Journal: bioRxiv

Article Title: Microbiome-derived hydroxyphenyl propanoates enhance antitumour immunity by potentiating gasdermin D activity in tumour-associated myeloid cells

doi: 10.64898/2026.04.23.720410

Figure Lengend Snippet: a, representation of the proteins with denaturing curves significantly altered (red) or unaltered (black) by 3,2-HPP treatment of THP1-Dual™ cells, using thermal proteome profiling (two combined experiments, proteomic coverage: 4301, NPARC test); b, protein-protein interaction network of proteins with denaturation curves altered by 3,2-HPP treatment (red) and the transcriptional regulators of 3, 2-HPP impacted genes in M3-9-M tumours (yellow); c, impact of 3,2-HPP treatment of THP1-Dual™ cells on the denaturation curve of GSDMD (two-combined experiments, NPARC test); d, NF-κB induction in THP1-Dual™ reporter cells treated with conditioned media harvested from WT or Gsdmd- KO THP1 cells treated with LPS ± HPP metabolites ± IL-1α and −1β neutralizing antibody (three combined experiments, one-way ANOVA with Bonferroni multiple comparison test); e-h, [secreted IL-1α and −1β] in conditioned media harvested from THP-1 WT or Gsdmd -KO cells, or human PBMCs, cultured for 16 hours in LPS ± HPPs (two combined experiments, one-way ANOVA with Bonferroni multiple comparison test); i-l, [secreted IL-1α and −1β] in conditioned media harvested from THP-1 WT vs. Gsdmd -KO cells, or human PBMCs, cultured for 16 hours in LPS and NG ± HPPs (two combined experiments, one-way ANOVA with Bonferroni multiple comparison test); m, western blot of THP-1 cells treated with LPS ± NG ± HPPs (representative of two experiments).

Article Snippet: To neutralize IL-1 receptor signalling, the following antibodies were used: anti-human IL-1α (1 μg/mL; clone 7D4; mabg-hil1a-3; InvivoGen), anti-human IL-1β (1 μg/mL; clone 4H5; mabg-hil1b-3; InvivoGen) and IgG1 isotype control (1 μg/mL; clone T8E5; mabg1-ctrlm; InvivoGen).

Techniques: Comparison, Cell Culture, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: Interleukin (IL)−1β levels are elevated in MK2/3 double-knockout (DKO) mice. ( a ) Il1b mRNA levels are increased in untreated and ( b ) IL-1α-treated (5 ng/mL, 1 h) MK2/3 -DKO bone marrow-derived macrophages (BMDMs) compared to wild type (WT). WT n = 14, DKO n = 13. ( c ) Basal IL-1β protein levels are elevated in MK2/3 -DKO BMDM. Elongation factor 2 (EF2) serves as a control. One representative Western blot of total WT n = 4, DKO n = 6. ( d ) The concentration of IL-1β is higher in the supernatant of untreated and ( e ) IL-1α (5 ng/mL, 4 h) + Nigericin (20 µM, 8 h) or ( f ) IL-1α + ATP (5 mM, 5.5 h)-treated MK2/3 -DKO BMDM than in WT. ( d , e ) WT n = 9, DKO n = 8. ( f ) n = 3/group. ( g ) The basal concentration of IL-1β is elevated in the serum of MK2/3 -DKO mice. n = 6 mice/group, whereby one sample/group was pooled from 3 mouse sera. Mean ± SEM, Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMDM (5 × 10 5 cells/well), i MK2 -KO or RAW 264.7 cells (both 2 × 10 5 cells/well) were seeded, and treated one day later with the indicated concentrations and durations of recombinant murine IL-1α (211-11A, PeproTech GmbH, Hamburg, Germany), LPS ( Escherichia coli 0127:B8; Sigma-Aldrich, Merck, St. Louis, MO, USA), Actinomycin D (Cayman Chemical Company, Ann-Arbor, MI, USA), B022 (MedChemExpress, Monmouth Junction, NJ, USA), BMS-345541 (Axon Medchem B.V., Groningen, Netherlands), CAY10657 (Cayman Chemical Company, Ann-Arbor, MI, USA), SML1160 (Sigma-Aldrich, Merck, St. Louis, MO, USA), IKK inhibitor XII (HPN-01, Sigma-Aldrich, Merck, St. Louis, MO, USA), sc-514 (Cayman Chemical Company, Ann-Arbor, MI, USA), T-5224 (Cayman Chemical Company, Ann-Arbor, MI, USA), Takinib (MedChemExpress, Monmouth Junction, NJ, USA) or Nutlin-3 (SCBT, Dallas, TX, USA).

