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

Journal: bioRxiv

Article Title: A Broad-Spectrum Chemokine Inhibitor Prevents Preterm Labor in Mice by Supressing Inflammation Induced by Intra-Amniotic Injection of Interleukin-1 alpha

doi: 10.64898/2026.03.04.709657

Figure Lengend Snippet: On GD15.5, pregnant mice [‘Vehicle’ group (n = 50) and ‘Broad Spectrum Chemokine Inhibitor’ (BSCI) group (n = 50)] were injected intravenously with the first dose of BSCI (10 mg/kg/day) or vehicle (saline). In 24 hrs, on GD16.5, pregnant mice received a second injection of BSCI or vehicle. At the same time, half of the vehicle group (n = 25) and half of the BSCI group (n = 25) received ultrasound-guided intra-amniotic infusions of IL-1α (IL-1α; 400 ng/amniotic sac). The second half of the vehicle group (n = 25) and half of the BSCI group (n = 25) received an infusion of sterile saline. The IL-1α injections were administered into each amniotic sac (number of fetuses was 5-9). (A) Long-term effect of BSCI. Pregnant mice were observed until delivery to record the PTB rate. All BSCI-treated animals from the IL-1α and saline groups that did not deliver preterm were given daily injections of BSCI. Mice from all four study groups (n=6/group) that carried the pregnancy to term were killed after delivery on GD19.5. The number of live pups per litter, birth weights, and placental weights were recorded. (B) Short-term effect of BSCI. In a replicate group of animals, pregnant BSCI-treated and vehicle-treated mice were sacrificed 2,8 and 24 hr post-IL-1α induction. On GD16.5, two hrs after IL-1α injection (t = 2 hr), the first six animals from each groups were killed, and maternal and fetal tissues were collected for analysis. After 8 hrs (t = 8 hr), six animals from each group were killed and maternal and fetal tissues were collected for analysis. The remaining animals (n=6/group) were killed during PTB, or exactly 24 hrs after the administration of IL-1α (t = 24 hr), and tissues were collected for biochemical and immunohistochemical evaluation.

Article Snippet: According to previous optimized conditions by Motomura et al. , ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 400 ng per 25 μl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Injection, Saline, Sterility, Immunohistochemical staining

Journal: bioRxiv

Article Title: A Broad-Spectrum Chemokine Inhibitor Prevents Preterm Labor in Mice by Supressing Inflammation Induced by Intra-Amniotic Injection of Interleukin-1 alpha

doi: 10.64898/2026.03.04.709657

Figure Lengend Snippet: (A) To induce in utero sterile inflammation, the alarmin IL-1α (400 ng/amniotic sac) was intra-amniotically administered to C57BL/6 dams under ultrasound guidance on gestational day (GD) 16.5. Dams were monitored until delivery, and neonatal survival and weight were recorded. (B) Gestational lengths are shown for each treatment group in time or GD. The dotted line indicates the threshold of term deliveries after GD 18. (C) Rates of preterm birth among dams injected with saline (n = 6), IL-1α (n = 8), BSCI (n = 5), and IL-1α + BSCI (n = 10) are shown as bar plots. (D) The fetal weights at delivery were calculated as an average of 3 pups per dam. Representative images show a fetus from each group. The dotted line indicates the threshold of viable pups. Scale bar represents 1 cm. Two-way ANOVA was utilized, followed by a Bonferroni post-test. Results were expressed as mean ± SEM. Significant difference is indicated by *** (P < 0.001).

Article Snippet: According to previous optimized conditions by Motomura et al. , ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 400 ng per 25 μl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: In Utero, Sterility, Injection, Saline

Journal: bioRxiv

Article Title: A Broad-Spectrum Chemokine Inhibitor Prevents Preterm Labor in Mice by Supressing Inflammation Induced by Intra-Amniotic Injection of Interleukin-1 alpha

doi: 10.64898/2026.03.04.709657

Figure Lengend Snippet: Pro-inflammatory (IL-1α, IL-6, IL-12p70, TNF-α, CSF2) and anti-inflammatory (IL-10) cytokines; protein expression was detected by multiplex magnetic bead assay following local IL-1α administration and treatment with BSCI. Shown are vehicle (white bars), BSCI-treated (striped bars), IL-1α-injected (grey bars), and IL-1α-injected BSCI-treated samples (striped grey bars), n =5–6/group. Two-way ANOVA was utilized, followed by a Bonferroni post-test. Results were expressed as mean ± SEM. Significant difference between groups is indicated by *** (P < 0.001).

