pma differentiated thp1  (InvivoGen)

Journal: Cell Death Discovery

Article Title: Pyroptosis leads to loss of centrosomal integrity in macrophages

doi: 10.1038/s41420-024-02093-1

Figure Lengend Snippet: THP1 ATCC cells were left untreated or treated with nigericin (10 µM) at the indicated time points to activate the NLRP3 inflammasome, then cell lysates and supernatants were analysed for PCNT, γ-tubulin, and β-actin protein expression by western blot ( A – F , N = 3). Lysates and supernatants were analysed for PCNT and γ-tubulin as well as loading control β-actin (42 kDa) ( D ). Whole well lysate with centrifugation step ( E ) and whole cell lysate with no centrifugation step ( F ) were also analysed for those proteins. Cell death was measured by LDH assay and shown as percentage relative to total cell death ( A ). Caspase-1 activity in supernatants was measured and shown as fold change relative to control ( B ). IL-18 in the supernatants was measured by ELISA ( C ). THP1 ATCC cells were stimulated with nigericin (10 µM) for 45 min or 90 min. G – K Immunofluorescence was used to analyse the centrosomal proteins including PCNT ( G ), γ-tubulin ( H ) and ninein ( I ) as well as ASC to determine the NLRP3 inflammasome activation. Percentages of ASC speck, PCNT, γ-tubulin or ninein positive cells relative to total cells ( J ) and PCNT, γ-tubulin or ninein positive cells in ASC positive cells ( K ) were quantified. N = 3 biologically independent experiments. For multiple comparisons, one‐way ANOVA with the Dunnett’s test for time response in THP1 ATCC cells was applied. Data was shown as mean ± S.D., * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001 were considered statistically significant when compared to vehicle treatment (time 0).

Article Snippet: PMA differentiated THP1 ATCC cells were left untreated or treated with MG132 (10 μM, 2 h), or E-64-D (20 μM, 2 h), or Ca-074Me (50 μM, 15 min), or pepstatin A (10 μM, 15 min) or bafilomycin A1 (100 μM, 15 min) before stimulation with nigericin (10 μM, 45 min) ( A – J , N = 3).

Techniques: Expressing, Western Blot, Control, Centrifugation, Lactate Dehydrogenase Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Activation Assay

Journal: Cell Death Discovery

Article Title: Pyroptosis leads to loss of centrosomal integrity in macrophages

doi: 10.1038/s41420-024-02093-1

Figure Lengend Snippet: THP1 Null2 and THP1 NLRP3−/− cells were stimulated with nigericin (10 µM, 45 min) ( A – D , N = 4). Lysates were analysed for PCNT as well as loading control β-actin by western blot ( A ). Densitometry analysis of relative protein expression of full-length PCNT-B (340 kDa) ( B ) and cleaved PCNT-B (275 kDa)/PCNT-A (220 kDa) ( C ) compared to the control (β-actin). Cell death was measured as LDH release and shown as percentage relative to total cell death ( D ). THP1 Null2 ( E – H ) or THP1 ATCC ( I – K ) cells were left untreated or treated with punicalagin (50 µM, 15 min) prior to treatment with ZVAD (50 µM, 40 min), after which cells were stimulated with nigericin (10 µM, 45 min) to activate the NLRP3 inflammasome ( N = 3). Lysates were analysed for PCNT protein levels as well as loading control β-actin ( E ). Relative expression of full-length PCNT-B ( F ) and cleaved PCNT-B/PCNT-A ( G ) compared to the control were quantified as described above. Cell death was measured as above ( H ). Immunofluorescence was used to analyse PCNT and ASC ( I ). Percentages of PCNT or ASC speck positive cells relative to total cells ( J ) and both PCNT and ASC positive cells or only ASC speck positive cells in total cells ( K ) were quantified by the ImageJ. N = 3–4 Biologically independent experiments. For multiple comparisons, two‐way ANOVA with the Tukey’s test for comparing nigericin treated THP1 Null2 and THP1 NLRP3−/− ( A – D ) and one‐way ANOVA with the Dunnett’s test in THP1 Null2 ( E – H ) and were applied. Data was shown as mean ± S.D., * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001 were considered statistically significant and compared to nigericin alone treatment.

