Journal: Journal of pharmacological sciences

Article Title: Sesamin promotes apoptosis and pyroptosis via autophagy to enhance antitumour effects on murine T-cell lymphoma.

doi: 10.1016/j.jphs.2021.08.001

Figure Lengend Snippet: Fig. 4. Sesamin obviously induced EL4 cell pyroptosis. (AeB). EL4 cells were stim- ulated with 10 mM, 20 mM and 40 mM sesamin for 72 h. Relative protein expression levels of pyroptotic markers were measured by western blotting (n ¼ 3 samples/ group). (C). Serum IL-1b levels in the different groups of mice were measured by ELISA (n ¼ 10 mice/group). (D). The supernatant IL-1b levels in the different groups of cells were measured by ELISA (n ¼ 6e9 mice/group). (E). Tumour tissues were fixed for IL- 1b and Cleaved-Caspase-1 immunohistochemistry staining. (F). The LDH activity in the different groups of cells was measured by an LDH release assay (n ¼ 5e6 samples/ group). (GeH). EL4 cells were stimulated with 10 mM MCC-950 before treatment with 20 mM seasamin. Relative protein expression levels of pyroptotic markers were measured by western blotting (n ¼ 3 samples/group). (I). The LDH activity and (J) cell viability in the different groups of cells were measured by LDH release assay and CCK8 assay (n ¼ 6 samples/group). (KeL). EL4 cells were stimulated with 10 mM Z-YVAD- FMK before treatment with 20 mM seasamin. Relative protein expression levels of pyroptotic markers were measured by western blotting (n ¼ 3 samples/group). (M). The LDH activity and (N) cell viability in the different groups of cells were measured by LDH release assay and CCK8 assay (n ¼ 6 samples/group). The data are representative of three independent experiments. Student's t-test was used for comparisons between two groups. Comparisons among three or more groups were made using one-way ANOVA (Tukey's test). *P < 0.05, **P < 0.01, ***P < 0.001, NS: no significance.

Article Snippet: Sesamin powder was initially dissolved in DMSO, and then diluted to final concentrations of 10 mM, 20 mM and 40 mM with RPMI-1640 medium.10 In some experiments, EL4 cells were stimulated with the Caspase-3 inhibitor Z-DEVE-FMK (10 mM, TargetMol, Shanghai, China), the NLRP3 inhibitor MCC950 (10 mM, TargetMol, Shanghai, China), the Caspase-1 inhibitor Z-YVAD-FMK (10 mM, TargetMol, Shanghai, China) and the autophagy inhibitor chloroquine (CQ, 10 mM, Sigma Aldrich, St. Louis, USA).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Activity Assay, Lactate Dehydrogenase Assay, CCK-8 Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Liver Sinusoidal Endothelial Cells Promote Metabolic Dysfunction-associated Steatohepatitis Progression via Interleukin-1R1-mediated Chemokine Production Induced by Macrophage-derived Interleukin-1β

doi: 10.1016/j.jcmgh.2025.101698

Figure Lengend Snippet: The impairment of autophagy in macrophages by fatty acids increases IL-1β secretion. ( A ) IL1B gene expression and the concentrations of IL-1β and caspase-1 in the supernatants of THP-1 macrophages cultured for 24 hours with or without LPS (25 ng/mL) and treated with either BSA or 400 μM PA (n = 4/group). ( B ) IL1B gene expression and the concentrations of IL-1β in the supernatants of THP-1 macrophages cultured for 24 hours with BSA or 400 μM PA and 25 ng/mL LPS in the presence or absence of 50 μM Z-YVAD-FMK (n = 4/group). ( C ) LC3 turnover assay to evaluate autophagic flux in THP-1 macrophages treated with PA and BFM (n = 3/group). ( D ) Expression of autophagy-related proteins in THP-1 macrophages treated with PA. ( E ) IL-1β and caspase-1 concentrations in the supernatants of THP-1 macrophages cultured for 24 hours with or without 25 ng/mL LPS and treated with either dimethyl sulfoxide (DMSO) or 50 nM BFM.

Article Snippet: THP-1 macrophages were pretreated for 1 hour with 50 μM Z-YVAD-FMK (S8507; Selleck, Houston, TX) before stimulation.

