bms 303141  (Merck & Co)

Journal: bioRxiv

Article Title: Ratiometric Fluorescent Protein Biosensors Reveal Citrate Dynamics and Cellular Heterogeneity

doi: 10.64898/2026.04.16.718871

Figure Lengend Snippet: A-B. Pseudo-colored images of HeLa cells expressing ratioCitron1Low ( A ) and ratioCitron1High ( B ) in cytosol before and after addition of citrate. Scale bar: 100 µm. C-D. Δ R / R and Δ F / F values of ratioCitron1Low ( C ) and ratioCitron1High ( D ) versus time upon the addition of citrate. HeLa cells were incubated with 4 μM digitonin for 10 mins before the experiment and a final concentration of 20 mM citrate was added at t = 0 (ratioCitron1Low n = 16, ratioCitron1High n = 19, mean ± SD). E. Time courses of Δ R / R ₀ of ratioCitron1 variants (ratioCitron1Low ( n = 20), ratioCitron1High ( n = 10), ratioCitron1Low-con ( n = 5), ratioCitron1High-con ( n = 5)) expressed in HeLa cells upon the citrate titration. HeLa cells expressing ratioCitron1 variants were treated with digitonin and citrate titration was carried out. The final concentration of citrate is 100 μM at 0 min, 1 mM at 5 min, 10 mM at 10 min (mean ± SD). F-G. Δ R / R values of ratioCitron1 variants expressed in cytosol ( F ) and in mitochondria (mito-ratioCiton1) ( G ) of HeLa cells upon the addition of the ACLY inhibitor of BMS-303141. H. Δ R / R values of ratioCitron1 variants expressed in mitochondria (mito-ratioCitron1) of HeLa cells upon the addition of MPC inhibitor UK-5099 (final concentration: 25 μM). Δ F/F = ( F on - F off )/ F off , and F with excitation 405 nm or 470 nm and emission 518/45 nm. Δ R / R = ( R on - R off )/ R off , and R = ( F with excitation 470 nm and emission 518/45 nm) / ( F with excitation 405 nm and emission 518/45 nm).

Article Snippet: For imaging the treatment with MPC inhibitor UK-5099 (MedChemExpress) and ACLY inhibitor BMS-303141 (MedChemExpress), Hank’s balanced salt solution (HBSS; Nacalai Tesque, 09735-75) and 10 mM HEPES (Nacalai Tesque, 177557-94) was used as imaging buffer.

Techniques: Expressing, Incubation, Concentration Assay, Titration

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: 4OI reduces expression of ATP-dependent citrate lyase required for HMPV replication. ( A ) 4OI reduces ATP-dependent citrate lyase ( ACLY) expression. MDMs were treated with 4OI (250 µM) or DMSO prior to infection with HMPV for 24 h. Expression levels of ACLY, FASN , and SCD1 mRNA were quantified relative to untreated, uninfected MDMs via qRT-PCR ( n ≥ 4). ( B ) ACLY inhibition reduces HMPV levels. MDMs were pretreated with 10 or 20 µM of the ACLY inhibitor BMS-303141 or DMSO prior to infection with HMPV for 24 h. HMPV N-gene mRNA was determined by qRT-PCR ( n = 4). Protein expression of HMPV N and GAPDH was analysed via immunoblotting of whole cell lysates ( n = 2). Protein levels were quantified by normalizing of band intensities against GAPDH and expressed as fold change compared to siNTC-transfected, infected MDMs. Multiple comparisons were analysed by a paired one-way ANOVA with Tukey post-hoc test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001 and ns, not significant. ( C ) HMPV stimulates increased level of neutral lipids in infected MDMs. MDMs were left uninfected (−) or infected with a MOI 1 of GFP-expressing recombinant HMPV for 24 h. HCS LipidTOX™ Deep Red neutral lipid stain was used to monitor lipids by confocal microscopy. Left panels: Representative images for each treatment showing HCS LipidTOX™ Deep Red (magenta) and HMPV (green). Right panel: Quantification of the LipidTOX™ signal per area. Signal was quantified from z-stacks of six fields of view per condition using the 20× numerical aperture yielding to ~2000 cells per condition and normalized to the area. Scalebar is adjusted to 100 µm ( n = 1). The data were assessed for normality and then compared with a non-parametric, two-tailed Mann–Whitney test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001, and ns, not significant.

