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anti p38  (Proteintech)
95
Proteintech anti p38
CIP exposure induced miscarriage by promoting senescence. (A and B) SA-β-Galactose staining of 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells and the quantification of SA-β-Galactose positive cells (n = 3 independent experiments, one-way ANOVA). (C) Western blot analysis of p16, p21, <t>p38,</t> and β-gal protein levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells and their relative quantification (n = 3 independent experiments). (D and E) SA-β-Galactose staining of HC and UM villous tissues and the quantification of positive intensity (n = 12, Student's t-test). (F) Western blot analysis of p16, p21, p38, and β-gal protein levels in HC and UM villous tissues (n = 12). (G and H) SA-β-Galactose staining of CIP-exposed mouse placental tissues and the quantification of positive intensity (each n = 6, one-way ANOVA). (I and J) Western blot analysis of p16, p21, p38, and β-gal protein levels in placental tissues of CIP-exposed mice and their relative quantification (n = 6, one-way ANOVA).
Anti P38, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti p38 beta mapk c28c2 monoclonal antibody  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc anti p38 beta mapk c28c2 monoclonal antibody
A. Schematic representation of the mechanism of <t>ZAKα/P38</t> stress kinases activation upon induction of Ribotoxic Stress Response (RSR). B. Phosphotag blotting of phosphorylated NLRP1 disordered Region (DR) in HEK293T expressing the NLRP1 DR construct (aa 86-275-GFP (described in A )) and exposed to all PP1/PP2A-targeting compounds identified in /B ) or to the known RSR inducer Anisomycin (1 µg/mL) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. C. Phosphotag blotting of phosphorylated full length NLRP1 in primary human keratinocytes exposed Dinophysis toxin (100 nM) for various time. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. D. Phosphotag blotting of phosphorylated ZAKα and NLRP1 disordered Region (DR) in WT or ZAK KO NTERT NLRP1 KO + 86-275-SNAP keratinocytes exposed to Dinophysis toxin (100nM), Cantharidin (5%M) or Val-boro-Pro (VbP, 10µM) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Plasma membrane permeabilization (SYTOX Green incorporation, 16 h) and IL-1β release evaluation (10 h) in WT, ZAKα or NLRP1 KO NTERT keratinocytes after exposure to Dinophysis toxin (100nM), Cantharidin (5%M), Okadaic acid (250nM) or Anisomycin (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. F. Immunoblotting and clonal selection (clone 3, red), fluorescence microscopy and associated quantifications of ASC-GFP specks in WT or ZAKα KO HEK293T ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or Anisomycin (1µM) for 5 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA.
Anti P38 Beta Mapk C28c2 Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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p38 alpha beta mapk a 12  (Santa Cruz Biotechnology)
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Santa Cruz Biotechnology p38 alpha beta mapk a 12
A. Schematic representation of the mechanism of <t>ZAKα/P38</t> stress kinases activation upon induction of Ribotoxic Stress Response (RSR). B. Phosphotag blotting of phosphorylated NLRP1 disordered Region (DR) in HEK293T expressing the NLRP1 DR construct (aa 86-275-GFP (described in A )) and exposed to all PP1/PP2A-targeting compounds identified in /B ) or to the known RSR inducer Anisomycin (1 µg/mL) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. C. Phosphotag blotting of phosphorylated full length NLRP1 in primary human keratinocytes exposed Dinophysis toxin (100 nM) for various time. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. D. Phosphotag blotting of phosphorylated ZAKα and NLRP1 disordered Region (DR) in WT or ZAK KO NTERT NLRP1 KO + 86-275-SNAP keratinocytes exposed to Dinophysis toxin (100nM), Cantharidin (5%M) or Val-boro-Pro (VbP, 10µM) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Plasma membrane permeabilization (SYTOX Green incorporation, 16 h) and IL-1β release evaluation (10 h) in WT, ZAKα or NLRP1 KO NTERT keratinocytes after exposure to Dinophysis toxin (100nM), Cantharidin (5%M), Okadaic acid (250nM) or Anisomycin (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. F. Immunoblotting and clonal selection (clone 3, red), fluorescence microscopy and associated quantifications of ASC-GFP specks in WT or ZAKα KO HEK293T ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or Anisomycin (1µM) for 5 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA.
P38 Alpha Beta Mapk A 12, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phospho p38 mapk thr180 tyr182  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182
A. Schematic representation of the mechanism of <t>ZAKα/P38</t> stress kinases activation upon induction of Ribotoxic Stress Response (RSR). B. Phosphotag blotting of phosphorylated NLRP1 disordered Region (DR) in HEK293T expressing the NLRP1 DR construct (aa 86-275-GFP (described in A )) and exposed to all PP1/PP2A-targeting compounds identified in /B ) or to the known RSR inducer Anisomycin (1 µg/mL) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. C. Phosphotag blotting of phosphorylated full length NLRP1 in primary human keratinocytes exposed Dinophysis toxin (100 nM) for various time. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. D. Phosphotag blotting of phosphorylated ZAKα and NLRP1 disordered Region (DR) in WT or ZAK KO NTERT NLRP1 KO + 86-275-SNAP keratinocytes exposed to Dinophysis toxin (100nM), Cantharidin (5%M) or Val-boro-Pro (VbP, 10µM) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Plasma membrane permeabilization (SYTOX Green incorporation, 16 h) and IL-1β release evaluation (10 h) in WT, ZAKα or NLRP1 KO NTERT keratinocytes after exposure to Dinophysis toxin (100nM), Cantharidin (5%M), Okadaic acid (250nM) or Anisomycin (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. F. Immunoblotting and clonal selection (clone 3, red), fluorescence microscopy and associated quantifications of ASC-GFP specks in WT or ZAKα KO HEK293T ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or Anisomycin (1µM) for 5 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA.
Phospho P38 Mapk Thr180 Tyr182, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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p38 beta mapk11  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology p38 beta mapk11
A. Schematic representation of the mechanism of <t>ZAKα/P38</t> stress kinases activation upon induction of Ribotoxic Stress Response (RSR). B. Phosphotag blotting of phosphorylated NLRP1 disordered Region (DR) in HEK293T expressing the NLRP1 DR construct (aa 86-275-GFP (described in A )) and exposed to all PP1/PP2A-targeting compounds identified in /B ) or to the known RSR inducer Anisomycin (1 µg/mL) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. C. Phosphotag blotting of phosphorylated full length NLRP1 in primary human keratinocytes exposed Dinophysis toxin (100 nM) for various time. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. D. Phosphotag blotting of phosphorylated ZAKα and NLRP1 disordered Region (DR) in WT or ZAK KO NTERT NLRP1 KO + 86-275-SNAP keratinocytes exposed to Dinophysis toxin (100nM), Cantharidin (5%M) or Val-boro-Pro (VbP, 10µM) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Plasma membrane permeabilization (SYTOX Green incorporation, 16 h) and IL-1β release evaluation (10 h) in WT, ZAKα or NLRP1 KO NTERT keratinocytes after exposure to Dinophysis toxin (100nM), Cantharidin (5%M), Okadaic acid (250nM) or Anisomycin (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. F. Immunoblotting and clonal selection (clone 3, red), fluorescence microscopy and associated quantifications of ASC-GFP specks in WT or ZAKα KO HEK293T ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or Anisomycin (1µM) for 5 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA.
P38 Beta Mapk11, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phosphorylated p38  (Proteintech)
97
Proteintech phosphorylated p38
Ectosomal‐calhm6 mitigates inflammation and enhances tissue repair via M2‐like macrophage polarization. A) After 24 h of LPS treatment, ectosomes were collected from the serum of wild‐type or Calhm6 knockout mice. These exosomes were used to treat wild‐type BMDMs, and the expression of M2‐like markers in the BMDMs was analyzed using RT‐PCR. B) RT‐PCR analysis of TNFA, NOS2, IL6, CHIL3, IL10, and ARG1 in wild‐type BMDMs incubated with or without ectosomes for 24 h from EV or OE‐Calhm6 cells. C) Immunoblot analysis of <t>phosphorylated</t> (p‐) <t>p38,</t> NFκB, IκBα, and β‐actin in wild‐type BMDMs stimulated for 0, 30, 60 min with LPS (200 ng mL −1 ) after incubation with ectosomal‐EV or ectosomal‐Calhm6 from EV or OE‐Calhm6 cells for 24 h. D,E) Morphology of EV or OE‐Calhm6 cells (D) and cell length (I) was measured by ImageJ ( n =304). Scale bars(E), 100 µm. F) RT‐PCR analysis of CHIL3, IL10, ARG1, TNFA, NOS2 and IL6 in EV or OE‐Calhm6 cells treated with or without BAPTA‐AM (20 µ m ) or Ca 2+ ‐free DMEM for 24 h. G) Calcium influx over time in EV or OE‐Calhm6 cells treated with LPS (200 ng mL −1 ) and stained by the Rhod2‐AM (4 µ m , 30 min). H,I) Diagram (H) of ectosomes by sequential ultracentrifugation for mass spectrometry from serum of wild‐type BMDMs treatment with LPS (200 ng mL −1 ) and IFNγ (10 ng mL −1 ) combined or IL‐4 (20 ng mL −1 ) for 0, 12 or 24 h; the indicated genes identified were shown in the table (I). J) Fluorescence microscopy of the colocalization (white arrows) of Calhm6 (green) and Tsg101 (red) in wild‐type BMDMs treated with or without LPS, IFNγ combined or IL‐4 for 12 h. Scale bars, 10 µm. K) Representative photographs of the wounds on days 0, 3, 6, and 10 after parawound injection of PBS (control), ectosomal‐EV, ectosomal‐Calhm6 in the mouse excisional wound splinting model. Scale bars, 2 mm. L) Measurement of wound area at indicated points. The percentage of wound closure was calculated as: (area of wound at time/area of original wound×100%. M) Immunofluorescence histochemistry of frozen section of wound area of F4/80 (red), CD206 (green), and counterstained with DAPI (blue) in each group as indicated above at day 10. Scale bars, 200 µm. N) Pearson's correlation coefficient values for colocalization of CD206 and F4/80 in the wound area. The average Pearson's correlation coefficients were calculated from eight randomly selected infected cells in each group ( n = 12 photos examined). O) Immunoblot analysis of Arg, Tgf‐β1, F4/80, and β‐actin in F4/80 + cells from the wound area for each group as indicated above at day 10. The data represent the mean ± S.D. ( n =3). NS, not significant ( p > 0.05); * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.00001 compared with control, Student's t‐test.
Phosphorylated P38, supplied by Proteintech, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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p38 alpha beta mapk  (Santa Cruz Biotechnology)
95
Santa Cruz Biotechnology p38 alpha beta mapk
Ectosomal‐calhm6 mitigates inflammation and enhances tissue repair via M2‐like macrophage polarization. A) After 24 h of LPS treatment, ectosomes were collected from the serum of wild‐type or Calhm6 knockout mice. These exosomes were used to treat wild‐type BMDMs, and the expression of M2‐like markers in the BMDMs was analyzed using RT‐PCR. B) RT‐PCR analysis of TNFA, NOS2, IL6, CHIL3, IL10, and ARG1 in wild‐type BMDMs incubated with or without ectosomes for 24 h from EV or OE‐Calhm6 cells. C) Immunoblot analysis of <t>phosphorylated</t> (p‐) <t>p38,</t> NFκB, IκBα, and β‐actin in wild‐type BMDMs stimulated for 0, 30, 60 min with LPS (200 ng mL −1 ) after incubation with ectosomal‐EV or ectosomal‐Calhm6 from EV or OE‐Calhm6 cells for 24 h. D,E) Morphology of EV or OE‐Calhm6 cells (D) and cell length (I) was measured by ImageJ ( n =304). Scale bars(E), 100 µm. F) RT‐PCR analysis of CHIL3, IL10, ARG1, TNFA, NOS2 and IL6 in EV or OE‐Calhm6 cells treated with or without BAPTA‐AM (20 µ m ) or Ca 2+ ‐free DMEM for 24 h. G) Calcium influx over time in EV or OE‐Calhm6 cells treated with LPS (200 ng mL −1 ) and stained by the Rhod2‐AM (4 µ m , 30 min). H,I) Diagram (H) of ectosomes by sequential ultracentrifugation for mass spectrometry from serum of wild‐type BMDMs treatment with LPS (200 ng mL −1 ) and IFNγ (10 ng mL −1 ) combined or IL‐4 (20 ng mL −1 ) for 0, 12 or 24 h; the indicated genes identified were shown in the table (I). J) Fluorescence microscopy of the colocalization (white arrows) of Calhm6 (green) and Tsg101 (red) in wild‐type BMDMs treated with or without LPS, IFNγ combined or IL‐4 for 12 h. Scale bars, 10 µm. K) Representative photographs of the wounds on days 0, 3, 6, and 10 after parawound injection of PBS (control), ectosomal‐EV, ectosomal‐Calhm6 in the mouse excisional wound splinting model. Scale bars, 2 mm. L) Measurement of wound area at indicated points. The percentage of wound closure was calculated as: (area of wound at time/area of original wound×100%. M) Immunofluorescence histochemistry of frozen section of wound area of F4/80 (red), CD206 (green), and counterstained with DAPI (blue) in each group as indicated above at day 10. Scale bars, 200 µm. N) Pearson's correlation coefficient values for colocalization of CD206 and F4/80 in the wound area. The average Pearson's correlation coefficients were calculated from eight randomly selected infected cells in each group ( n = 12 photos examined). O) Immunoblot analysis of Arg, Tgf‐β1, F4/80, and β‐actin in F4/80 + cells from the wound area for each group as indicated above at day 10. The data represent the mean ± S.D. ( n =3). NS, not significant ( p > 0.05); * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.00001 compared with control, Student's t‐test.
P38 Alpha Beta Mapk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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p38 mapk  (Novus Biologicals)
93
Novus Biologicals p38 mapk
Fig. 7. B–F indicates the effects of aerobic exercise on aortic <t>P38MAPK,</t> pp38MAPK, pp38MAPK/P38MAPK, collagen II, and IL-6 protein expression in AS rats, compared with Con group, * p < 0.05 and **p < 0.01; compared with AS group # p < 0.05 and ##p < 0.01.
P38 Mapk, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phospho p38 mapk  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc phospho p38 mapk
Fig. 7. B–F indicates the effects of aerobic exercise on aortic <t>P38MAPK,</t> pp38MAPK, pp38MAPK/P38MAPK, collagen II, and IL-6 protein expression in AS rats, compared with Con group, * p < 0.05 and **p < 0.01; compared with AS group # p < 0.05 and ##p < 0.01.
Phospho P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