Techniques: Gene Expression, Cell Culture, LC3 Turnover Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

doi: 10.1074/jbc.RA120.014467

Figure Lengend Snippet: Impact of oxidants on protein phosphorylation in ARVMs. A, ARVMs were exposed to vehicle (control), NCA (100 µmol/liter, 30 min), AS (300 µmol/liter, 15 min), CXL-1020 (300 µmol/liter, 15 min), H2O2 (100 µmol/liter, 10 min), DIA (500 µmol/liter, 10 min), or ISO (10 nmol/liter, 10 min). Cell lysates were resolved by SDS-PAGE and gels were stained for phosphoproteins by ProQ-Diamond. Total protein was visualized by SYPRO Ruby and Coomassie staining. B, phosphorylation of cMyBP-C (pSer-273, pSer-282, and Ser-302), PLN (pSer-16), and cTnI (pSer-23/24) in response to oxidant exposure was investigated by immunoblotting (IB) and phospho-specific antibodies. Scatter plots summarize data of at least 7 (phospho-cMyBP-C), 4 (phospho-PLN), or 5 (phospho-cTnI) independent experiments normalized to α-actinin and expressed as % of signal induced by ISO. *, p < 0.05; ***, p < 0.001 for comparison with vehicle control by one-way ANOVA and Dunnett's post-test (F and p values): a, pSer-273 cMyBP-C: F = 79.70, p < 0.0001; b, pSer-282 cMyBP-C: F = 142.4, p < 0.0001; c, pSer-302 cMyBP-C: F = 15.27, p < 0.0001; d, pSer-16 PLB: F = 259.1, p < 0.0001; e, pcTnI: F = 1.246, p = 0.3192). C, ARVMs were treated with the indicated concentrations of NCA (for 30 min), DIA (for 10 min), CXL-1020 (for 15 min), and AS (for 15 min) or were exposed to vehicle (DMSO for 30 min or NaOH for 15 min) or ISO (10 nmol/liter, 10 min). Phosphorylation of cMyBP-C (pSer-282) and PLN (pSer-16) was analyzed by IB using the respective phospho-specific antibodies. Protein loading was assessed using an antibody against α-actinin. Blots are representative of three independent experiments. ns, not significant.

Article Snippet: OA and the anti-pSer-282 cMyBP-C antibody (dilution IB 1:1000; rabbit polyclonal; number ALX-215-057-R050) was from Enzo Life Sciences.

Techniques: SDS Page, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

doi: 10.1074/jbc.RA120.014467

Figure Lengend Snippet: Time-dependent impact of NCA on PKA-RI dimerization and protein translocation in ARVMs. A, ARVMs were treated with vehicle for 100 min (0 min NCA), NCA (100 µmol/liter) for 3, 10, 30, and 100 min, or ISO (10 nmol/liter) for 10 min. PKA-RI and cMyBP-C phosphorylation at Ser-282 were detected in whole lysates by IB analysis under NR or R conditions, respectively. Scatter plots summarize RI dimer signal fold-change compared with control sample (0 min NCA) and cMyBP-C phosphorylation as % of ISO-response from 4 independent experiments. Band intensities were normalized to α-actinin. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for comparison with vehicle control (0 min NCA) by one-way ANOVA with Dunnett's post-test (RI dimer: F = 8.23, p = 0.0003; pSer-282 cMyBP-C: F = 107.5, p < 0.0001). B, ARVMs were exposed to vehicle for 100 min (0 min NCA), NCA (100 µmol/liter) for 3, 10, 30, and 100 min or ISO (10 nmol/liter) for 10 min and subcellular fractionation was performed under NR conditions. Phosphorylation of cMyBP-C at Ser-282, as well as the presence of PKA-RI, PKA-C, B56α, and PP2A-C were detected in input lysates and Triton-insoluble fractions. Phosphorylation of cMyBP-C at Ser-282 (n = 6) normalized to Coomassie stain signals (not shown) is presented as % of the band intensity induced by ISO. PKA-RI (n = 4), PKA-C (n = 6), B56α (n = 5), and PP2A-C (n = 6) signals from Triton-insoluble fractions normalized to the corresponding inputs are shown as fold-change of the control signal (0 min NCA). *, p < 0.05; **, p < 0.01; ***, p < 0.001 for comparison with the corresponding vehicle control (sample 0 min) by one-way ANOVA with Dunnett's post-test (F and p values): a, PKA-RI, F = 15.18, p < 0.0001; b, PKA-C, F = 15.61, p < 0.0001; c, B56α, F = 4.78, p = 0.0036; d, PP2A-C, F = 9.16, p < 0.0001; e, pSer-282 cMyBP-C, F = 67.33, p < 0.0001). ns, not significant.