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Expressing, Infection, Quantitative RT-PCR, Inhibition, Western Blot, Transfection, Recombinant, Staining, Confocal Microscopy, Two Tailed Test, MANN-WHITNEY

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: Exogenously added 4OI, but not itaconate or citraconate, reduces HMPV levels in human MDMs. (A–C) MDMs were preincubated with 100 or 250 µM 4OI or its vehicle, DMSO, prior to HMPV infection for 24 h. Levels of HMPV N-gene mRNA were quantified by qRT-PCR ( A ; n ≥ 5), HMPV N and GAPDH protein levels were analysed by immunoblotting ( B ; n ≥ 5), while infectious HMPV/HMPV titers in MDM supernatants was determined as TCID 50 /ml ( C ; n = 5). (D, E) MDMs were treated with itaconate (ITA; 0.5, 5, 10, and 20 mM) prior to HMPV infection for 24 h. Levels of HMPV N-gene mRNA were quantified by qRT-PCR ( D ; n ≥ 2), while HMPV N and GAPDH proteins were analysed by immunoblotting ( E ; n ≥ 2). (F, G) MDMs were treated with citraconate (CIT; 1, 10, 20, and 50 mM) prior to HMPV infection for 24 h. Levels of the HMPV N-gene mRNA were quantified by qRT-PCR ( F ; n = 4), while HMPV N and GAPDH protein levels were analysed by immunoblotting ( G ; n = 2). Protein levels were quantified by normalizing band intensities against GAPDH and expressed as fold change compared to untreated (i.e. no itaconate variant) but infected MDMs. Multiple comparisons were analysed by a paired onw-way ANOVA with Tukey post-hoc test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001, and ns, not significant.

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Variant Assay

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: The Nrf2 pathway is induced by 4OI, but not by itaconate or citraconate, and limits HMPV in human macrophages. MDMs were preincubated with ( A ) 100 and 250 µM of 4OI or DMSO, ( B ) 0.5, 5, 10, and 20 mM of itaconate, and ( C ) 1, 10, 20, and 50 mM of citraconate prior to infection with HMPV for 24 h. (A–C) Levels of HO1 and NQO1 mRNA were quantified by qRT-PCR. The expression of HO1 and GAPDH protein was analysed using immunoblotting of whole cell lysates. ( A ) n ≥ 5 for mRNA and protein; ( B ) n ≥ 2 for mRNA and protein; ( C ) n = 4 for mRNA and n = 2 for protein. HO1 and NQO1 mRNA levels were assessed relative to untreated, uninfected MDMs for all experiments. HO1 protein levels were quantified by normalization of band intensities against GAPDH and expressed as fold change compared to untreated, infected MDMs. ( D ) MDMs were transfected with 20 nM NFE2L2 (Nrf2) siRNA or siNTC and infected with HMPV for 24 h. Nrf2, HMPV N protein, and GAPDH were analysed by immunoblotting of whole cell lysates and quantified ( n = 4). Nrf2 protein level is presented as fold change relative to siNTC-treated uninfected MDMs (left panel), while HMPV N-protein is presented as relative to siNTC-treated HMPV-infected MDMs (right panel). In panels (A–C), multiple comparisons were analysed by a paired onw-way ANOVA with Tukey post-hoc test, in panel (D) unpaired Student’s t - test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001 and ns, not significant.

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Infection, Quantitative RT-PCR, Expressing, Western Blot, Transfection

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: HMPV induction of IRG1 in human macrophages is dependent on type I IFN. ( A ) MDMs were infected with HMPV for the indicated time points. HMPV N gene or IRG1 mRNA levels were determined by qRT-PCR analysis ( n = 3), while protein levels of IRG1, HMPV N, and GAPDH were analysed by immunoblotting ( n = 2). IRG1 protein levels shown are for the same donor sample used in Fig. of , thus panels showing HMPV N and GAPDH levels being identical to the ones shown . ( B ) MDMs were treated with 500 ng/ml LPS for 2 h. IRG1 mRNA levels were assessed by qRT-PCR and normalized to untreated (−) control ( n = 5). ( C ) MDMs were transfected with 10 μg/ml poly IC for 6 or 24 h. IRG1 mRNA levels were assessed by qRT-PCR and normalized to untreated (−) cells ( n ≥ 2). ( D ) MDMs were pre-incubated (30 min) or not with 10 μg/ml of neutralizing IFNAR antibody before infection with HMPV for 24 h. IRG1 mRNA expression was determined using qRT-PCR and normalized to untreated (−) cells ( n = 4). ( E ) MDMs were treated with 100 U/ml recIFN-β for 24 h. IRG1 mRNA expression was determined using qRT-PCR and normalized to untreated (−) cells ( n = 4). ( F ) MDMs were preincubated with the JAK1/JAK2 inhibitor ruxolitinib (5 or 10 μM) prior to infection with HMPV for 18 h or treatment with IFN-β for 3 h. Protein levels in whole cell lysates were analysed for IRG1, STAT1 (Tyr701), STAT1, and GAPDH via immunoblot ( n = 2).