Article Snippet: According to previous optimized conditions by Motomura et al. , ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 400 ng per 25 μl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Expressing, Multiplex Assay, Injection

Journal: bioRxiv

Article Title: A Broad-Spectrum Chemokine Inhibitor Prevents Preterm Labor in Mice by Supressing Inflammation Induced by Intra-Amniotic Injection of Interleukin-1 alpha

doi: 10.64898/2026.03.04.709657

Figure Lengend Snippet: (A) Pro-inflammatory (Il6, Il1β, Tnfα, Csf2/Gm-scf) and anti-inflammatory (Il10) cytokines; (B) Chemokines Ccl2 (Mcp1), Ccl4 (Mip1β), Cxcl1 (KC/Groα), and Cxcl2 (Mip2α) transcripts were detected by Real-Time RT-PCR in mouse liver. Shown are samples from vehicle (white bars), BSCI-treated (striped bars), IL-1α-injected (grey bars), and IL-1α-injected + BSCI-treated pregnant animals (striped grey bars), n = 5–6/group. Two-way ANOVA was utilized, followed by a Bonferroni post-test. Results were expressed as mean ± SEM. Significant difference is indicated by * (P < 0.05), ** (P < 0.01) and *** (P < 0.001).

Article Snippet: According to previous optimized conditions by Motomura et al. , ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 400 ng per 25 μl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Quantitative RT-PCR, Injection

Journal: bioRxiv

Article Title: A Broad-Spectrum Chemokine Inhibitor Prevents Preterm Labor in Mice by Supressing Inflammation Induced by Intra-Amniotic Injection of Interleukin-1 alpha

doi: 10.64898/2026.03.04.709657

Figure Lengend Snippet: (A) Pro-inflammatory (Il6, Il1β, Tnfα, Csf2/Gmscf) and anti-inflammatory (Il10) cytokines; (B) Chemokines (Ccl2/Mcp1, Ccl4/Mip1β, Cxcl1/KC/Groα and Cxcl2/Mip2α); (C) Pro-labor genes Nfkb1, Ptgs2/Cox2, Akr1c18/20αHSD, and Gja1/Cx43 transcripts were detected by Real-Time RT-PCR. (D) Pro-inflammatory cytokines (CSF2, CSF3, IL-6) and chemokines (CCL2, CCL3, CCL4, CXCL1, Eotaxin) proteins were detected by multiplex magnetic bead assay (BioRad). Shown are vehicle (white bars), BSCI-treated (striped bars), IL-1α-injected (grey bars), and IL-1α-injected BSCI-treated samples (striped grey bars), n = 5–6/group. Two-way ANOVA was utilized, followed by a Bonferroni post-test. Results were expressed as mean ± SEM. Significant difference is indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001).

Article Snippet: According to previous optimized conditions by Motomura et al. , ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 400 ng per 25 μl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Quantitative RT-PCR, Multiplex Assay, Injection

Journal: bioRxiv

Article Title: A Broad-Spectrum Chemokine Inhibitor Prevents Preterm Labor in Mice by Supressing Inflammation Induced by Intra-Amniotic Injection of Interleukin-1 alpha

doi: 10.64898/2026.03.04.709657

Figure Lengend Snippet: (A) Pro-inflammatory (Il6, Il1β, Tnfα, Csf2) and anti-inflammatory (Il10) cytokines; (B) Chemokines (Ccl2/Mcp1, Ccl4/Mip1β, Cxcl1/KC/Groα and Cxcl2/Mip2α) mRNA levels were detected by Real-Time RT-PCR. (C) Pro-inflammatory (CSF2, CSF3, IL-6, IL-12(p40), IL-1β, TNF-α), anti-inflammatory (IL-10) cytokines; and (D) chemokines (CCL2, CCL3, CCL4, CCL5, CXCL1, Eotaxin) protein levels were detected by multiplex magnetic bead assay. Shown are vehicle (white bars), BSCI-treated (striped bars), IL-1α-injected (grey bars), and IL-1α-injected BSCI-treated samples (striped grey bars), n=5–6/group. Two-way ANOVA was utilized, followed by a Bonferroni post-test. Results were expressed as mean ± SEM. Significant difference is indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001).