Article Snippet: OA and the anti-pSer-282 cMyBP-C antibody (dilution IB 1:1000; rabbit polyclonal; number ALX-215-057-R050) was from Enzo Life Sciences.

Techniques: Translocation Assay, Fractionation, Staining

Journal: The Journal of Biological Chemistry

Article Title: Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

doi: 10.1074/jbc.RA120.014467

Figure Lengend Snippet: Oxidation-mediated effects on PKA and PP2A-C activity in vitro. A, active PKA catalytic subunit (PKA-C) was preincubated for 10 min with either ATP (100 µmol/liter), H89 (25 µmol/liter), or assay buffer (Ø) and then exposed to vehicle (control) or 100 µmol/liter NCA for 30 min. Then, the samples that initially did not contain ATP were supplemented with 100 µmol/liter of ATP and the in vitro kinase reaction was initiated by adding 100 pmol of recombinant His6-tagged C1-M-C2 cMyBP-C to each sample. Samples that did not contain kinase served as additional controls. Substrate phosphorylation and content was assessed under R conditions (sample reduction with 10% (v/v) β-mercaptoethanol) by IB using antibodies against pSer-282-cMyBP-C and the His6 tag. The presence and oxidation of PKA-C was monitored under NR conditions by IB using an anti-PKA-C antibody. Coomassie stain of the substrate blot is also shown as additional loading reference. The scatter plot summarizes results for C1-M-C2 phosphorylation from 4 independent experiments. Data are expressed as % of the signal of the vehicle control after ATP preincubation. **, p < 0.01; ***, p < 0.001 for comparison with the corresponding assay buffer (Ø)-pretreated sample; #, p < 0.05; ###, p < 0.001 for comparison with corresponding samples with and without NCA treatment by two-way ANOVA with Bonferroni post-test (interaction, F = 7.07, p = 0.0054; preincubation buffer, F = 31.64, p < 0.0001; treatment: F = 22.50, p = 0.0002). B, active recombinant human PP2A-C was incubated with vehicle (DMSO), NCA (100 μmol/liter), CXL-1020 (300 μmol/liter), or OA (10 nmol/liter), and the substrate DiFMUP (500 µmol/liter). Fluorescence reflecting phosphatase activity was monitored over time (representative traces). For each treatment, a phosphatase-free blank sample was included in the measurement. The table shows the activity of active recombinant PP2A-C at each condition, calculated from the slope of the linear regression of averaged RFU values and expressed as ΔRFU/min or % of the activity calculated under control conditions (n = 4-5 experiments). Measured fluorescence was normalized to blank values and expressed as % of the control signal. C, the data from B were reanalyzed to reflect the HNO donor incubation times in cell culture experiments. The scatter plots represent average phosphatase activity expressed as ΔRFU/min calculated from the slope of a linear curve fit after 15 min of CXL-1020 or 30 min of NCA treatment (n = 4-5 experiments) to reflect the cell culture conditions. *, p < 0.05; **, p < 0.01 two-tailed Student's t test for comparison of each time point with the corresponding vehicle control; ns, not significant.

Article Snippet: OA and the anti-pSer-282 cMyBP-C antibody (dilution IB 1:1000; rabbit polyclonal; number ALX-215-057-R050) was from Enzo Life Sciences.