Article Snippet: PMA differentiated THP1 ATCC cells were left untreated or treated with MG132 (10 μM, 2 h), or E-64-D (20 μM, 2 h), or Ca-074Me (50 μM, 15 min), or pepstatin A (10 μM, 15 min) or bafilomycin A1 (100 μM, 15 min) before stimulation with nigericin (10 μM, 45 min) ( A – J , N = 3).

Techniques: Control, Western Blot, Expressing, Immunofluorescence

Journal: Cell Death Discovery

Article Title: Pyroptosis leads to loss of centrosomal integrity in macrophages

doi: 10.1038/s41420-024-02093-1

Figure Lengend Snippet: THP1 GFP-NLRP3 cells were left untreated or treated with ZVAD (50 µM, 40 min) or MCC950 (10 µM, 15 min) prior to treatment with nigericin (10 µM, 45 min) to activate the NLRP3 inflammasome ( A – D ). Images show NLRP3 (green), ASC (Red) and PCNT (purple). Nuclei are shown in blue. ASC speck positive cells were quantified and plotted as percentages versus total number of cells ( B ). Percentages of cells with NLRP3 at centrosome relative to cells with centrosome and cells with no PCNT in total cells were calculated respectively ( C , D ). THP1 GFP-NLRP3 cells were left untreated or treated with MCC950 (10 µM, 15 min) before stimulation with nigericin (10 µM) at different time points as indicated ( E , F ). Percentages of cells with ASC specks, or with NLRP3 and PCNT, or with no PCNT in total cells were calculated. 300 cells were counted and analysed per experiment, N = 3, Biologically independent experiments.

Article Snippet: PMA differentiated THP1 ATCC cells were left untreated or treated with MG132 (10 μM, 2 h), or E-64-D (20 μM, 2 h), or Ca-074Me (50 μM, 15 min), or pepstatin A (10 μM, 15 min) or bafilomycin A1 (100 μM, 15 min) before stimulation with nigericin (10 μM, 45 min) ( A – J , N = 3).

Techniques:

Journal: Cell Death Discovery

Article Title: Pyroptosis leads to loss of centrosomal integrity in macrophages

doi: 10.1038/s41420-024-02093-1

Figure Lengend Snippet: THP1 Null2 cells were left untreated or treated with YVAD (50 µM, 40 min), then stimulated with nigericin (10 µM, 45 min) ( A – F , N = 3). Lysates were analysed for PCNT as well as loading control β-actin by western blot ( A ). Relative expression of full-length PCNT-B and cleaved PCNT-B/PCNT-A compared to the β-actin was quantified respectively ( B , C ). Cell death ( D ), caspase-1 activity ( E ) and IL-18 ( F ) were measured as described above. THP1 ATCC and THP1 Caspase-1−/− cells were directly stimulated with nigericin (10 µM, 45 min) ( G – J , N = 4). Lysates were analysed for PCNT, caspase-1 and caspase-3 as well as loading control β-actin ( G ). Cell death ( H ), caspase-1 activity in supernatants ( I ) and IL-18 release ( J ) were measured. N = 3–4 biologically independent experiments. For multiple comparisons, one‐way ANOVA with the Dunnett’s test for YVAD in THP1 Null2 cells ( B – F ) and two‐way ANOVA with the Tukey’s test for comparing nigericin treated THP1 ATCC and THP1 Caspase-1−/− cells (I-K)were applied. Data was shown as mean ± S.D., * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001 were considered statistically significant and compared to nigericin alone treatment.

Article Snippet: PMA differentiated THP1 ATCC cells were left untreated or treated with MG132 (10 μM, 2 h), or E-64-D (20 μM, 2 h), or Ca-074Me (50 μM, 15 min), or pepstatin A (10 μM, 15 min) or bafilomycin A1 (100 μM, 15 min) before stimulation with nigericin (10 μM, 45 min) ( A – J , N = 3).