CIP exposure induced miscarriage by promoting senescence. (A and B) SA-β-Galactose staining of 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells and the quantification of SA-β-Galactose positive cells (n = 3 independent experiments, one-way ANOVA). (C) Western blot analysis of p16, p21, p38, and β-gal protein levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells and their relative quantification (n = 3 independent experiments). (D and E) SA-β-Galactose staining of HC and UM villous tissues and the quantification of positive intensity (n = 12, Student's t-test). (F) Western blot analysis of p16, p21, p38, and β-gal protein levels in HC and UM villous tissues (n = 12). (G and H) SA-β-Galactose staining of CIP-exposed mouse placental tissues and the quantification of positive intensity (each n = 6, one-way ANOVA). (I and J) Western blot analysis of p16, p21, p38, and β-gal protein levels in placental tissues of CIP-exposed mice and their relative quantification (n = 6, one-way ANOVA).

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: CIP exposure induced miscarriage by promoting senescence. (A and B) SA-β-Galactose staining of 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells and the quantification of SA-β-Galactose positive cells (n = 3 independent experiments, one-way ANOVA). (C) Western blot analysis of p16, p21, p38, and β-gal protein levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells and their relative quantification (n = 3 independent experiments). (D and E) SA-β-Galactose staining of HC and UM villous tissues and the quantification of positive intensity (n = 12, Student's t-test). (F) Western blot analysis of p16, p21, p38, and β-gal protein levels in HC and UM villous tissues (n = 12). (G and H) SA-β-Galactose staining of CIP-exposed mouse placental tissues and the quantification of positive intensity (each n = 6, one-way ANOVA). (I and J) Western blot analysis of p16, p21, p38, and β-gal protein levels in placental tissues of CIP-exposed mice and their relative quantification (n = 6, one-way ANOVA).

Article Snippet: The primary antibodies contained anti-MFF (17090-1-AP, proteintech, dilution 1:1000, RRID: AB_2142463 ), anti-TRIM21 (67136-1-Ig, proteintech, dilution 1:1000, RRID: AB_2882435 ), anti-GLB1 (66586-1-Ig, proteintech, dilution 1:1000, RRID: AB_2881946 ), anti-p38 (80821-2-RR, proteintech, dilution 1:1000, RRID: AB_3670489 ), anti-p21 (ab109199, abcam, dilution 1:1000, RRID: AB_10861551 ), anti-p16 (ab189034, abcam, dilution 1:1000, RRID: RRID: AB_2737282), and anti-GAPDH (#5174, Cell Signalling Technology, dilution 1:1000, RRID: AB_10622025 ).

Techniques: Staining, Western Blot, Quantitative Proteomics

Further validate the roles of senescence in CIP-induced mouse miscarriage. (A) Schematic diagram of CIP-exposed mouse model with D + Q treatment. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 5 mg/kg/3 d D and 50 mg/kg/3 d Q (with 10% PEG-400 as solvent). (B and C) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with D + Q treatment (each n = 6, one-way ANOVA). (D) SA-β-Galactose staining of placental tissues of 160 mg/kg/d CIP-exposed mice with D + Q treatment (n = 6). (E) The quantification of positive intensity of SA-β-Galactose staining of placental tissues in 160 mg/kg/d CIP-exposed mice with D + Q treatment (each n = 6, one-way ANOVA). (F and G) Western blot analysis of p16, p21, p38, and β-gal protein levels in placental tissues of 160 mg/kg/d CIP-exposed mice with D + Q treatment and their relative quantification (each n = 6, one-way ANOVA).

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: Further validate the roles of senescence in CIP-induced mouse miscarriage. (A) Schematic diagram of CIP-exposed mouse model with D + Q treatment. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 5 mg/kg/3 d D and 50 mg/kg/3 d Q (with 10% PEG-400 as solvent). (B and C) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with D + Q treatment (each n = 6, one-way ANOVA). (D) SA-β-Galactose staining of placental tissues of 160 mg/kg/d CIP-exposed mice with D + Q treatment (n = 6). (E) The quantification of positive intensity of SA-β-Galactose staining of placental tissues in 160 mg/kg/d CIP-exposed mice with D + Q treatment (each n = 6, one-way ANOVA). (F and G) Western blot analysis of p16, p21, p38, and β-gal protein levels in placental tissues of 160 mg/kg/d CIP-exposed mice with D + Q treatment and their relative quantification (each n = 6, one-way ANOVA).

