rabbit anti pro il 1b  (Novus Biologicals)

Journal: Frontiers in immunology

Article Title: NLRP3 Inflammasome Promotes the Progression of Acute Myeloid Leukemia via IL-1β Pathway.

doi: 10.3389/fimmu.2021.661939

Figure Lengend Snippet: FIGURE 1 | NLRP3 inflammasome is over-expressed and highly activated in AML, which plays leukemia-promoting effects in vitro. (A) The western blot results of NLRP3, NF-kB, caspase-1, pro-IL-1b and ASC in BM-MNCs from ND AML patients (n=3) and controls (n=3). (B) The concentrations of IL-1b and IL-18 in BM supernatant from ND AML patients (n=70) in comparison to controls (n=15). (C) The Western blot results of pNF-kB, pro-caspase-1, pro-IL-1b, cleaved caspase-1 and cleaved IL-1b were showed in primary leukemia cells after different treatment. b-actin is used as a loading control. (D) LPS stimulated the secretion of IL-1b and IL-18 into the supernatants from cultured leukemia cells. (E) CCK8 analysis was performed to detect the proliferation of leukemia cells 24, 48 and 72 hours after LPS stimulation (n=10). (F) The quantified apoptosis rate was shown (n=10). (G) The western blot results of PARP, C-myc and Bcl-2 in primary leukemia cells after LPS stimulation. (H) CCK8 analysis for the proliferation of primary leukemia cells with or without LPS 24, 48 and 72 hours after treatment with ADR, DNR (n=10). (I) The apoptosis results of primary leukemia cells with or without LPS after treatment with ADR, DNR (n=6). (J) The expression of NLRP3 in U937 cell line was enhanced after being transfected with lentivirus (n=3). (K) The IC50 value of ADR for U937 cells was higher after upregulating NLRP3 expression (n=3). (L) PARP, C-myc and Bcl-2 in primary leukemia cells after LPS stimulation (n=1). b-actin is used as a loading control. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Primary antibodies against NLRP3 (#15101), NF-kB p65(# 8242), phospho-NF-kB p65 (NF-kB pp65) (#3033), caspase-1 (#3866), pro-IL-1b (#12703), cleaved IL-1b (#83186), Bcl-2 (#3498), C-myc (#5605), and cleaved PARP (#9548) used for Western blot were obtained from Cell Signaling Technology (Beverly, MA, USA); and antibodies against caspase-9 (ab202068), b-tubulin (ab0039), cleaved caspase-3 (ab13847), BAX (ab199677) and b-actin (ab227387) were purchased from Abcam (Cambridge, UK).

Techniques: In Vitro, Western Blot, Comparison, Control, Cell Culture, Expressing, Transfection

Journal: Frontiers in immunology

Article Title: NLRP3 Inflammasome Promotes the Progression of Acute Myeloid Leukemia via IL-1β Pathway.

doi: 10.3389/fimmu.2021.661939

Figure Lengend Snippet: FIGURE 3 | Inactivation of NLRP3 inflammasome by caspase-1 or NF-kB inhibitor suppresses AML leukemia cells in vitro. (A) The concentration of IL-1b (n=5) and IL-18 (n=4) in supernatants of cultured leukemia cells following Z-YVAD-FMK treatment. (B) Cell proliferation was analyzed by CCK8 assay in primary leukemia cells 24, 48 and 72 hours after treatment with LPS or/and Z-YVAD-FMK (n=8). (C) The apoptosis rate of leukemia cells in primary leukemia cells 48 hours after treatment with LPS or/and Z-YVAD-FMK (n=5). (D) Representative scatter plots of apoptosis. (E) ELISA results of secreted IL-1b (n=3) and IL-18 (n=4) by AML primary leukemia cells after treatment with Bay11-7082. (F) Cell proliferation was analyzed by CCK8 assay in primary leukemia cells 24, 48 and 72 hours after treatment with LPS or/and Bay11-7082 (n=7). (G) The apoptosis rate of leukemia cells in primary leukemia cells 48 hours after treatment with LPS or/and Bay11-7082 (n=3). (H) Representative scatter plots of apoptosis. (I) The results of NLRP3 mRNA expression in THP1 cells after being transfected with lentivirus (n=3). (J) The IC50 values of ADR for THP1 cells after downregulating NLRP3 expression (n=3). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Primary antibodies against NLRP3 (#15101), NF-kB p65(# 8242), phospho-NF-kB p65 (NF-kB pp65) (#3033), caspase-1 (#3866), pro-IL-1b (#12703), cleaved IL-1b (#83186), Bcl-2 (#3498), C-myc (#5605), and cleaved PARP (#9548) used for Western blot were obtained from Cell Signaling Technology (Beverly, MA, USA); and antibodies against caspase-9 (ab202068), b-tubulin (ab0039), cleaved caspase-3 (ab13847), BAX (ab199677) and b-actin (ab227387) were purchased from Abcam (Cambridge, UK).

Techniques: In Vitro, Concentration Assay, Cell Culture, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Expressing, Transfection

Journal: Frontiers in immunology

Article Title: NLRP3 Inflammasome Promotes the Progression of Acute Myeloid Leukemia via IL-1β Pathway.

doi: 10.3389/fimmu.2021.661939

Figure Lengend Snippet: FIGURE 4 | Knockout of NLRP3 attenuates leukemia burden in AML mice. (A) Representative photographs of spleen from WT mice (n=3) and NLRP3-/- mice (n=4). (B) The weight of spleens of WT mice (n=3) and NLRP3-/- mice (n=4). (C) The leukemia cells (GFP+ cells) in spleen and bone marrow from WT mice (n=3) and NLRP3-/- mice (n=4). (D) Hematoxylin and eosin-stained histopathology sections of a representative spleen and bone marrow from WT and NLRP3-/- mice (100× or400×). (E) The mRNA and protein expressions of IL-1b by qRT-PCR and western- blot in bone marrow of WT and NLRP3 -/- mice. *P < 0.05.