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Control, Transfection, Incubation, Expressing

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: TBK1 and NF-κB positively regulate IRG1 levels in HMPV-infected human macrophages. MDMs were incubated with 5 or 10 μM of the TBK1 inhibitor BX795 for 30 min before infection with HMPV for 24 h. ( A ) IRG1 and IFN-β mRNA levels ( n ≥ 3) were analysed by qRT-PCR (left panels), while in panel ( B ) IRG1 and GAPDH protein levels were determined by immunoblotting, quantified, and presented with SD relative to uninfected cells treated with DMSO ( n = 2; right panel). (C–K) MDMs were transfected with siRNAs targeting the NF-κB subunit RELA (C–E), IRF1 (F–H), RIPK3 (I–K), or control siRNA (siNTC) before infection with HMPV for 6, 9, or 24 h and analysis of IRG1 mRNA levels by qRT-PCR or IRG1, RelA/p65, IRF1, RIPK3, or GAPDH protein levels by immunoblotting. IRG1 mRNA levels were assessed relative to siNTC-transfected, uninfected MDMs for all experiments. ( C, E ) IRG1 mRNA after 6 h ( n = 3) or 24 h HMPV ( n = 3). ( D ) Protein levels of IRG1, RelA/p65, and GAPDH ( n = 3) after 9 h HMPV. ( F, H ) IRG1 mRNA after 6 h ( n = 4) or 24 h of infection ( n = 3). ( G ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( I, K ) IRG1 mRNA after 6 h ( n = 3) or 24 h of infection ( n = 3). ( J ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( L ) MDMs were incubated with 100 nM of the RIPK3 inhibitor GSK 872 for 1 h before infection with HMPV for 24 h. IRG1 mRNA levels were normalized relative to uninfected cells treated with DMSO ( n = 2). Single comparison between control (NTC) siRNA and target-siRNA conditions were calculated using paired t -test with Tukey post-hoc test. * P < .05, ** P < .01, *** P < .001, **** P < .0001; ns = non-significant. See also

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Infection, Incubation, Quantitative RT-PCR, Western Blot, Transfection, Control, Comparison

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: 4OI, itaconate, and citraconate differ in regulating IRG1 and the IFN-β response upon HMPV infection. MDMs were preincubated with ( A ) 100 and 250 µM of 4OI or DMSO, ( B ) 0.5, 5, 10, and 20 mM of itaconate, and ( C ) 1, 10, 20, and 50 mM of citraconate prior to infection with HMPV for 24 h. Levels of IRG1 and IFNB mRNA (left panels) were quantified by qRT-PCR. The expression of STAT1 (Tyr701), STAT1, IRG1, and GAPDH protein (right panels) was analysed using immunoblotting of whole-cell lysates and quantification STAT1 (Tyr701) expression. ( A ) n ≥ 5 for mRNA and protein; ( B ) n ≥ 2 for mRNA and protein; and ( C ) n = 4 for mRNA and n = 2 for protein. IFNB and IRG1 mRNA levels were assessed relative to untreated, uninfected MDMs for all experiments. Protein levels were quantified by normalization of band intensities against GAPDH and was expressed as fold change compared to untreated, uninfected MDMs. Multiple comparisons were analysed by a paired onw-way ANOVA with Tukey post-hoc test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001, and ns, not significant.

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Infection, Quantitative RT-PCR, Expressing, Western Blot

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: 4OI reduces expression of ATP-dependent citrate lyase required for HMPV replication. ( A ) 4OI reduces ATP-dependent citrate lyase ( ACLY) expression. MDMs were treated with 4OI (250 µM) or DMSO prior to infection with HMPV for 24 h. Expression levels of ACLY, FASN , and SCD1 mRNA were quantified relative to untreated, uninfected MDMs via qRT-PCR ( n ≥ 4). ( B ) ACLY inhibition reduces HMPV levels. MDMs were pretreated with 10 or 20 µM of the ACLY inhibitor BMS-303141 or DMSO prior to infection with HMPV for 24 h. HMPV N-gene mRNA was determined by qRT-PCR ( n = 4). Protein expression of HMPV N and GAPDH was analysed via immunoblotting of whole cell lysates ( n = 2). Protein levels were quantified by normalizing of band intensities against GAPDH and expressed as fold change compared to siNTC-transfected, infected MDMs. Multiple comparisons were analysed by a paired one-way ANOVA with Tukey post-hoc test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001 and ns, not significant. ( C ) HMPV stimulates increased level of neutral lipids in infected MDMs. MDMs were left uninfected (−) or infected with a MOI 1 of GFP-expressing recombinant HMPV for 24 h. HCS LipidTOX™ Deep Red neutral lipid stain was used to monitor lipids by confocal microscopy. Left panels: Representative images for each treatment showing HCS LipidTOX™ Deep Red (magenta) and HMPV (green). Right panel: Quantification of the LipidTOX™ signal per area. Signal was quantified from z-stacks of six fields of view per condition using the 20× numerical aperture yielding to ~2000 cells per condition and normalized to the area. Scalebar is adjusted to 100 µm ( n = 1). The data were assessed for normality and then compared with a non-parametric, two-tailed Mann–Whitney test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001, and ns, not significant.

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Expressing, Infection, Quantitative RT-PCR, Inhibition, Western Blot, Transfection, Recombinant, Staining, Confocal Microscopy, Two Tailed Test, MANN-WHITNEY