Techniques: Control, Western Blot, Expressing, Activity Assay

Journal: Cell Death Discovery

Article Title: Pyroptosis leads to loss of centrosomal integrity in macrophages

doi: 10.1038/s41420-024-02093-1

Figure Lengend Snippet: THP1 ATCC and THP1 GSDMD−/− cells were left untreated or treated with ZVAD (50 µM, 40 min) or Z-DEVD (20 µM, 2 h), after which cells were stimulated with nigericin (10 µM, 45 min) to activate the NLRP3 inflammasome ( A – F , N = 3 biologically independent experiments). Cell death was measured as described above and shown as percentage relative to total cell death ( A , D ). Caspase-1 activity was measured by caspase-1 assay and shown as fold change relative to control ( B , E ). Lysates were analysed for PCNT, caspase-1 and caspase-3 as well as loading control β-actin ( C , F ). One‐way ANOVA with the Dunnett’s test analysis. Data was shown as mean ± S.D., * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001 were considered statistically significant when compared to nigericin alone treatment.

Article Snippet: PMA differentiated THP1 ATCC cells were left untreated or treated with MG132 (10 μM, 2 h), or E-64-D (20 μM, 2 h), or Ca-074Me (50 μM, 15 min), or pepstatin A (10 μM, 15 min) or bafilomycin A1 (100 μM, 15 min) before stimulation with nigericin (10 μM, 45 min) ( A – J , N = 3).

Techniques: Activity Assay, Control

Journal: Cell Death Discovery

Article Title: Pyroptosis leads to loss of centrosomal integrity in macrophages

doi: 10.1038/s41420-024-02093-1

Figure Lengend Snippet: LPS (1 µg/ml, 4 h) primed THP1 ATCC cells were left untreated or treated with ionomycin (10 µM, 1 h) ( A – E ). Lysates were analysed for PCNT as well as loading control β-actin ( A – C ). Cell death was measured as LDH release and shown as percentage relative to total cell death ( D ). Caspase-1 activity in supernatants was shown as fold change relative to control ( E ; N). LPS (1 µg/ml, 4 h) primed THP1 ATCC cells were left untreated or pre-treated with calpeptin (40 µM, 15 min), then stimulated with nigericin to activate the NLRP3 inflammasome ( F – L ). Lysates were analysed for PCNT as well as loading control β-actin ( F – H ). Cell death ( I ), caspase-1 activity ( J ), IL-18 ( K ) and IL1α ( L ) were measured. For blots ( A , F ), N = 2. Otherwise, N ≥ 3, Biologically independent experiments. T-test analysis ( B – E ) and one‐way ANOVA with the Dunnett’s test analysis ( G – J ). Data was shown as mean ± S.D., * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001 were considered statistically significant when compared to nigericin alone treatment.

Article Snippet: PMA differentiated THP1 ATCC cells were left untreated or treated with MG132 (10 μM, 2 h), or E-64-D (20 μM, 2 h), or Ca-074Me (50 μM, 15 min), or pepstatin A (10 μM, 15 min) or bafilomycin A1 (100 μM, 15 min) before stimulation with nigericin (10 μM, 45 min) ( A – J , N = 3).

Techniques: Control, Activity Assay

Journal: Cell Death Discovery

Article Title: Pyroptosis leads to loss of centrosomal integrity in macrophages

doi: 10.1038/s41420-024-02093-1

Figure Lengend Snippet: PMA differentiated THP1 ATCC cells were left untreated or treated with MG132 (10 µM, 2 h), or E-64-D (20 µM, 2 h), or Ca-074Me (50 µM, 15 min), or pepstatin A (10 µM, 15 min) or bafilomycin A1 (100 µM, 15 min) before stimulation with nigericin (10 µM, 45 min) ( A – J , N = 3). Lysates were analysed for PCNT as well as loading control β-actin by western blot ( A , C , E , G , I ). Cell death ( B , D , F , H , J ) was measured as percentage of LDH release. Data was shown as mean ± S.D. and one‐way ANOVA with the Dunnett’s test analysis performed. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001 were considered statistically significant when compared to nigericin alone treatment.