Techniques: Activity Assay, In Vitro, Recombinant, Staining, Incubation, Fluorescence, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: Papilloma-pseudovirus eradicates intestinal tumours and triples the lifespan of Apc Min/+ mice

doi: 10.1038/ncomms15004

Figure Lengend Snippet: ( a ) The levels of secreted IL-1β (left panel) or IL-18 (right panel) from small intestinal macrophages that were primed with LPS followed by stimulation with indicated stimuli. Results are shown as mean±s.d. ( b ) IL-1β levels from LPS-primed WT, NLRP3-KO, ASC-KO, Casp1-KO or NLRC4-KO immortalized BMDMs that were activated by the stimuli as indicated. Results are shown as mean±s.d. ( c ) IL-1β levels from LPS-primed BMDMs that were stimulated with indicated stimuli. Results are shown as mean±s.d. ( d ) IL-1β levels from LPS-primed WT or AIM2/IFI204 deficient BMDMs that were transfected with DNA plasmids as indicated. Results are shown as mean±s.d. All data are representative of three independent experiments.

Article Snippet: Caspase-1 inhibitor Z-YVAD-FMK and caspase-3 inhibitor Z-DEVD-FMK were from Enzo Life Sciences.

Techniques: Transfection

Journal: Nature Communications

Article Title: Papilloma-pseudovirus eradicates intestinal tumours and triples the lifespan of Apc Min/+ mice

doi: 10.1038/ncomms15004

Figure Lengend Snippet: ( a – c ) Intestinal polyp numbers of hCEA-Tg/Apc Min/+ mice ( n =10–15, including 5–7 male and 5–8 female mice per group) that were pretreated with caspase-1 or -3 inhibitors followed by immunization of VLP-hCEA ( a ) VLP-pUMVC3 ( b ) or VLP ( c ). Results are shown as mean±s.d. Analysis of variance (ANOVA) was performed to determine the statistical significance. *** P <0.001; n.s., not significant. Data are representative of two independent experiments. ( d ) The 14-week-old Apc Min/+ mice were orally immunized with VLP-hCEA or PBS control. After 24 and 48 h, mice were killed and intestinal tissues were collected and separated microscopically as nontumour or tumour tissues. Cellular proteins were extracted and immunoblotted for caspase-1 or β-actin. Data are representative of three independent experiments.

Article Snippet: Caspase-1 inhibitor Z-YVAD-FMK and caspase-3 inhibitor Z-DEVD-FMK were from Enzo Life Sciences.

Techniques:

Journal: Nature Communications

Article Title: Papilloma-pseudovirus eradicates intestinal tumours and triples the lifespan of Apc Min/+ mice

doi: 10.1038/ncomms15004

Figure Lengend Snippet: ( a , b ) The 14-week-old Apc Min/+ mice were orally immunized with VLP-GFP. At 48 h post immunization, the polyps of small intestines, which were separated microscopically from normal intestinal tissues, were collected and stained with anti-EpCAM, anti-GFP antibody and anti-F4/80 antibodies, respectively. Arrows indicate cells with colocalization of EpCAM/GFP or F4/80/GFP. Data are representative of three independent experiments. Scale bars, 100 μm. ( c , d ) The 14-week-old Apc Min/+ mice were orally immunized with VLP-hCEA. At 48 h post immunization, the polyps of small intestines, which were separated microscopically from normal intestinal tissues, were collected and stained with anti-EpCAM, anti-F4/80 antibodies and FAM-FLICA-Casp1 probe, respectively. Arrows indicate cells with colocalization of EpCAM/FLICA or F4/80/FLICA. Data are representative of three independent experiments. Scale bars, 100 μm. ( e , f ) The 14-week-old Apc Min/+ mice were orally immunized with VLP-hCEA. At 48 h post immunization, the polyps of small intestines, which were separated microscopically from normal intestinal tissues, were collected and co-stained with anti-EpCAM ( e ) or anti-F4/80 ( f ) antibodies and TUNEL, respectively. Arrows indicate dying intestinal tumour cells or macrophages. Data are representative of three independent experiments. Scale bars, 100 μm.

Article Snippet: Caspase-1 inhibitor Z-YVAD-FMK and caspase-3 inhibitor Z-DEVD-FMK were from Enzo Life Sciences.

