 0.05, ** p < 0.005; *** p < 0.0005).

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Gene Expression