Article Snippet: PMA differentiated THP1 ATCC cells were left untreated or treated with MG132 (10 μM, 2 h), or E-64-D (20 μM, 2 h), or Ca-074Me (50 μM, 15 min), or pepstatin A (10 μM, 15 min) or bafilomycin A1 (100 μM, 15 min) before stimulation with nigericin (10 μM, 45 min) ( A – J , N = 3).

Techniques: Control, Western Blot

Journal: Frontiers in Immunology

Article Title: Negative Regulation of RNF90 on RNA Virus-Triggered Antiviral Immune Responses Targeting MAVS

doi: 10.3389/fimmu.2021.730483

Figure Lengend Snippet: RNF90 overexpression inhibits RNA virus-triggered innate immune responses. (A) PMA-THP1 cells were stimulated with HSV-1 (MOI = 1) or VSV (MOI = 1) for 8 h. Afterwards, the cells were lysed for immunoblot assays. (B) PMA-THP1 cells were transfected with 1 μg/ml HSV60, 2.5 μg/ml poly(I:C), or 1 μg/ml cGAMP (B) for 8 h. Afterwards, the cells were lysed for immunoblot assays. (C, D) HEK293T cells were transfected with an empty vector (Vec) or RNF90 plasmid. At 24 h after transfection, HEK293T cells were treated with poly(I:C) (2.5 μg/ml) (C) or VSV (MOI = 1) (D) for indicated time periods. Then the cells were lysed for real-time PCR analysis. β-actin was used as a loading control in all the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Phorbol-12-myristate-13-acetate (PMA)-differentiated THP1 (PMA-THP1, a human macrophage-like cell line) cells referred to THP1 cells that were pretreated with 100ng/ml PMA for 24 h. All cells were supplemented with 10% FBS (Gibco), 4 mM L-glutamine, 100μg/ml penicillin, and 100U/ml streptomycin under humidified conditions with 5% CO 2 at 37°C.

Techniques: Over Expression, Virus, Western Blot, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Control

Journal: Frontiers in Immunology

Article Title: Negative Regulation of RNF90 on RNA Virus-Triggered Antiviral Immune Responses Targeting MAVS

doi: 10.3389/fimmu.2021.730483

Figure Lengend Snippet: RNF90 knockdown promotes RNA virus-triggered innate immune responses. (A) PMA-THP1 cells were transfected with control siRNA (SC) or RNF90-specific siRNA (R2 and R3). At 24 h after transfection, the cells were infected with VSV (MOI = 1) for 8 h and then lysed for immunoblot assay. (B, C) PMA-THP1 cells were transfected with control siRNA (SC) or RNF90-specific siRNA (R3). 24 h later, PMA-THP1 cells were treated with poly(I:C) (2.5 μg/ml) for 8 h (B) or VSV (MOI = 1) (C) for indicated time periods. Then the cells were lysed for real-time PCR analysis. (D) PMA-THP1 cells were transfected with control siRNA (SC) or RNF90-specific siRNA (R2, R3). At 24 h after transfection, the cells were infected with VSV (MOI = 1) for 8 h. Then, the cells were lysed for immunoblot analysis. (E) PMA-THP1 cells were transfected with control siRNA (SC) or RNF90-specific siRNA (R3). At 24 h after transfection, the cells were infected with VSV (MOI = 1) for 24 h. The titers of VSV were determined by standard plaque assay. β-actin was used as a loading control in all the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Phorbol-12-myristate-13-acetate (PMA)-differentiated THP1 (PMA-THP1, a human macrophage-like cell line) cells referred to THP1 cells that were pretreated with 100ng/ml PMA for 24 h. All cells were supplemented with 10% FBS (Gibco), 4 mM L-glutamine, 100μg/ml penicillin, and 100U/ml streptomycin under humidified conditions with 5% CO 2 at 37°C.