Article Snippet: Primary antibodies against NLRP3 (#15101), NF-kB p65(# 8242), phospho-NF-kB p65 (NF-kB pp65) (#3033), caspase-1 (#3866), pro-IL-1b (#12703), cleaved IL-1b (#83186), Bcl-2 (#3498), C-myc (#5605), and cleaved PARP (#9548) used for Western blot were obtained from Cell Signaling Technology (Beverly, MA, USA); and antibodies against caspase-9 (ab202068), b-tubulin (ab0039), cleaved caspase-3 (ab13847), BAX (ab199677) and b-actin (ab227387) were purchased from Abcam (Cambridge, UK).

Techniques: Knock-Out, Staining, Histopathology, Quantitative RT-PCR, Western Blot

Journal: Frontiers in immunology

Article Title: NLRP3 Inflammasome Promotes the Progression of Acute Myeloid Leukemia via IL-1β Pathway.

doi: 10.3389/fimmu.2021.661939

Figure Lengend Snippet: FIGURE 5 | The leukemia-promoting effect induced by NLRP3 activation acts through IL-1b but not IL-18. (A) Bayesian network model diagram was designed according to qRT-PCR results of NLRP3 inflammasome in controls and ND AML patients. (B) The mRNA expressions of NLRP3 inflammasome components caspase-1 and IL-1b in favorable (n=13) and intermediate/poor-risk (n=32) groups. (C) The mRNA expressions of NLRP3 inflammasome components caspase-1, IL-1b, NLRP3 and NF-kB in THP-1 cells were compared before and after LPS stimulation. (D) The concentrations of IL-1b and IL-18 in supernatants of cultured THP-1 cells following LPS or LPS+ATP treatment. (E) The apoptosis rate of primary leukemia cells after being co-cultured with LPS-activated THP1 cells with or without adding anti-IL-1b or anti-IL-18 antibody (n=6). (F) Representative scatter plots of apoptosis. (G) CCK8 assay was applied to analyze the proliferation of primary leukemia cells after adding IL-1b or/and IL-18 for 24, 48, 72 and 96 hours (n=21). (H) Flow cytometry analysis of Annexin V-FITC/PI-staining method was performed to analyze apoptosis of primary leukemia cells after adding IL-1b or/and IL-18 for 48 hours (n=24). (I) Results were plotted as the percentage of cells in each quadrant. (J) Cell apoptosis was analyzed by flow cytometry 48 hours after adding ADR (200 mg/L) or DNR (20 mg/L) with or without IL-1b or IL-18 (n=8). (K) Representative scatter plots of flow cytometry. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Primary antibodies against NLRP3 (#15101), NF-kB p65(# 8242), phospho-NF-kB p65 (NF-kB pp65) (#3033), caspase-1 (#3866), pro-IL-1b (#12703), cleaved IL-1b (#83186), Bcl-2 (#3498), C-myc (#5605), and cleaved PARP (#9548) used for Western blot were obtained from Cell Signaling Technology (Beverly, MA, USA); and antibodies against caspase-9 (ab202068), b-tubulin (ab0039), cleaved caspase-3 (ab13847), BAX (ab199677) and b-actin (ab227387) were purchased from Abcam (Cambridge, UK).

Techniques: Activation Assay, Quantitative RT-PCR, Cell Culture, CCK-8 Assay, Flow Cytometry, Staining, Cytometry

Journal: Frontiers in immunology

Article Title: NLRP3 Inflammasome Promotes the Progression of Acute Myeloid Leukemia via IL-1β Pathway.

doi: 10.3389/fimmu.2021.661939

Figure Lengend Snippet: FIGURE 6 | Down-regulation of endogenous IL-1b suppresses the growth of leukemia cells. The mRNA expression of IL-1b was determined by qRT-PCR in THP1 cells after IL-1b siRNA transfection (n=3) (A) and the protein level of IL-1b by western blot in THP1 cells after IL-1b siRNA transfection was also shown (n=3) (B). The proliferation results by CCK8 assay for leukemia cells 24, 48, 72 hours after knocking down IL-1b by siRNA transfection (n=3) (C) and treatment with drugs (n=3) (D). The apoptosis of THP1 cells was quantified 48 hours after IL-1b knockdown by siRNA (n=3) (E) and the representative scatter plots of apoptosis (F). (G) The western blot results of pro-caspase-9, cleaved caspase-9, and b-tubulin is a loading control. Representative western blot bands of Bcl-2, BAX, cleaved caspase-3, and GAPDH is a loading control (n=3). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Primary antibodies against NLRP3 (#15101), NF-kB p65(# 8242), phospho-NF-kB p65 (NF-kB pp65) (#3033), caspase-1 (#3866), pro-IL-1b (#12703), cleaved IL-1b (#83186), Bcl-2 (#3498), C-myc (#5605), and cleaved PARP (#9548) used for Western blot were obtained from Cell Signaling Technology (Beverly, MA, USA); and antibodies against caspase-9 (ab202068), b-tubulin (ab0039), cleaved caspase-3 (ab13847), BAX (ab199677) and b-actin (ab227387) were purchased from Abcam (Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, CCK-8 Assay, Knockdown, Control