Article Snippet: The primary antibodies contained anti-MFF (17090-1-AP, proteintech, dilution 1:1000, RRID: AB_2142463 ), anti-TRIM21 (67136-1-Ig, proteintech, dilution 1:1000, RRID: AB_2882435 ), anti-GLB1 (66586-1-Ig, proteintech, dilution 1:1000, RRID: AB_2881946 ), anti-p38 (80821-2-RR, proteintech, dilution 1:1000, RRID: AB_3670489 ), anti-p21 (ab109199, abcam, dilution 1:1000, RRID: AB_10861551 ), anti-p16 (ab189034, abcam, dilution 1:1000, RRID: RRID: AB_2737282), and anti-GAPDH (#5174, Cell Signalling Technology, dilution 1:1000, RRID: AB_10622025 ).

Techniques: Saline, Solvent, Staining, Western Blot, Quantitative Proteomics

CIP exposure caused senescence by down-regulating MFF. (A) Wayne diagram of the differentially expressed protein (DEPs) in the intersections of UM vs HC villous tissues, 160 vs 0 mg/kg/d CIP-exposed mouse placenta tissues, and 40 vs 0 μg/mL CIP-exposed HTR-8/SVneo cells with difference >1.3-fold and p < 0.05. (B) RT-qPCR analysis of MFF mRNA levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells (n = 3 independent experiments, one-way ANOVA). (C) Western blot analysis of MFF protein levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells and its relative quantification (n = 3 independent experiments, one-way ANOVA). (D and E) SA-β-Galactose staining of HTR-8/SVneo cells with MFF overexpression (Student's t-test) or knockdown (one-way ANOVA) and the quantification with SA-β-Galactose positive cells (n = 3 independent experiments). (F and G) Western blot analysis of p16, p21, p38, and β-gal protein levels in HTR-8/SVneo cells with with MFF overexpression (Student's t-test) or knockdown (one-way ANOVA) and their relative quantification (n = 3 independent experiments). (H) CCK8 assay analysis of cell viability in 40 μg/mL CIP-exposed HTR-8/SVneo cells with MFF overexpression for 0, 12, 24, 36, or 48 h (n = 3 independent experiments, one-way ANOVA). (I and J) SA-β-Galactose staining of 40 μg/mL CIP-exposed HTR-8/SVneo cells with MFF overexpression and the quantification of SA-β-Galactose positive cells (n = 3 independent experiments, one-way ANOVA). (K and L) Western blot analysis of p16, p21, p38, and β-gal protein levels in 40 μg/mL CIP-exposed HTR-8/SVneo cells with MFF overexpression and their relative quantification (n = 3 independent experiments, one-way ANOVA).

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: CIP exposure caused senescence by down-regulating MFF. (A) Wayne diagram of the differentially expressed protein (DEPs) in the intersections of UM vs HC villous tissues, 160 vs 0 mg/kg/d CIP-exposed mouse placenta tissues, and 40 vs 0 μg/mL CIP-exposed HTR-8/SVneo cells with difference >1.3-fold and p < 0.05. (B) RT-qPCR analysis of MFF mRNA levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells (n = 3 independent experiments, one-way ANOVA). (C) Western blot analysis of MFF protein levels in 0, 1, 10, 20, or 40 μg/mL CIP-exposed HTR-8/SVneo cells and its relative quantification (n = 3 independent experiments, one-way ANOVA). (D and E) SA-β-Galactose staining of HTR-8/SVneo cells with MFF overexpression (Student's t-test) or knockdown (one-way ANOVA) and the quantification with SA-β-Galactose positive cells (n = 3 independent experiments). (F and G) Western blot analysis of p16, p21, p38, and β-gal protein levels in HTR-8/SVneo cells with with MFF overexpression (Student's t-test) or knockdown (one-way ANOVA) and their relative quantification (n = 3 independent experiments). (H) CCK8 assay analysis of cell viability in 40 μg/mL CIP-exposed HTR-8/SVneo cells with MFF overexpression for 0, 12, 24, 36, or 48 h (n = 3 independent experiments, one-way ANOVA). (I and J) SA-β-Galactose staining of 40 μg/mL CIP-exposed HTR-8/SVneo cells with MFF overexpression and the quantification of SA-β-Galactose positive cells (n = 3 independent experiments, one-way ANOVA). (K and L) Western blot analysis of p16, p21, p38, and β-gal protein levels in 40 μg/mL CIP-exposed HTR-8/SVneo cells with MFF overexpression and their relative quantification (n = 3 independent experiments, one-way ANOVA).

Article Snippet: The primary antibodies contained anti-MFF (17090-1-AP, proteintech, dilution 1:1000, RRID: AB_2142463 ), anti-TRIM21 (67136-1-Ig, proteintech, dilution 1:1000, RRID: AB_2882435 ), anti-GLB1 (66586-1-Ig, proteintech, dilution 1:1000, RRID: AB_2881946 ), anti-p38 (80821-2-RR, proteintech, dilution 1:1000, RRID: AB_3670489 ), anti-p21 (ab109199, abcam, dilution 1:1000, RRID: AB_10861551 ), anti-p16 (ab189034, abcam, dilution 1:1000, RRID: RRID: AB_2737282), and anti-GAPDH (#5174, Cell Signalling Technology, dilution 1:1000, RRID: AB_10622025 ).

Techniques: Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Staining, Over Expression, Knockdown, CCK-8 Assay

CIP exposure down-regulated Mff protein levels to cause placental senescence and thus induced mouse miscarriage in CIP-exposed mouse model. (A and B) Western blot analysis of murine Mff protein levels in placental tissues of CIP-exposed mice and its relative quantification (n = 6, one-way ANOVA). (C) RT-qPCR analysis of Mff mRNA levels in placental tissues of CIP-exposed mice (n = 6, one-way ANOVA). (D) Schematic diagram of CIP-exposed mouse model with Mff overexpression. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 10 mg/kg/3 d pcDNA3.1-Mff for overexpression of Mff (with empty vector pcDNA3.1 as control). (E and F) Embryo resorption (indicated by red arrows, e) and the average miscarriage rates (f) in 160 mg/kg/d CIP-exposed mice with Mff overexpression (each n = 6, one-way ANOVA). (G and H) SA-β-Galactose staining of placental tissues of 160 mg/kg/d CIP-exposed mice with Mff overexpression and the relative quantification of positive intensity (each n = 6, one-way ANOVA). (I and J) Western blot analysis of murine Mff, p16, p21, p38, and β-gal protein levels in placental tissues of 160 mg/kg/d CIP-exposed mice with Mff overexpression and their relative quantification (each n = 6, one-way ANOVA). (K) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with Mff overexpression (n = 6, one-way ANOVA). (L) The NAD + /NADH ratios in total placental tissues of 160 mg/kg/d CIP-exposed mice with Mff overexpression (n = 6, one-way ANOVA). (M) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with Mff overexpression (n = 6, one-way ANOVA).

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: CIP exposure down-regulated Mff protein levels to cause placental senescence and thus induced mouse miscarriage in CIP-exposed mouse model. (A and B) Western blot analysis of murine Mff protein levels in placental tissues of CIP-exposed mice and its relative quantification (n = 6, one-way ANOVA). (C) RT-qPCR analysis of Mff mRNA levels in placental tissues of CIP-exposed mice (n = 6, one-way ANOVA). (D) Schematic diagram of CIP-exposed mouse model with Mff overexpression. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 10 mg/kg/3 d pcDNA3.1-Mff for overexpression of Mff (with empty vector pcDNA3.1 as control). (E and F) Embryo resorption (indicated by red arrows, e) and the average miscarriage rates (f) in 160 mg/kg/d CIP-exposed mice with Mff overexpression (each n = 6, one-way ANOVA). (G and H) SA-β-Galactose staining of placental tissues of 160 mg/kg/d CIP-exposed mice with Mff overexpression and the relative quantification of positive intensity (each n = 6, one-way ANOVA). (I and J) Western blot analysis of murine Mff, p16, p21, p38, and β-gal protein levels in placental tissues of 160 mg/kg/d CIP-exposed mice with Mff overexpression and their relative quantification (each n = 6, one-way ANOVA). (K) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with Mff overexpression (n = 6, one-way ANOVA). (L) The NAD + /NADH ratios in total placental tissues of 160 mg/kg/d CIP-exposed mice with Mff overexpression (n = 6, one-way ANOVA). (M) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with Mff overexpression (n = 6, one-way ANOVA).

Article Snippet: The primary antibodies contained anti-MFF (17090-1-AP, proteintech, dilution 1:1000, RRID: AB_2142463 ), anti-TRIM21 (67136-1-Ig, proteintech, dilution 1:1000, RRID: AB_2882435 ), anti-GLB1 (66586-1-Ig, proteintech, dilution 1:1000, RRID: AB_2881946 ), anti-p38 (80821-2-RR, proteintech, dilution 1:1000, RRID: AB_3670489 ), anti-p21 (ab109199, abcam, dilution 1:1000, RRID: AB_10861551 ), anti-p16 (ab189034, abcam, dilution 1:1000, RRID: RRID: AB_2737282), and anti-GAPDH (#5174, Cell Signalling Technology, dilution 1:1000, RRID: AB_10622025 ).

Techniques: Western Blot, Quantitative Proteomics, Quantitative RT-PCR, Over Expression, Saline, Plasmid Preparation, Control, Staining

TRIM21 was involved in CIP-caused mitochondrial dysfunctions and senescence in trophoblast cells. (A) CCK8 assay analysis of cell viability in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown for 0, 12, 24, 36, or 48 h (n = 3 independent experiments, one-way ANOVA). (B and C) SA-β-Galactose staining of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and the quantification of SA-β-Galactose positive cells (n = 3 independent experiments, one-way ANOVA). (D and E) The protein levels of p16, p21, p38, and β-gal in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and their relative quantification (n = 3 independent experiments, one-way ANOVA). (F) The relative mtDNA copy number in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (G and H) The relative NAD + /NADH ratios in total lysates (G) or mitochondria (H) of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (I) The relative quantification of MMP levels in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (J and K) The representative image of ROS fluorescent signal in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and the relative quantification of ROS levels (n = 3 independent experiments, one-way ANOVA). (L) The relative quantification of ROS levels in mitochondria of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA).