Techniques: Double Knockout, Derivative Assay, Control, Western Blot, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: The level of IL-1β is increased in immortalized MK2 -KO (i MK2 -KO) cells. ( a ) i MK2 -KO cells transduced with an empty vector (+ GFP ) showed elevated levels of Il1b mRNA compared to MK2 -rescued cells (+ MK2 ) (right, n = 8), similar to those observed in MK2/3 -DKO and WT bone-marrow-derived macrophages (BMDMs) (left; WT n = 9, DKO n = 8). ( b ) i MK2 -KO + GFP cells show increased Il1b mRNA after IL-1α treatment (5 ng/mL) compared to MK2 -rescued cells. n = 3. ( c ) RAW cells treated with MK2 siRNA have higher Il1b mRNA levels compared to the control after IL-1α stimulation. ( d ) Similar to MK2 , rescuing MK3 decreases the level of Il1b mRNA in resting or ( e ) IL-1α (5 ng/mL, 1 h)-treated i MK2 -KO cells, ( f ) as well as the basal level of IL-1β protein. Histone H3 serves as a control. ( a , c ) Student’s t -test, ( b ) 2W-RM-ANOVA with Bonferroni posttests, ( d , e ) 1W-ANOVA with Tukey’s Multiple Comparison Test, mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMDM (5 × 10 5 cells/well), i MK2 -KO or RAW 264.7 cells (both 2 × 10 5 cells/well) were seeded, and treated one day later with the indicated concentrations and durations of recombinant murine IL-1α (211-11A, PeproTech GmbH, Hamburg, Germany), LPS ( Escherichia coli 0127:B8; Sigma-Aldrich, Merck, St. Louis, MO, USA), Actinomycin D (Cayman Chemical Company, Ann-Arbor, MI, USA), B022 (MedChemExpress, Monmouth Junction, NJ, USA), BMS-345541 (Axon Medchem B.V., Groningen, Netherlands), CAY10657 (Cayman Chemical Company, Ann-Arbor, MI, USA), SML1160 (Sigma-Aldrich, Merck, St. Louis, MO, USA), IKK inhibitor XII (HPN-01, Sigma-Aldrich, Merck, St. Louis, MO, USA), sc-514 (Cayman Chemical Company, Ann-Arbor, MI, USA), T-5224 (Cayman Chemical Company, Ann-Arbor, MI, USA), Takinib (MedChemExpress, Monmouth Junction, NJ, USA) or Nutlin-3 (SCBT, Dallas, TX, USA).

Techniques: Transduction, Plasmid Preparation, Derivative Assay, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: The non-canonical NF-κB pathway is activated in i MK2 -KO cells (mRNA). ( a ) Inhibition of the canonical NF-κB pathway using the IKKβ inhibitors Takinib (10 µM, 2 h) and sc-514 (10 µM, 2 h) reduced Il1b mRNA levels in IL-1α- and LPS-treated i MK2 -KO cells, but did not affect basal Il1b levels. Inhibiting IKKα and IKKβ with HPN-01 (10 µM, 2 h) reduced Il1b mRNA levels in untreated (UT) and IL-1α (5 ng/mL, 1 h)- or LPS-stimulated cells (100 ng/mL, 1 h). Inhibition of the non-canonical NF-κB pathway by IKKα inhibitor B022 (5 µM, 2 h) mainly reduced basal Il1b mRNA. ( b , c ) IL-1β protein level is reduced in resting i MK2 -KO cells after treatment with B022 (5 µM, 7 h). GAPDH serves as a control. ( d ) Il1b mRNA is reduced in MK2/3 -DKO BMDMs after treatment with B022 (5 µM, 2 h). ( e ) The level of Map3k14 mRNA is increased in i MK2 -KO + GFP . ( f ) Relb mRNA level is increased in UT i MK2 -KO + GFP cells. ( g ) Relb and ( h ) Nfkb2 mRNA levels are increased in IL-1α (5 ng/mL, 1 h)-stimulated i MK2 -KO + GFP cells. ( i ) Basal Traf2 mRNA is reduced in i MK2 -KO + GFP cells. ( j ) The Traf3 mRNA level is not changed significantly. ( a ) 1W-ANOVA with Tukey’s Multiple Comparison Test, ( c – j ) Student’s t -test, mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMDM (5 × 10 5 cells/well), i MK2 -KO or RAW 264.7 cells (both 2 × 10 5 cells/well) were seeded, and treated one day later with the indicated concentrations and durations of recombinant murine IL-1α (211-11A, PeproTech GmbH, Hamburg, Germany), LPS ( Escherichia coli 0127:B8; Sigma-Aldrich, Merck, St. Louis, MO, USA), Actinomycin D (Cayman Chemical Company, Ann-Arbor, MI, USA), B022 (MedChemExpress, Monmouth Junction, NJ, USA), BMS-345541 (Axon Medchem B.V., Groningen, Netherlands), CAY10657 (Cayman Chemical Company, Ann-Arbor, MI, USA), SML1160 (Sigma-Aldrich, Merck, St. Louis, MO, USA), IKK inhibitor XII (HPN-01, Sigma-Aldrich, Merck, St. Louis, MO, USA), sc-514 (Cayman Chemical Company, Ann-Arbor, MI, USA), T-5224 (Cayman Chemical Company, Ann-Arbor, MI, USA), Takinib (MedChemExpress, Monmouth Junction, NJ, USA) or Nutlin-3 (SCBT, Dallas, TX, USA).