Techniques: Staining, TUNEL Assay

Journal: mBio

Article Title: Human Cytomegalovirus Immediate Early 86-kDa Protein Blocks Transcription and Induces Degradation of the Immature Interleukin-1β Protein during Virion-Mediated Activation of the AIM2 Inflammasome

doi: 10.1128/mBio.02510-18

Figure Lengend Snippet: Secretion of IL-1β in response to live and UV-inactivated HCMV requires the AIM2 inflammasome. (A) Immunoblot assay for caspase-1, ASC, NLRP3, IFI16, AIM2, and GAPDH in THP-1 cells transduced with Cas9 and guide RNA (gRNA) targeting HUMCYC pseudogene or in indicated CRISPR-generated knockout cells. (B) IL-1β secretion in THP-1 cells with the HUMCYC pseudogene or gene encoding an indicated protein deleted was measured following mock treatment (white) or treatment with live (gray) or UV-inactivated (black) HCMV (HCMV-TB40/E; MOI of 3) for 18 h. Values are averages ± SEM from three biological replicates. (C) IL-1β secretion in differentiated THP-1 cells following mock treatment or treatment with UV-HCMV (UV) alone or in combination with caspase-1 inhibitor (YVAD), caspase-4 inhibitor (LEVD), caspase-5 inhibitor (WEHD), or caspase-8 inhibitor (IETD) for 18 h. Values are averages ± SEM from three replicates. One-way ANOVA with Bonferroni correction was performed. **, P < 0.01.

Article Snippet: Caspase-1 inhibitor carboxybenzoxy-Tyr-Val-Ala-Asp-fluoromethylketone (Z-YVAD-FMK), capase-4 inhibitor carboxybenzoxy-Leu-Glu-Val-Asp-fluoromethylketone (Z-LEVD-FMK), caspase-5 inhibitor carboxybenzoxy-Trp-Glu-His-Asp-fluoromethylketone (Z-WEHD-FMK), and caspase-8 inhibitor carboxybenzoxy-Ile-Glu-Thr-Asp-fluoromethylketone (Z-IETD-FMK) were all obtained from Enzo Life Science and used at a concentration of 2 μM.

Techniques: Western Blot, Transduction, CRISPR, Generated, Knock-Out

Journal: mBio

Article Title: Human Cytomegalovirus Immediate Early 86-kDa Protein Blocks Transcription and Induces Degradation of the Immature Interleukin-1β Protein during Virion-Mediated Activation of the AIM2 Inflammasome

doi: 10.1128/mBio.02510-18

Figure Lengend Snippet: HCMV IE72 and IE86 do not inhibit UV-HCMV-induced caspase-1 cleavage, IL-18 secretion, or GSDMD cleavage. (A) Immunoblot assay for pro-IL-1β, pro-caspase-1, active caspase-1, IE72/IE86, and GAPDH in THP-1-IE72 and THP-1-IE86 cells either left untreated (mock) or treated with UV-inactivated HCMV-TB40/E (MOI of 3) for 18 h in the presence or absence of doxycycline. (B) IL-18 secretion in THP-1-IE86 cells left untreated (mock) or infected with UV-inactivated HCMV-TB40/E (MOI of 3) for 18 h in the presence (gray) or absence (black) of doxycycline. Values are averages ± SEM from three biological replicates. One-way ANOVA with Bonferroni correction was performed. n.s., not significant. (C) Immunoblot assay for total and cleaved (*) GSDMD and GAPDH in THP-1-IE86 cells either untreated (M) or infected with UV-inactivated (UV) HCMV-TB40/E (MOI of 3) for 18 h in the absence (−) or presence (+) of doxycycline.

Article Snippet: Caspase-1 inhibitor carboxybenzoxy-Tyr-Val-Ala-Asp-fluoromethylketone (Z-YVAD-FMK), capase-4 inhibitor carboxybenzoxy-Leu-Glu-Val-Asp-fluoromethylketone (Z-LEVD-FMK), caspase-5 inhibitor carboxybenzoxy-Trp-Glu-His-Asp-fluoromethylketone (Z-WEHD-FMK), and caspase-8 inhibitor carboxybenzoxy-Ile-Glu-Thr-Asp-fluoromethylketone (Z-IETD-FMK) were all obtained from Enzo Life Science and used at a concentration of 2 μM.

Techniques: Western Blot, Infection