Article Snippet: According to previous optimized conditions by Motomura et al. , ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 400 ng per 25 μl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Quantitative RT-PCR, Multiplex Assay, Injection

Journal: bioRxiv

Article Title: A Broad-Spectrum Chemokine Inhibitor Prevents Preterm Labor in Mice by Supressing Inflammation Induced by Intra-Amniotic Injection of Interleukin-1 alpha

doi: 10.64898/2026.03.04.709657

Figure Lengend Snippet: Pro-inflammatory (IL-6, TNF-α, CSF2) and anti-inflammatory (IL-10) cytokines; protein expression was detected by multiplex magnetic bead assay following local IL-1α administration and treatment with BSCI. Shown are vehicle (white bars), BSCI-treated (striped bars), IL-1α-injected (grey bars), and IL-1α-injected BSCI-treated samples (striped grey bars), n = 5–6/group. Two-way ANOVA was utilized, followed by a Bonferroni post-test. Results were expressed as mean ± SEM. Significant difference is indicated by * (P < 0.05).

Article Snippet: According to previous optimized conditions by Motomura et al. , ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 400 ng per 25 μl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Expressing, Multiplex Assay, Injection

Journal: bioRxiv

Article Title: A Broad-Spectrum Chemokine Inhibitor Prevents Preterm Labor in Mice by Supressing Inflammation Induced by Intra-Amniotic Injection of Interleukin-1 alpha

doi: 10.64898/2026.03.04.709657

Figure Lengend Snippet: (A) Pro-inflammatory (Il6, Il1β, Tnfα, Csf2/Gmscf) and anti-inflammatory (Il10) cytokines; (B) Chemokines (Ccl2 (Mcp1), Ccl4 (Mip1β), Cxcl1 (KC or Groα), and Cxcl2 (Mip2α)) mRNA expression was detected by Real-Time RT-PCR. Shown are vehicle (white bars), BSCI-treated (striped bars), IL-1α-injected (grey bars), and IL-1α-injected BSCI-treated samples (striped grey bars), n = 5–6/group. Two-way ANOVA was utilized, followed by a Bonferroni post-test. Results were expressed as mean ± SEM. Significant difference is indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001).

Article Snippet: According to previous optimized conditions by Motomura et al. , ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 400 ng per 25 μl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Expressing, Quantitative RT-PCR, Injection

Journal: bioRxiv

Article Title: A Broad-Spectrum Chemokine Inhibitor Prevents Preterm Labor in Mice by Supressing Inflammation Induced by Intra-Amniotic Injection of Interleukin-1 alpha

doi: 10.64898/2026.03.04.709657

Figure Lengend Snippet: (A) A UCSC bedgraph of transcript reads mapped to the mm10 genome demonstrating an increase in expression of the chemokine Cxcl2 in response to IL-1α, and repression of Cxcl2 expression with BSCI pre-treatment at 24 hrs (n=3). (B) Principal component analyses demonstrating similarity in transcript profiles (RNASeq) between saline vehicle control (V/V), BSCI/V, IL-1α/BSCI, and the independent clustering of IL-1α/V. (C) Bar graph showing comparison of differentially expressed genes identified (abs. fold change ≥ 2; padj < 0.05; Y-axis) in V/BSCI, IL-1α/V, and IL-1α/BSCI compared to V/V control. The number of differentially expressed genes is on the Y-axis and listed above each bar, while differentially expressed genes common between conditions are shown below the X-axis. (D) An example of a UCSC bedgraph track generated from ATAC-seq data mapped to the mm10 genome demonstrating an increase in accessibility around the chemokine Cxcl2 in response to IL-1α, and repression of this increase in accessibility by BSCI (n=3). Bars under UCSC tracks identify where ATAC-seq peaks were called using macs2. (E) Principal component analyses demonstrating similarity in chromatin accessibility (ATACseq) between V/V, V/BSCI, IL-1α/BSCI, and the independent clustering of IL-1α/V. (F) Volcano plot showing regions of chromatin gaining and losing accessibility between IL-1α/Vehicle and V/V and (G) BSCI/IL-1α and V/V. Y-axis is -log 10 (padj) and X-axis log 2 (foldchange); dashed lines represent significance thresholds (abs. fold change ≥ 2; padj < 0.05). Samples in grey did not meet criteria for significance.