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: TRIM21 was involved in CIP-caused mitochondrial dysfunctions and senescence in trophoblast cells. (A) CCK8 assay analysis of cell viability in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown for 0, 12, 24, 36, or 48 h (n = 3 independent experiments, one-way ANOVA). (B and C) SA-β-Galactose staining of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and the quantification of SA-β-Galactose positive cells (n = 3 independent experiments, one-way ANOVA). (D and E) The protein levels of p16, p21, p38, and β-gal in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and their relative quantification (n = 3 independent experiments, one-way ANOVA). (F) The relative mtDNA copy number in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (G and H) The relative NAD + /NADH ratios in total lysates (G) or mitochondria (H) of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (I) The relative quantification of MMP levels in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA). (J and K) The representative image of ROS fluorescent signal in 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown and the relative quantification of ROS levels (n = 3 independent experiments, one-way ANOVA). (L) The relative quantification of ROS levels in mitochondria of 40 μg/mL CIP-exposed HTR-8/SVneo cells with TRIM21 knockdown (n = 3 independent experiments, one-way ANOVA).

Article Snippet: The primary antibodies contained anti-MFF (17090-1-AP, proteintech, dilution 1:1000, RRID: AB_2142463 ), anti-TRIM21 (67136-1-Ig, proteintech, dilution 1:1000, RRID: AB_2882435 ), anti-GLB1 (66586-1-Ig, proteintech, dilution 1:1000, RRID: AB_2881946 ), anti-p38 (80821-2-RR, proteintech, dilution 1:1000, RRID: AB_3670489 ), anti-p21 (ab109199, abcam, dilution 1:1000, RRID: AB_10861551 ), anti-p16 (ab189034, abcam, dilution 1:1000, RRID: RRID: AB_2737282), and anti-GAPDH (#5174, Cell Signalling Technology, dilution 1:1000, RRID: AB_10622025 ).

Techniques: CCK-8 Assay, Knockdown, Staining, Quantitative Proteomics

Verification of murine Trim21/Mff signalling in CIP-exposed mouse placental tissues. (A) Murine Trim21 mRNA levels in placental tissues of CIP-exposed mice (n = 6, one-way ANOVA). (B and C) Murine Trim21 protein levels in placental tissues of CIP-exposed mice and its relative quantification (n = 6, one-way ANOVA). (D) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in placental tissues of 160 mg/kg/d CIP-exposed mice, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (E) Schematic diagram of CIP-exposed mouse model with Trim21 knockdown. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 10 mg/kg/3 d AS-Trim 21 (with AS–NC as control). (F and G) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6, one-way ANOVA). (H) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (I) The quantification of positive intensity of SA-β-Galactose staining of placental tissues in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6, one-way ANOVA). (J) The protein levels of p16, p21, p38, and β-gal in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6). (K) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA). (L) The NAD + /NADH ratios in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA). (M) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA).

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: Verification of murine Trim21/Mff signalling in CIP-exposed mouse placental tissues. (A) Murine Trim21 mRNA levels in placental tissues of CIP-exposed mice (n = 6, one-way ANOVA). (B and C) Murine Trim21 protein levels in placental tissues of CIP-exposed mice and its relative quantification (n = 6, one-way ANOVA). (D) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in placental tissues of 160 mg/kg/d CIP-exposed mice, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (E) Schematic diagram of CIP-exposed mouse model with Trim21 knockdown. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 10 mg/kg/3 d AS-Trim 21 (with AS–NC as control). (F and G) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6, one-way ANOVA). (H) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (I) The quantification of positive intensity of SA-β-Galactose staining of placental tissues in 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6, one-way ANOVA). (J) The protein levels of p16, p21, p38, and β-gal in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (each n = 6). (K) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA). (L) The NAD + /NADH ratios in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA). (M) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with Trim21 knockdown (n = 6, one-way ANOVA).

Article Snippet: The primary antibodies contained anti-MFF (17090-1-AP, proteintech, dilution 1:1000, RRID: AB_2142463 ), anti-TRIM21 (67136-1-Ig, proteintech, dilution 1:1000, RRID: AB_2882435 ), anti-GLB1 (66586-1-Ig, proteintech, dilution 1:1000, RRID: AB_2881946 ), anti-p38 (80821-2-RR, proteintech, dilution 1:1000, RRID: AB_3670489 ), anti-p21 (ab109199, abcam, dilution 1:1000, RRID: AB_10861551 ), anti-p16 (ab189034, abcam, dilution 1:1000, RRID: RRID: AB_2737282), and anti-GAPDH (#5174, Cell Signalling Technology, dilution 1:1000, RRID: AB_10622025 ).

Techniques: Quantitative Proteomics, Knockdown, Saline, Control, Staining

Miscarriage treatment. (A) Schematic diagram of CIP-exposed mouse model with F + Q treatment. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 50 mg/kg/2 d F and 10 mg/kg/2 d Q. (B–D) The protein levels of Trim21 and Mff in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment and their relative quantification (each n = 6, Student's t-test). (E and F) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6, Student's t-test). (G) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in 160 mg/kg/d CIP-exposed mice with F + Q treatment, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (H) The quantification of positive intensity of SA-β-Galactose staining of placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6, Student's t-test). (I) The protein levels of p16, p21, p38, and β-gal in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6). (J) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (K) The NAD + /NADH ratios in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (L) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (M) The proposed mechanisms.

Journal: eBioMedicine

Article Title: Environmental ciprofloxacin triggers pregnancy loss: senescence-driven miscarriage via TRIM21-mediated MFF degradation

doi: 10.1016/j.ebiom.2026.106146

Figure Lengend Snippet: Miscarriage treatment. (A) Schematic diagram of CIP-exposed mouse model with F + Q treatment. Pregnant mice were treated with saline or 160 mg/kg/d CIP and also treated with 50 mg/kg/2 d F and 10 mg/kg/2 d Q. (B–D) The protein levels of Trim21 and Mff in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment and their relative quantification (each n = 6, Student's t-test). (E and F) Embryo resorption (indicated by red arrows) and the average miscarriage rates in 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6, Student's t-test). (G) IP assay analysis of the protein levels of Trim21 and Mff-Ub that were pulled down by Mff in 160 mg/kg/d CIP-exposed mice with F + Q treatment, with Mff and Trim21 protein levels in tissue lysates (each n = 6). (H) The quantification of positive intensity of SA-β-Galactose staining of placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6, Student's t-test). (I) The protein levels of p16, p21, p38, and β-gal in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (each n = 6). (J) The relative mtDNA copy number in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (K) The NAD + /NADH ratios in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (L) The MMP levels in placental tissues of 160 mg/kg/d CIP-exposed mice with F + Q treatment (n = 6, Student's t-test). (M) The proposed mechanisms.

Article Snippet: The primary antibodies contained anti-MFF (17090-1-AP, proteintech, dilution 1:1000, RRID: AB_2142463 ), anti-TRIM21 (67136-1-Ig, proteintech, dilution 1:1000, RRID: AB_2882435 ), anti-GLB1 (66586-1-Ig, proteintech, dilution 1:1000, RRID: AB_2881946 ), anti-p38 (80821-2-RR, proteintech, dilution 1:1000, RRID: AB_3670489 ), anti-p21 (ab109199, abcam, dilution 1:1000, RRID: AB_10861551 ), anti-p16 (ab189034, abcam, dilution 1:1000, RRID: RRID: AB_2737282), and anti-GAPDH (#5174, Cell Signalling Technology, dilution 1:1000, RRID: AB_10622025 ).

Techniques: Saline, Quantitative Proteomics, Staining

A. Schematic representation of the mechanism of ZAKα/P38 stress kinases activation upon induction of Ribotoxic Stress Response (RSR). B. Phosphotag blotting of phosphorylated NLRP1 disordered Region (DR) in HEK293T expressing the NLRP1 DR construct (aa 86-275-GFP (described in A )) and exposed to all PP1/PP2A-targeting compounds identified in /B ) or to the known RSR inducer Anisomycin (1 µg/mL) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. C. Phosphotag blotting of phosphorylated full length NLRP1 in primary human keratinocytes exposed Dinophysis toxin (100 nM) for various time. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. D. Phosphotag blotting of phosphorylated ZAKα and NLRP1 disordered Region (DR) in WT or ZAK KO NTERT NLRP1 KO + 86-275-SNAP keratinocytes exposed to Dinophysis toxin (100nM), Cantharidin (5%M) or Val-boro-Pro (VbP, 10µM) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Plasma membrane permeabilization (SYTOX Green incorporation, 16 h) and IL-1β release evaluation (10 h) in WT, ZAKα or NLRP1 KO NTERT keratinocytes after exposure to Dinophysis toxin (100nM), Cantharidin (5%M), Okadaic acid (250nM) or Anisomycin (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. F. Immunoblotting and clonal selection (clone 3, red), fluorescence microscopy and associated quantifications of ASC-GFP specks in WT or ZAKα KO HEK293T ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or Anisomycin (1µM) for 5 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA.