Techniques: Inhibition, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: The non-canonical NF-κB pathway is activated in i MK2 -KO cells (protein). ( a – c ) Compared to i MK2 - KO + MK2 cells, untreated (UT) i MK2 -KO + GFP cells have higher levels of the non-canonical proteins RelB and NF-κB2 in nuclear and cytoplasmic fractions, but not of the canonical RelA and NF-κB1 proteins. Following IL-1α treatment (5 ng/mL, 2 h), the nuclear fraction of i MK2 -KO cells showed elevated protein levels of RelB, NF-κB2, RelA, and NF-κB1. p53 and GAPDH serve as controls for successful nuclear/cytoplasmic separation. EF2 acts as a general control, used for normalization. ( d , e ) The basal protein level of TRAF2 is reduced in whole-cell lysis of i MK2 -KO + GFP cells, whereas TRAF3 and cIAP1/2 are not changed. ( f ) The protein level of c-Rel is increased in i MK2 -KO+ GFP cells. Examples of Western blots from different gels are shown. Mean ± SEM, Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMDM (5 × 10 5 cells/well), i MK2 -KO or RAW 264.7 cells (both 2 × 10 5 cells/well) were seeded, and treated one day later with the indicated concentrations and durations of recombinant murine IL-1α (211-11A, PeproTech GmbH, Hamburg, Germany), LPS ( Escherichia coli 0127:B8; Sigma-Aldrich, Merck, St. Louis, MO, USA), Actinomycin D (Cayman Chemical Company, Ann-Arbor, MI, USA), B022 (MedChemExpress, Monmouth Junction, NJ, USA), BMS-345541 (Axon Medchem B.V., Groningen, Netherlands), CAY10657 (Cayman Chemical Company, Ann-Arbor, MI, USA), SML1160 (Sigma-Aldrich, Merck, St. Louis, MO, USA), IKK inhibitor XII (HPN-01, Sigma-Aldrich, Merck, St. Louis, MO, USA), sc-514 (Cayman Chemical Company, Ann-Arbor, MI, USA), T-5224 (Cayman Chemical Company, Ann-Arbor, MI, USA), Takinib (MedChemExpress, Monmouth Junction, NJ, USA) or Nutlin-3 (SCBT, Dallas, TX, USA).

Techniques: Control, Lysis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: The non-canonical NF-κB pathway is activated in i MK2 -KO cells. RNA sequencing revealed increased levels of mRNA for components of the IL-1β and non-canonical NF-κB pathways, as well as for targets of the non-canonical NF-κB pathway, in i MK2 -KO + GFP cells. This finding was reinforced by IL-1α treatment (5 ng/mL, 1 h).

Article Snippet: BMDM (5 × 10 5 cells/well), i MK2 -KO or RAW 264.7 cells (both 2 × 10 5 cells/well) were seeded, and treated one day later with the indicated concentrations and durations of recombinant murine IL-1α (211-11A, PeproTech GmbH, Hamburg, Germany), LPS ( Escherichia coli 0127:B8; Sigma-Aldrich, Merck, St. Louis, MO, USA), Actinomycin D (Cayman Chemical Company, Ann-Arbor, MI, USA), B022 (MedChemExpress, Monmouth Junction, NJ, USA), BMS-345541 (Axon Medchem B.V., Groningen, Netherlands), CAY10657 (Cayman Chemical Company, Ann-Arbor, MI, USA), SML1160 (Sigma-Aldrich, Merck, St. Louis, MO, USA), IKK inhibitor XII (HPN-01, Sigma-Aldrich, Merck, St. Louis, MO, USA), sc-514 (Cayman Chemical Company, Ann-Arbor, MI, USA), T-5224 (Cayman Chemical Company, Ann-Arbor, MI, USA), Takinib (MedChemExpress, Monmouth Junction, NJ, USA) or Nutlin-3 (SCBT, Dallas, TX, USA).