Article Snippet: According to previous optimized conditions by Motomura et al. , ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 400 ng per 25 μl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Expressing, RNA sequencing, Saline, Control, Comparison, Generated

Journal: bioRxiv

Article Title: A Broad-Spectrum Chemokine Inhibitor Prevents Preterm Labor in Mice by Supressing Inflammation Induced by Intra-Amniotic Injection of Interleukin-1 alpha

doi: 10.64898/2026.03.04.709657

Figure Lengend Snippet: Volcano plots showing differentially expressed proteins identified by mass spectrometry, with each dot representing one protein. In both panels, red dots indicate significantly increased (upregulated) proteins and blue dots indicate significantly decreased (downregulated) proteins. (A) Comparison of IL-1α/vehicle (IL-1α/V) vs vehicle/vehicle (V/V) tissue, with selected downregulated proteins annotated. (B) Comparison of IL-1α/BSCI vs IL-1α/vehicle (IL-1α/V) tissue, with selected upregulated proteins annotated. Gene Ontology (GO) enrichment analyses of differentially expressed proteins: (C) IL-1α/vehicle (IL-1α/V) vs vehicle (V/V) and (D) IL-1α/BSCI vs IL-1α/vehicle. In both, enriched biological processes are shown by fold enrichment, with point size reflecting enrichment magnitude and color representing statistical significance (−log10[FDR]). (E) Bar graphs showing raw mass spectrometry intensity values of selected extracellular matrix (ECM) proteins across V/V, IL-1α/V, and IL-1α/BSCI groups, with particular emphasis on fibrillar collagen-associated proteins (including COL3A1, COL1A1, and COL1A2). Two-way ANOVA was utilized, followed by a Bonferroni post-test. Results were expressed as mean ± SEM. Significant difference is indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001).

Article Snippet: According to previous optimized conditions by Motomura et al. , ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 400 ng per 25 μl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Mass Spectrometry, Comparison

Journal: bioRxiv

Article Title: A Broad-Spectrum Chemokine Inhibitor Prevents Preterm Labor in Mice by Supressing Inflammation Induced by Intra-Amniotic Injection of Interleukin-1 alpha

doi: 10.64898/2026.03.04.709657

Figure Lengend Snippet: Macrophages were identified using anti-F4/80 antibody (yellow), using anti-CD86 for pro-inflammatory M1 macrophages (red), and using anti-CD206 for homeostatic M2 macrophages (green). Bright field (BF, top row) images were used to mask the borders between the myometrium and the decidua. (A) Shown are the representative immunofluorescence images with negative control inlets (images taken with respective IgG controls for each antibody). (B) Qupath software was used to quantify the number of macrophages in the myometrium and decidua. The bar graphs show quantification of vehicle (white bars), BSCI-treated (striped bars), IL-1α-injected (grey bars), and IL-1α-injected BSCI-treated samples (striped grey bars), n = 5–6/group. Two-way ANOVA was utilized, followed by a Bonferroni post-test. Results were expressed as mean ± SEM. Significant difference is indicated by ** (P < 0.01) and *** (P < 0.001).

Article Snippet: According to previous optimized conditions by Motomura et al. , ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 400 ng per 25 μl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Immunofluorescence, Negative Control, Software, Injection