Journal: bioRxiv

Article Title: A TAK1-Driven NLRP1 Inflammasome Pathway Revealed by Phosphatase-Targeting Environmental Toxins

doi: 10.64898/2026.01.23.701233

Figure Lengend Snippet: A. Schematic representation of the mechanism of ZAKα/P38 stress kinases activation upon induction of Ribotoxic Stress Response (RSR). B. Phosphotag blotting of phosphorylated NLRP1 disordered Region (DR) in HEK293T expressing the NLRP1 DR construct (aa 86-275-GFP (described in A )) and exposed to all PP1/PP2A-targeting compounds identified in /B ) or to the known RSR inducer Anisomycin (1 µg/mL) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. C. Phosphotag blotting of phosphorylated full length NLRP1 in primary human keratinocytes exposed Dinophysis toxin (100 nM) for various time. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. D. Phosphotag blotting of phosphorylated ZAKα and NLRP1 disordered Region (DR) in WT or ZAK KO NTERT NLRP1 KO + 86-275-SNAP keratinocytes exposed to Dinophysis toxin (100nM), Cantharidin (5%M) or Val-boro-Pro (VbP, 10µM) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Plasma membrane permeabilization (SYTOX Green incorporation, 16 h) and IL-1β release evaluation (10 h) in WT, ZAKα or NLRP1 KO NTERT keratinocytes after exposure to Dinophysis toxin (100nM), Cantharidin (5%M), Okadaic acid (250nM) or Anisomycin (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. F. Immunoblotting and clonal selection (clone 3, red), fluorescence microscopy and associated quantifications of ASC-GFP specks in WT or ZAKα KO HEK293T ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or Anisomycin (1µM) for 5 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA.

Article Snippet: Antibodies used in this study were anti-Gasdermin D (E8G3F) Monoclonal antibody (1:1000, Cell signaling, 97558S); anti-Caspase 1 (p20) (human) mAb (Bally-1) antibody (1:500, Adipogen, AG-20B-0048); anti-DFNA5/Gasdermin E (EPR19859) Monoclonal antibody (1:1000,Abcam, ab215191); anti-Caspase 3 antibody (1:500, Cell signaling, 9662S); anti-Human IL-1β/IL-1F2 Polyclonal antibody (1:500, R&D systems, AF-201-NA); anti-Cleaved IL-1β (Asp116) Monoclonal antibody (1:750, Cell signaling, 83186S); anti-Tubulin-α antibody (1:1000, Abcam, ab4074); anti-PP1alpha Polyclonal antibody (1:1000, Invitrogen, PA5-119781); anti-PP1CB-Specific antibody (1:1000, ProteinTech, PR-55136-AP-150); anti-PP1CC antibody (1:1000, ProteinTech, PR-11082-1-AP-150); PP2A C Subunit antibody (1:500, Cell signaling, 2038S); anti-Purified NLRP1 (N-terminal) antibody (1:300, Biolegend, 679802); anti-NLRP1 (C-terminal) Polyclonal antibody (1:500,Abcam, ab36852); anti-GFP antibody (1:1000, Abcam, ab6673); anti-SNAP/CLIP-tag Monoclonal antibody (1:1000, ProteinTech, 6F9-100); anti-ZAKα Polyclonal antibody (1:1000, Bethyl Laboratories, A301-993A); anti-P38 MAPK antibody (1:1000, Cell signaling, 9212S); anti-Phospho-p38 (Thr180/Tyr182) (D3F9) Monoclonal antibody (1:1000, Cell signaling, 4511S); anti-Puromycine antibody Clone 12D10 (1:1000, Sigma-Aldrich, MABE343); anti-TAK1 antibody (1:1000, Cell signaling, 4505S); anti-Phospho-TAK1 (Ser412) antibody (1:1000, Cell signaling, 9339S); anti-Phospho-TAK1 (Thr184/187) 90C7 Monoclonal antibody (1:1000, Cell signaling, 4508S); anti-P38 alpha/MAPK14 antibody (E229) (1:1000, Abcam, ab170099); anti-P38 MAPK beta Monoclonal antibody (1:1000, Invitrogen, MA514-950); anti-P38 beta MAPK (C28C2) Monoclonal antibody (1:1000, Cell Signaling, 2339S); anti-P38 gamma/MAPK12 antibody (EPR6528N) (1:1000, Abcam, ab205926); anti-P38 gamma MAPK antibody (1:1000, Cell Signaling, 2307S); anti-Human P38 delta antibody (1:1000, R&D system, AF1519); anti-P38δ MAPK (10A8) mAb (1:1000, Cell Signaling, 2308S); anti-GAPDH antibody (1:1000, GeneTex, GTX100118); Goat anti-rabbit HRP secondary antibody (1:5000, Advansta R-05072-500); Goat anti-mouse HRP secondary antibody (1:5000, Advansta, R-05071-500); Goat anti-rat IgG H&L (HRP) (1:5000, Abcam, ab97057); Goat IgG HRP-conjugated Antibody (1:5000, Biotechne, HAF109).

Techniques: Activation Assay, Expressing, Construct, Western Blot, Clinical Proteomics, Membrane, Selection, Fluorescence, Microscopy, Staining

A. Pamgene analysis of activated Serine/threonine kinases in primary human keratinocytes exposed to Dinophysis toxin (100nM) for 2 hours and subsequent determination of IL-1β release in WT NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) in presence/absence of inhibitors of identified kinases in. For all kinases, inhibitors were used at 10µM. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least two times. B. Phosphotag blotting of phosphorylated P38 kinase isoforms in NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour in presence/absence of the Pan P38 inhibitor Doramapimod (10µM). Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. C. Immunoblotting of P38, ZAKα, NLRP1 and phosphorylated P38 kinases in WT, ZAKα KO or NLRP1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. D. Immunoblotting characterization of the P38 isoform genetic knockdown (CRISPR-Cas9) and of the subsequent IL-1β release in WT, P38δ KO, P38α/β dKO, or P38α/β/δ TKO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) for 8 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. E. Western blot showing NLRP1 (anti-NLRP1 N-terminal antibody (aa 1–323)) and associated fluorescence microscopy/quantifications of ASC-GFP specks in HEK293 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for important 38 phosphorylation sites (S107A, TST112-114AAA and TST178-180AAA) after 10 h of exposure to Dinophysis toxin (100nM), Okadaic acid (250nM), Cantharidin (5µM) or Val-boro-Pro (VbP, 10µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of three independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.

Journal: bioRxiv

Article Title: A TAK1-Driven NLRP1 Inflammasome Pathway Revealed by Phosphatase-Targeting Environmental Toxins

doi: 10.64898/2026.01.23.701233

Figure Lengend Snippet: A. Pamgene analysis of activated Serine/threonine kinases in primary human keratinocytes exposed to Dinophysis toxin (100nM) for 2 hours and subsequent determination of IL-1β release in WT NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) in presence/absence of inhibitors of identified kinases in. For all kinases, inhibitors were used at 10µM. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least two times. B. Phosphotag blotting of phosphorylated P38 kinase isoforms in NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour in presence/absence of the Pan P38 inhibitor Doramapimod (10µM). Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. C. Immunoblotting of P38, ZAKα, NLRP1 and phosphorylated P38 kinases in WT, ZAKα KO or NLRP1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. D. Immunoblotting characterization of the P38 isoform genetic knockdown (CRISPR-Cas9) and of the subsequent IL-1β release in WT, P38δ KO, P38α/β dKO, or P38α/β/δ TKO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) for 8 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. E. Western blot showing NLRP1 (anti-NLRP1 N-terminal antibody (aa 1–323)) and associated fluorescence microscopy/quantifications of ASC-GFP specks in HEK293 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for important 38 phosphorylation sites (S107A, TST112-114AAA and TST178-180AAA) after 10 h of exposure to Dinophysis toxin (100nM), Okadaic acid (250nM), Cantharidin (5µM) or Val-boro-Pro (VbP, 10µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of three independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.

Article Snippet: Antibodies used in this study were anti-Gasdermin D (E8G3F) Monoclonal antibody (1:1000, Cell signaling, 97558S); anti-Caspase 1 (p20) (human) mAb (Bally-1) antibody (1:500, Adipogen, AG-20B-0048); anti-DFNA5/Gasdermin E (EPR19859) Monoclonal antibody (1:1000,Abcam, ab215191); anti-Caspase 3 antibody (1:500, Cell signaling, 9662S); anti-Human IL-1β/IL-1F2 Polyclonal antibody (1:500, R&D systems, AF-201-NA); anti-Cleaved IL-1β (Asp116) Monoclonal antibody (1:750, Cell signaling, 83186S); anti-Tubulin-α antibody (1:1000, Abcam, ab4074); anti-PP1alpha Polyclonal antibody (1:1000, Invitrogen, PA5-119781); anti-PP1CB-Specific antibody (1:1000, ProteinTech, PR-55136-AP-150); anti-PP1CC antibody (1:1000, ProteinTech, PR-11082-1-AP-150); PP2A C Subunit antibody (1:500, Cell signaling, 2038S); anti-Purified NLRP1 (N-terminal) antibody (1:300, Biolegend, 679802); anti-NLRP1 (C-terminal) Polyclonal antibody (1:500,Abcam, ab36852); anti-GFP antibody (1:1000, Abcam, ab6673); anti-SNAP/CLIP-tag Monoclonal antibody (1:1000, ProteinTech, 6F9-100); anti-ZAKα Polyclonal antibody (1:1000, Bethyl Laboratories, A301-993A); anti-P38 MAPK antibody (1:1000, Cell signaling, 9212S); anti-Phospho-p38 (Thr180/Tyr182) (D3F9) Monoclonal antibody (1:1000, Cell signaling, 4511S); anti-Puromycine antibody Clone 12D10 (1:1000, Sigma-Aldrich, MABE343); anti-TAK1 antibody (1:1000, Cell signaling, 4505S); anti-Phospho-TAK1 (Ser412) antibody (1:1000, Cell signaling, 9339S); anti-Phospho-TAK1 (Thr184/187) 90C7 Monoclonal antibody (1:1000, Cell signaling, 4508S); anti-P38 alpha/MAPK14 antibody (E229) (1:1000, Abcam, ab170099); anti-P38 MAPK beta Monoclonal antibody (1:1000, Invitrogen, MA514-950); anti-P38 beta MAPK (C28C2) Monoclonal antibody (1:1000, Cell Signaling, 2339S); anti-P38 gamma/MAPK12 antibody (EPR6528N) (1:1000, Abcam, ab205926); anti-P38 gamma MAPK antibody (1:1000, Cell Signaling, 2307S); anti-Human P38 delta antibody (1:1000, R&D system, AF1519); anti-P38δ MAPK (10A8) mAb (1:1000, Cell Signaling, 2308S); anti-GAPDH antibody (1:1000, GeneTex, GTX100118); Goat anti-rabbit HRP secondary antibody (1:5000, Advansta R-05072-500); Goat anti-mouse HRP secondary antibody (1:5000, Advansta, R-05071-500); Goat anti-rat IgG H&L (HRP) (1:5000, Abcam, ab97057); Goat IgG HRP-conjugated Antibody (1:5000, Biotechne, HAF109).