Techniques: RNA Sequencing

Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: The MK2 kinase activity is not involved, but the MK2 C-terminus is important. ( a ) The rescued MK2 kinase-inactive mutant, MK2K79R, reduces Il1b mRNA levels to a degree comparable to that of the rescued MK2 in untreated (UT) and ( b ) IL-1α-treated (5 ng/mL, 1 h) i MK2 -KO macrophages. ( c ) i MK2 -KO cells have lower levels of the p38α protein. These levels can be restored by rescuing MK2 , but not by rescuing a MK2 mutant lacking the C-terminus MK2-Δ365–386 . ( d ) MK2-Δ365-386 does not affect the Il1b mRNA levels in IL-1α-treated cells. 1W-ANOVA with Tukey’s Multiple Comparison Test, mean ± SEM, ** p < 0.01, *** p < 0.001.

Article Snippet: BMDM (5 × 10 5 cells/well), i MK2 -KO or RAW 264.7 cells (both 2 × 10 5 cells/well) were seeded, and treated one day later with the indicated concentrations and durations of recombinant murine IL-1α (211-11A, PeproTech GmbH, Hamburg, Germany), LPS ( Escherichia coli 0127:B8; Sigma-Aldrich, Merck, St. Louis, MO, USA), Actinomycin D (Cayman Chemical Company, Ann-Arbor, MI, USA), B022 (MedChemExpress, Monmouth Junction, NJ, USA), BMS-345541 (Axon Medchem B.V., Groningen, Netherlands), CAY10657 (Cayman Chemical Company, Ann-Arbor, MI, USA), SML1160 (Sigma-Aldrich, Merck, St. Louis, MO, USA), IKK inhibitor XII (HPN-01, Sigma-Aldrich, Merck, St. Louis, MO, USA), sc-514 (Cayman Chemical Company, Ann-Arbor, MI, USA), T-5224 (Cayman Chemical Company, Ann-Arbor, MI, USA), Takinib (MedChemExpress, Monmouth Junction, NJ, USA) or Nutlin-3 (SCBT, Dallas, TX, USA).

Techniques: Activity Assay, Mutagenesis, Comparison

Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: p38α inactivates the non-canonical NF-κB pathway independent of the kinase activity. ( a – c ) Overexpression of p38α in i MK2 -KO cells increases basal TRAF2 and reduces basal RelB protein levels. ( d ) Overexpression of p38α and kinase inactive mutant p38-AGF reduce basal Il1b and ( e ) Map3k14 mRNA and ( f ) increase basal Traf2 mRNA in resting i MK2 -KO cells. ( g ) Relb and ( h ) Nfkb2 mRNA are reduced in IL-1α-treated (5 ng/mL, 1 h) i MK2 -KO+ p38α and +p38-AGF cells. ( b , c ) Student’s t -test, ( d – h ) 1W-ANOVA followed by Tukey’s Multiple Comparison Test, mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMDM (5 × 10 5 cells/well), i MK2 -KO or RAW 264.7 cells (both 2 × 10 5 cells/well) were seeded, and treated one day later with the indicated concentrations and durations of recombinant murine IL-1α (211-11A, PeproTech GmbH, Hamburg, Germany), LPS ( Escherichia coli 0127:B8; Sigma-Aldrich, Merck, St. Louis, MO, USA), Actinomycin D (Cayman Chemical Company, Ann-Arbor, MI, USA), B022 (MedChemExpress, Monmouth Junction, NJ, USA), BMS-345541 (Axon Medchem B.V., Groningen, Netherlands), CAY10657 (Cayman Chemical Company, Ann-Arbor, MI, USA), SML1160 (Sigma-Aldrich, Merck, St. Louis, MO, USA), IKK inhibitor XII (HPN-01, Sigma-Aldrich, Merck, St. Louis, MO, USA), sc-514 (Cayman Chemical Company, Ann-Arbor, MI, USA), T-5224 (Cayman Chemical Company, Ann-Arbor, MI, USA), Takinib (MedChemExpress, Monmouth Junction, NJ, USA) or Nutlin-3 (SCBT, Dallas, TX, USA).

Techniques: Activity Assay, Over Expression, Mutagenesis, Comparison