Techniques: Knockdown, Virus, Transfection, Control, Infection, Western Blot, Real-time Polymerase Chain Reaction, Plaque Assay

Journal: Frontiers in Immunology

Article Title: Negative Regulation of RNF90 on RNA Virus-Triggered Antiviral Immune Responses Targeting MAVS

doi: 10.3389/fimmu.2021.730483

Figure Lengend Snippet: RNF90 inhibits RNA virus-triggered innate immune responses independent of STING Signaling. (A) WT and STING-deficient (KO) PMA-THP1 cells were lysed for immunoblot analysis. β-actin served as a loading control. (B) STING-deficient (KO) PMA-THP1 cells were transfected with control siRNA (SC) or RNF90-specific siRNA (R3) for 24 h and then transfected with poly (I:C) or infected with VSV for 8 h. Then, the cells were lysed for real-time PCR assays. The data are representative of three independent experiments and are presented as mean ± SEM. ** P < 0.01, *** P < 0.001.

Article Snippet: Phorbol-12-myristate-13-acetate (PMA)-differentiated THP1 (PMA-THP1, a human macrophage-like cell line) cells referred to THP1 cells that were pretreated with 100ng/ml PMA for 24 h. All cells were supplemented with 10% FBS (Gibco), 4 mM L-glutamine, 100μg/ml penicillin, and 100U/ml streptomycin under humidified conditions with 5% CO 2 at 37°C.

Techniques: Virus, Western Blot, Control, Transfection, Infection, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Negative Regulation of RNF90 on RNA Virus-Triggered Antiviral Immune Responses Targeting MAVS

doi: 10.3389/fimmu.2021.730483

Figure Lengend Snippet: RNF90 interacts with MAVS. (A) HEK293T cells were transfected with an IFN-β luciferase reporter, together with RIG-I, MAVS, TBK1, or IRF3-5D and the empty vector (Vec) or the RNF90 plasmid. At 24h after transfection, the cells were lysed for luciferase assay and immunoblot analysis. β-actin served as a loading control. ** P < 0.01. (B) HEK293T cells were transfected with HA-RNF90, together with the empty vector (-) or Flag-MAVS plasmid. At 24 h after transfection, the cells were lysed and subjected to immunoprecipitation (IP) and immunoblot (IB) analysis. (C) HEK293T cells were transfected with Flag-RNF90, at 24 h after transfection, HEK293T cells were stimulated with poly(I:C) (2.5 μg/ml) or left untreated for another 8 h. Immunofluorescence was performed using anti-Flag (green) and anti-MAVS (red). Mitochondria and Nuclei were stained with Mito Tracker or DAPI, respectively. Scale bars, 10 μm. (D, E) PMA-THP1 cells (D) or HaCaT cells (E) were infected with VSV (MOI = 1) for indicated time points, and then the cell lysates were subjected to immunoprecipitation (IP) and immunoblot (IB) analysis as indicated. (F) A schematic presentation of full-length MAVS and its mutants. (G) HEK293T cells were transfected with plasmids as indicated. At 24 h after transfection, the cell lysates were subjected to immunoprecipitation (IP) and immunoblot (IB) analysis as indicated. (H) A schematic presentation of full-length RNF90 and its mutants. (I) HEK293T cells were transfected with plasmids as indicated. At 24 h after transfection, the cell lysates were subjected to immunoprecipitation (IP) and immunoblot (IB) analysis as indicated. The data are representative of three independent experiments.

Article Snippet: Phorbol-12-myristate-13-acetate (PMA)-differentiated THP1 (PMA-THP1, a human macrophage-like cell line) cells referred to THP1 cells that were pretreated with 100ng/ml PMA for 24 h. All cells were supplemented with 10% FBS (Gibco), 4 mM L-glutamine, 100μg/ml penicillin, and 100U/ml streptomycin under humidified conditions with 5% CO 2 at 37°C.

Techniques: Transfection, Luciferase, Plasmid Preparation, Western Blot, Control, Immunoprecipitation, Immunofluorescence, Staining, Infection