Techniques: Western Blot, Knockdown, CRISPR, Fluorescence, Microscopy, Plasmid Preparation, Construct, Phospho-proteomics

A. Quantifications of ASC-GFP specks in HEK293 ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or not for 6 h in presence or absence of various MAPK inhibitors (10µM). TAK1 inhibitor; HS-276, ZAKα inhibitor; PLX4720, TAOK inhibitor; CP-43, MLKL inhibitor; necro sulfonamide, ASK1 inhibitor; GS-444217, DLK/LZK inhibitor; DN-1289, RIPK3 inhibitor; GSK-872. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. B. Determination of the IL-1β release in WT, P38α/β/δ TKO, TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of P38, TAK1, cleaved GSDMD and IL-1β and of the subsequent IL-1β release in WT, P38α/β/δ TKO, TAK1 KO, P38α/β/δ TKO/TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Phosphotag blotting of phosphorylated P38, TAK1 and NLRP1-DR-SNAP in WT, P38α/β/δ TKO, TAK1 KO or P38α/β/δ TKO/TAK1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Phosphotag immunoblotting of phosphorylated recombinant NLRP1 full-length protein or immunoprecipitated GFP-tagged NLRP1-DR incubated with recombinant TAK1-TAB1 fusion or P38α kinases for 60 minutes in presence/absence of lambda phosphatase. Immunoblots show proteins from one experiment performed at least three times. F. Fluorescence microscopy quantifications of ASC-GFP specks in WT, P38α/β/δ TKO or in P38α/β/δ TKO/TAK1 KO HEK293 ASC-GFP/NLRP1 reporter cells after 6 h of exposure to Dinophysis toxin (100nM) or Val-boro-Pro (VbP, 10µM). ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.

Journal: bioRxiv

Article Title: A TAK1-Driven NLRP1 Inflammasome Pathway Revealed by Phosphatase-Targeting Environmental Toxins

doi: 10.64898/2026.01.23.701233

Figure Lengend Snippet: A. Quantifications of ASC-GFP specks in HEK293 ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or not for 6 h in presence or absence of various MAPK inhibitors (10µM). TAK1 inhibitor; HS-276, ZAKα inhibitor; PLX4720, TAOK inhibitor; CP-43, MLKL inhibitor; necro sulfonamide, ASK1 inhibitor; GS-444217, DLK/LZK inhibitor; DN-1289, RIPK3 inhibitor; GSK-872. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. B. Determination of the IL-1β release in WT, P38α/β/δ TKO, TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of P38, TAK1, cleaved GSDMD and IL-1β and of the subsequent IL-1β release in WT, P38α/β/δ TKO, TAK1 KO, P38α/β/δ TKO/TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Phosphotag blotting of phosphorylated P38, TAK1 and NLRP1-DR-SNAP in WT, P38α/β/δ TKO, TAK1 KO or P38α/β/δ TKO/TAK1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Phosphotag immunoblotting of phosphorylated recombinant NLRP1 full-length protein or immunoprecipitated GFP-tagged NLRP1-DR incubated with recombinant TAK1-TAB1 fusion or P38α kinases for 60 minutes in presence/absence of lambda phosphatase. Immunoblots show proteins from one experiment performed at least three times. F. Fluorescence microscopy quantifications of ASC-GFP specks in WT, P38α/β/δ TKO or in P38α/β/δ TKO/TAK1 KO HEK293 ASC-GFP/NLRP1 reporter cells after 6 h of exposure to Dinophysis toxin (100nM) or Val-boro-Pro (VbP, 10µM). ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.

Article Snippet: Antibodies used in this study were anti-Gasdermin D (E8G3F) Monoclonal antibody (1:1000, Cell signaling, 97558S); anti-Caspase 1 (p20) (human) mAb (Bally-1) antibody (1:500, Adipogen, AG-20B-0048); anti-DFNA5/Gasdermin E (EPR19859) Monoclonal antibody (1:1000,Abcam, ab215191); anti-Caspase 3 antibody (1:500, Cell signaling, 9662S); anti-Human IL-1β/IL-1F2 Polyclonal antibody (1:500, R&D systems, AF-201-NA); anti-Cleaved IL-1β (Asp116) Monoclonal antibody (1:750, Cell signaling, 83186S); anti-Tubulin-α antibody (1:1000, Abcam, ab4074); anti-PP1alpha Polyclonal antibody (1:1000, Invitrogen, PA5-119781); anti-PP1CB-Specific antibody (1:1000, ProteinTech, PR-55136-AP-150); anti-PP1CC antibody (1:1000, ProteinTech, PR-11082-1-AP-150); PP2A C Subunit antibody (1:500, Cell signaling, 2038S); anti-Purified NLRP1 (N-terminal) antibody (1:300, Biolegend, 679802); anti-NLRP1 (C-terminal) Polyclonal antibody (1:500,Abcam, ab36852); anti-GFP antibody (1:1000, Abcam, ab6673); anti-SNAP/CLIP-tag Monoclonal antibody (1:1000, ProteinTech, 6F9-100); anti-ZAKα Polyclonal antibody (1:1000, Bethyl Laboratories, A301-993A); anti-P38 MAPK antibody (1:1000, Cell signaling, 9212S); anti-Phospho-p38 (Thr180/Tyr182) (D3F9) Monoclonal antibody (1:1000, Cell signaling, 4511S); anti-Puromycine antibody Clone 12D10 (1:1000, Sigma-Aldrich, MABE343); anti-TAK1 antibody (1:1000, Cell signaling, 4505S); anti-Phospho-TAK1 (Ser412) antibody (1:1000, Cell signaling, 9339S); anti-Phospho-TAK1 (Thr184/187) 90C7 Monoclonal antibody (1:1000, Cell signaling, 4508S); anti-P38 alpha/MAPK14 antibody (E229) (1:1000, Abcam, ab170099); anti-P38 MAPK beta Monoclonal antibody (1:1000, Invitrogen, MA514-950); anti-P38 beta MAPK (C28C2) Monoclonal antibody (1:1000, Cell Signaling, 2339S); anti-P38 gamma/MAPK12 antibody (EPR6528N) (1:1000, Abcam, ab205926); anti-P38 gamma MAPK antibody (1:1000, Cell Signaling, 2307S); anti-Human P38 delta antibody (1:1000, R&D system, AF1519); anti-P38δ MAPK (10A8) mAb (1:1000, Cell Signaling, 2308S); anti-GAPDH antibody (1:1000, GeneTex, GTX100118); Goat anti-rabbit HRP secondary antibody (1:5000, Advansta R-05072-500); Goat anti-mouse HRP secondary antibody (1:5000, Advansta, R-05071-500); Goat anti-rat IgG H&L (HRP) (1:5000, Abcam, ab97057); Goat IgG HRP-conjugated Antibody (1:5000, Biotechne, HAF109).

Techniques: Staining, Western Blot, Recombinant, Immunoprecipitation, Incubation, Fluorescence, Microscopy

A-C . Hematoxylin (H) & Eosin (E) or ASC immunobiological staining showing P38 and TAK1-dependent histological/inflammasome changes caused by Dinophysis toxin (250 nM), Okadaic acid (600 nM) and Cantharidin (10µM) exposure for 24 hours. When specified, pan P38 kinase inhibitor Doramapimod (10µM) and TAK1 inhibitor HS-276 (20µM) were used. Associated quantification of IL-1β release, the dermal–epidermal layer detachment and the percentage of ASC specks in the Human skin explants. P values indicated in figure, one-way ANOVA. Images are representative of two (A) and three (B) biological replicates. Scale bar = 50 µm.

Journal: bioRxiv

Article Title: A TAK1-Driven NLRP1 Inflammasome Pathway Revealed by Phosphatase-Targeting Environmental Toxins

doi: 10.64898/2026.01.23.701233

Figure Lengend Snippet: A-C . Hematoxylin (H) & Eosin (E) or ASC immunobiological staining showing P38 and TAK1-dependent histological/inflammasome changes caused by Dinophysis toxin (250 nM), Okadaic acid (600 nM) and Cantharidin (10µM) exposure for 24 hours. When specified, pan P38 kinase inhibitor Doramapimod (10µM) and TAK1 inhibitor HS-276 (20µM) were used. Associated quantification of IL-1β release, the dermal–epidermal layer detachment and the percentage of ASC specks in the Human skin explants. P values indicated in figure, one-way ANOVA. Images are representative of two (A) and three (B) biological replicates. Scale bar = 50 µm.

Article Snippet: Antibodies used in this study were anti-Gasdermin D (E8G3F) Monoclonal antibody (1:1000, Cell signaling, 97558S); anti-Caspase 1 (p20) (human) mAb (Bally-1) antibody (1:500, Adipogen, AG-20B-0048); anti-DFNA5/Gasdermin E (EPR19859) Monoclonal antibody (1:1000,Abcam, ab215191); anti-Caspase 3 antibody (1:500, Cell signaling, 9662S); anti-Human IL-1β/IL-1F2 Polyclonal antibody (1:500, R&D systems, AF-201-NA); anti-Cleaved IL-1β (Asp116) Monoclonal antibody (1:750, Cell signaling, 83186S); anti-Tubulin-α antibody (1:1000, Abcam, ab4074); anti-PP1alpha Polyclonal antibody (1:1000, Invitrogen, PA5-119781); anti-PP1CB-Specific antibody (1:1000, ProteinTech, PR-55136-AP-150); anti-PP1CC antibody (1:1000, ProteinTech, PR-11082-1-AP-150); PP2A C Subunit antibody (1:500, Cell signaling, 2038S); anti-Purified NLRP1 (N-terminal) antibody (1:300, Biolegend, 679802); anti-NLRP1 (C-terminal) Polyclonal antibody (1:500,Abcam, ab36852); anti-GFP antibody (1:1000, Abcam, ab6673); anti-SNAP/CLIP-tag Monoclonal antibody (1:1000, ProteinTech, 6F9-100); anti-ZAKα Polyclonal antibody (1:1000, Bethyl Laboratories, A301-993A); anti-P38 MAPK antibody (1:1000, Cell signaling, 9212S); anti-Phospho-p38 (Thr180/Tyr182) (D3F9) Monoclonal antibody (1:1000, Cell signaling, 4511S); anti-Puromycine antibody Clone 12D10 (1:1000, Sigma-Aldrich, MABE343); anti-TAK1 antibody (1:1000, Cell signaling, 4505S); anti-Phospho-TAK1 (Ser412) antibody (1:1000, Cell signaling, 9339S); anti-Phospho-TAK1 (Thr184/187) 90C7 Monoclonal antibody (1:1000, Cell signaling, 4508S); anti-P38 alpha/MAPK14 antibody (E229) (1:1000, Abcam, ab170099); anti-P38 MAPK beta Monoclonal antibody (1:1000, Invitrogen, MA514-950); anti-P38 beta MAPK (C28C2) Monoclonal antibody (1:1000, Cell Signaling, 2339S); anti-P38 gamma/MAPK12 antibody (EPR6528N) (1:1000, Abcam, ab205926); anti-P38 gamma MAPK antibody (1:1000, Cell Signaling, 2307S); anti-Human P38 delta antibody (1:1000, R&D system, AF1519); anti-P38δ MAPK (10A8) mAb (1:1000, Cell Signaling, 2308S); anti-GAPDH antibody (1:1000, GeneTex, GTX100118); Goat anti-rabbit HRP secondary antibody (1:5000, Advansta R-05072-500); Goat anti-mouse HRP secondary antibody (1:5000, Advansta, R-05071-500); Goat anti-rat IgG H&L (HRP) (1:5000, Abcam, ab97057); Goat IgG HRP-conjugated Antibody (1:5000, Biotechne, HAF109).

Techniques: Staining

Ectosomal‐calhm6 mitigates inflammation and enhances tissue repair via M2‐like macrophage polarization. A) After 24 h of LPS treatment, ectosomes were collected from the serum of wild‐type or Calhm6 knockout mice. These exosomes were used to treat wild‐type BMDMs, and the expression of M2‐like markers in the BMDMs was analyzed using RT‐PCR. B) RT‐PCR analysis of TNFA, NOS2, IL6, CHIL3, IL10, and ARG1 in wild‐type BMDMs incubated with or without ectosomes for 24 h from EV or OE‐Calhm6 cells. C) Immunoblot analysis of phosphorylated (p‐) p38, NFκB, IκBα, and β‐actin in wild‐type BMDMs stimulated for 0, 30, 60 min with LPS (200 ng mL −1 ) after incubation with ectosomal‐EV or ectosomal‐Calhm6 from EV or OE‐Calhm6 cells for 24 h. D,E) Morphology of EV or OE‐Calhm6 cells (D) and cell length (I) was measured by ImageJ ( n =304). Scale bars(E), 100 µm. F) RT‐PCR analysis of CHIL3, IL10, ARG1, TNFA, NOS2 and IL6 in EV or OE‐Calhm6 cells treated with or without BAPTA‐AM (20 µ m ) or Ca 2+ ‐free DMEM for 24 h. G) Calcium influx over time in EV or OE‐Calhm6 cells treated with LPS (200 ng mL −1 ) and stained by the Rhod2‐AM (4 µ m , 30 min). H,I) Diagram (H) of ectosomes by sequential ultracentrifugation for mass spectrometry from serum of wild‐type BMDMs treatment with LPS (200 ng mL −1 ) and IFNγ (10 ng mL −1 ) combined or IL‐4 (20 ng mL −1 ) for 0, 12 or 24 h; the indicated genes identified were shown in the table (I). J) Fluorescence microscopy of the colocalization (white arrows) of Calhm6 (green) and Tsg101 (red) in wild‐type BMDMs treated with or without LPS, IFNγ combined or IL‐4 for 12 h. Scale bars, 10 µm. K) Representative photographs of the wounds on days 0, 3, 6, and 10 after parawound injection of PBS (control), ectosomal‐EV, ectosomal‐Calhm6 in the mouse excisional wound splinting model. Scale bars, 2 mm. L) Measurement of wound area at indicated points. The percentage of wound closure was calculated as: (area of wound at time/area of original wound×100%. M) Immunofluorescence histochemistry of frozen section of wound area of F4/80 (red), CD206 (green), and counterstained with DAPI (blue) in each group as indicated above at day 10. Scale bars, 200 µm. N) Pearson's correlation coefficient values for colocalization of CD206 and F4/80 in the wound area. The average Pearson's correlation coefficients were calculated from eight randomly selected infected cells in each group ( n = 12 photos examined). O) Immunoblot analysis of Arg, Tgf‐β1, F4/80, and β‐actin in F4/80 + cells from the wound area for each group as indicated above at day 10. The data represent the mean ± S.D. ( n =3). NS, not significant ( p > 0.05); * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.00001 compared with control, Student's t‐test.

Journal: Advanced Science

Article Title: Calhm6 Governs Macrophage Polarization Through Chp1‐Camk4‐Creb1 Axis and Ectosomal Delivery in Inflammatory Responses

doi: 10.1002/advs.202502395

Figure Lengend Snippet: Ectosomal‐calhm6 mitigates inflammation and enhances tissue repair via M2‐like macrophage polarization. A) After 24 h of LPS treatment, ectosomes were collected from the serum of wild‐type or Calhm6 knockout mice. These exosomes were used to treat wild‐type BMDMs, and the expression of M2‐like markers in the BMDMs was analyzed using RT‐PCR. B) RT‐PCR analysis of TNFA, NOS2, IL6, CHIL3, IL10, and ARG1 in wild‐type BMDMs incubated with or without ectosomes for 24 h from EV or OE‐Calhm6 cells. C) Immunoblot analysis of phosphorylated (p‐) p38, NFκB, IκBα, and β‐actin in wild‐type BMDMs stimulated for 0, 30, 60 min with LPS (200 ng mL −1 ) after incubation with ectosomal‐EV or ectosomal‐Calhm6 from EV or OE‐Calhm6 cells for 24 h. D,E) Morphology of EV or OE‐Calhm6 cells (D) and cell length (I) was measured by ImageJ ( n =304). Scale bars(E), 100 µm. F) RT‐PCR analysis of CHIL3, IL10, ARG1, TNFA, NOS2 and IL6 in EV or OE‐Calhm6 cells treated with or without BAPTA‐AM (20 µ m ) or Ca 2+ ‐free DMEM for 24 h. G) Calcium influx over time in EV or OE‐Calhm6 cells treated with LPS (200 ng mL −1 ) and stained by the Rhod2‐AM (4 µ m , 30 min). H,I) Diagram (H) of ectosomes by sequential ultracentrifugation for mass spectrometry from serum of wild‐type BMDMs treatment with LPS (200 ng mL −1 ) and IFNγ (10 ng mL −1 ) combined or IL‐4 (20 ng mL −1 ) for 0, 12 or 24 h; the indicated genes identified were shown in the table (I). J) Fluorescence microscopy of the colocalization (white arrows) of Calhm6 (green) and Tsg101 (red) in wild‐type BMDMs treated with or without LPS, IFNγ combined or IL‐4 for 12 h. Scale bars, 10 µm. K) Representative photographs of the wounds on days 0, 3, 6, and 10 after parawound injection of PBS (control), ectosomal‐EV, ectosomal‐Calhm6 in the mouse excisional wound splinting model. Scale bars, 2 mm. L) Measurement of wound area at indicated points. The percentage of wound closure was calculated as: (area of wound at time/area of original wound×100%. M) Immunofluorescence histochemistry of frozen section of wound area of F4/80 (red), CD206 (green), and counterstained with DAPI (blue) in each group as indicated above at day 10. Scale bars, 200 µm. N) Pearson's correlation coefficient values for colocalization of CD206 and F4/80 in the wound area. The average Pearson's correlation coefficients were calculated from eight randomly selected infected cells in each group ( n = 12 photos examined). O) Immunoblot analysis of Arg, Tgf‐β1, F4/80, and β‐actin in F4/80 + cells from the wound area for each group as indicated above at day 10. The data represent the mean ± S.D. ( n =3). NS, not significant ( p > 0.05); * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.00001 compared with control, Student's t‐test.

Article Snippet: Anti‐β‐Actin (66009‐1‐Ig), anti‐TGF‐beta1 (21898‐1‐AP), anti‐F4/80 (29414‐1‐AP) and antibody to phosphorylated p38 (28796‐1‐AP) were from Proteintech.

Techniques: Knock-Out, Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Western Blot, Staining, Mass Spectrometry, Fluorescence, Microscopy, Injection, Control, Immunofluorescence, Infection

Calhm6 promotes Creb1 activation and M2‐like polarization of macrophages via the Chp1‐CaMK4 axis. A) Identification of Calhm6, Chp1, and CaMK4 by mass spectrometry in a Flag‐tagged Calhm6‐precipitation assay in cell lysates of OE‐Calhm6 cells, untreated or treated with IL‐4 (20 ng mL −1 ) or CaCl 2 (2 m m ). B) RT‐PCR of TNFA, NOS2, IL6, CHIL3, IL10, and ARG1 in Sh‐Scr or Sh‐Chp1 cells treated with LPS, IFNγ combined, or IL‐4 (dose as above). C) Immunoblot analysis of phosphorylated (p‐) p38, NFκB, IκBα, and β‐actin in total lysates of Raw264.7 cells treated with Scramble‐shRNA (Sh‐Scr) or Chp1‐specific shRNA (Sh‐Chp1), left treated for 0, 30, 60 min with LPS (200 ng mL −1 ) or LTA (100 ng mL −1 ). D,E) Seahorse analysis of OCR (D) and ECAR (E) in BMDMs from Sh‐Scr or Sh‐Chp1 cells. F) Immunoblot analysis of (p‐) Stat1, IκBα, Creb1, Stat3, Stat6, and β‐actin in Sh‐Scr or Sh‐Chp1 cells stimulated for 0, 30 min with LPS (200 ng mL −1 ) and IFNγ (10 ng mL −1 ) combined or IL‐4 (20 ng mL −1 ). G) Immunoblot analysis of p‐Creb1 and Chp1, CaMK4, β‐actin in Raw264.7 cells expressing Flag‐tagged CaMK4 and Myc‐tagged Chp1. H) HeLa cells were transfected with plasmids expressing Myc‐tagged CaMK4 and HA‐tagged Chp1. Before fixing the cells, they were treated with CaCl 2 , BAPTA‐AM, or Ca 2+ ‐free DMEM for 3 h. Confocal microscopy shows the co‐localization of Chp1 (red) with CaMK4 (green) at the cell periphery. Images shown are representative of approximately 20 cells. Scale bar, 20 µm. I) Immunoassay of 293T cells expressing various combinations (above lanes) of Flag‐tagged CaMK4 and Myc‐tagged Chp1, and after being treated with PBS or the CaCl 2 , BAPTA‐AM, or Ca 2+ ‐free DMEM for 3 h, assessed in anti‐Flag immunoprecipitates or total cell lysates, probed with various tag antibodies; below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. J) Immunoassays of 293T cells expressing various combinations (above lanes) of Flag‐tagged Chp1 and the fragment of GFP‐tagged CaMK4 as indicated; immunoprecipitation with anti‐Flag and analysis by immunoblot with the tag antibodies; below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. K) Immunoassays (as in J) of 293T cells expressing various combinations (above lanes) of Flag‐tagged CaMK4 and the fragment of GFP‐tagged Chp1 as indicated. L) Phos‐tag analysis of 293T cells expressing Flag‐tagged wild‐type Chp1 or Chp1 (S99A) and HA‐tagged CaMK4 (above lanes). M) Immunoassay of lysates of 293T cells expressing various combinations (above lanes) of Flag‐tagged wild‐type Chp1 or Chp1 (S99A) and HA‐tagged CaMK4, immunoprecipitated with anti‐Flag and analyzed by immunoblot with anti‐HA (α‐HA) or anti‐Flag (α‐Flag); below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. N) Immunoblot analysis of (p‐) and total CaMK4 and β‐actin in BMDMs treated with LPS, IFNγ combined, or IL‐4 for 30 min. O) Immunoblot analysis of (p‐) and total CaMK4 and β‐actin in BMDMs treated with IL‐4 for 30 min after pretreatment with BAPTA‐AM (20 µ m ) or Ca 2+ ‐free DMEM for 3 h. NS, not significant ( p > 0.05); * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with control, Student's t‐test.

Journal: Advanced Science

Article Title: Calhm6 Governs Macrophage Polarization Through Chp1‐Camk4‐Creb1 Axis and Ectosomal Delivery in Inflammatory Responses

doi: 10.1002/advs.202502395

Figure Lengend Snippet: Calhm6 promotes Creb1 activation and M2‐like polarization of macrophages via the Chp1‐CaMK4 axis. A) Identification of Calhm6, Chp1, and CaMK4 by mass spectrometry in a Flag‐tagged Calhm6‐precipitation assay in cell lysates of OE‐Calhm6 cells, untreated or treated with IL‐4 (20 ng mL −1 ) or CaCl 2 (2 m m ). B) RT‐PCR of TNFA, NOS2, IL6, CHIL3, IL10, and ARG1 in Sh‐Scr or Sh‐Chp1 cells treated with LPS, IFNγ combined, or IL‐4 (dose as above). C) Immunoblot analysis of phosphorylated (p‐) p38, NFκB, IκBα, and β‐actin in total lysates of Raw264.7 cells treated with Scramble‐shRNA (Sh‐Scr) or Chp1‐specific shRNA (Sh‐Chp1), left treated for 0, 30, 60 min with LPS (200 ng mL −1 ) or LTA (100 ng mL −1 ). D,E) Seahorse analysis of OCR (D) and ECAR (E) in BMDMs from Sh‐Scr or Sh‐Chp1 cells. F) Immunoblot analysis of (p‐) Stat1, IκBα, Creb1, Stat3, Stat6, and β‐actin in Sh‐Scr or Sh‐Chp1 cells stimulated for 0, 30 min with LPS (200 ng mL −1 ) and IFNγ (10 ng mL −1 ) combined or IL‐4 (20 ng mL −1 ). G) Immunoblot analysis of p‐Creb1 and Chp1, CaMK4, β‐actin in Raw264.7 cells expressing Flag‐tagged CaMK4 and Myc‐tagged Chp1. H) HeLa cells were transfected with plasmids expressing Myc‐tagged CaMK4 and HA‐tagged Chp1. Before fixing the cells, they were treated with CaCl 2 , BAPTA‐AM, or Ca 2+ ‐free DMEM for 3 h. Confocal microscopy shows the co‐localization of Chp1 (red) with CaMK4 (green) at the cell periphery. Images shown are representative of approximately 20 cells. Scale bar, 20 µm. I) Immunoassay of 293T cells expressing various combinations (above lanes) of Flag‐tagged CaMK4 and Myc‐tagged Chp1, and after being treated with PBS or the CaCl 2 , BAPTA‐AM, or Ca 2+ ‐free DMEM for 3 h, assessed in anti‐Flag immunoprecipitates or total cell lysates, probed with various tag antibodies; below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. J) Immunoassays of 293T cells expressing various combinations (above lanes) of Flag‐tagged Chp1 and the fragment of GFP‐tagged CaMK4 as indicated; immunoprecipitation with anti‐Flag and analysis by immunoblot with the tag antibodies; below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. K) Immunoassays (as in J) of 293T cells expressing various combinations (above lanes) of Flag‐tagged CaMK4 and the fragment of GFP‐tagged Chp1 as indicated. L) Phos‐tag analysis of 293T cells expressing Flag‐tagged wild‐type Chp1 or Chp1 (S99A) and HA‐tagged CaMK4 (above lanes). M) Immunoassay of lysates of 293T cells expressing various combinations (above lanes) of Flag‐tagged wild‐type Chp1 or Chp1 (S99A) and HA‐tagged CaMK4, immunoprecipitated with anti‐Flag and analyzed by immunoblot with anti‐HA (α‐HA) or anti‐Flag (α‐Flag); below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. N) Immunoblot analysis of (p‐) and total CaMK4 and β‐actin in BMDMs treated with LPS, IFNγ combined, or IL‐4 for 30 min. O) Immunoblot analysis of (p‐) and total CaMK4 and β‐actin in BMDMs treated with IL‐4 for 30 min after pretreatment with BAPTA‐AM (20 µ m ) or Ca 2+ ‐free DMEM for 3 h. NS, not significant ( p > 0.05); * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with control, Student's t‐test.

Article Snippet: Anti‐β‐Actin (66009‐1‐Ig), anti‐TGF‐beta1 (21898‐1‐AP), anti‐F4/80 (29414‐1‐AP) and antibody to phosphorylated p38 (28796‐1‐AP) were from Proteintech.

Techniques: Activation Assay, Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction, Western Blot, shRNA, Expressing, Transfection, Confocal Microscopy, Immunoprecipitation, Control

Fig. 7. B–F indicates the effects of aerobic exercise on aortic P38MAPK, pp38MAPK, pp38MAPK/P38MAPK, collagen II, and IL-6 protein expression in AS rats, compared with Con group, * p < 0.05 and **p < 0.01; compared with AS group # p < 0.05 and ##p < 0.01.

Journal: Experimental gerontology

Article Title: Aerobic exercise modulates aortic chondrogenesis and calcification via 5-methoxytryptophan and P38MAPK in atherosclerotic rats.

doi: 10.1016/j.exger.2025.112722

Figure Lengend Snippet: Fig. 7. B–F indicates the effects of aerobic exercise on aortic P38MAPK, pp38MAPK, pp38MAPK/P38MAPK, collagen II, and IL-6 protein expression in AS rats, compared with Con group, * p < 0.05 and **p < 0.01; compared with AS group # p < 0.05 and ##p < 0.01.

Article Snippet: The PVDF transmembrane was transfected with the addition of primary antibodies pp38 MAPK (1:1000, ab4822, abcam), p38 MAPK (1:1000, NB100–56446, NOVUS), collagen II (1:1000, ab34712, abcam), IL-6 (1:1000, NBP2–89149, NOVUS), incubated for 12 h, secondary antibody incubated for 2 h, ECL developed, fixed, exposed and scanned to process the gray value of each band.

Techniques: Expressing