Journal: iScience

Article Title: Downregulation of SPRED1 drives keratinocyte hyperproliferation in psoriasis via activation of the ERK-RSK1 signaling axis

doi: 10.1016/j.isci.2026.116812

Figure Lengend Snippet: SPRED1 regulates NHEK cell proliferation via indirect interaction with ERK (A) Co-immunoprecipitation (CoIP) analysis of SPRED1-ERK interaction in NHEK cells transfected with pLVX-Flag-spred1. (B) qRT-PCR analysis of ERK mRNA expression in five experimental groups treated with ERK agonist Ro67-7476 or inhibitors PD98059/SCH772984. Statistical significance was assessed by one-way analysis of variance (ANOVA). (C and D) Western blot analysis of p -ERK protein expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (E and F) Immunofluorescence staining of p -ERK. Statistical significance was assessed by Brown-Forsythe corrected ANOVA. Scale bars, 20 μm. (G and H) CCK-8 assays for keratinocyte viability in five groups. Statistical significance was assessed by one-way ANOVA followed by Bonferroni correction. (I and J) EdU staining for keratinocyte proliferation. Statistical significance was assessed by one-way analysis of variance (ANOVA). Scale bars, 200 μm. (K) qRT-PCR analysis of PCNA mRNA expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (L and M) Western blot analysis of PCNA protein expression. Statistical significance was assessed by one-way analysis of variance (ANOVA). (N and O) Immunofluorescence staining of PCNA. Statistical significance was assessed by Brown-Forsythe corrected ANOVA. Scale bars, 40 μm. All experiments were performed in triplicate. N = 6 for all. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: ERK inhibitor PD98059 , MCE , Cat No. HY-12028.

Techniques: Immunoprecipitation, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, CCK-8 Assay

Journal: The Journal of Experimental Medicine

Article Title: Interferon-λ drives renal fibrosis by coordinating epithelial–fibroblast crosstalk

doi: 10.1084/jem.20251858

Figure Lengend Snippet: Type I and II IFNs differ from type III IFN in their regulation of TGF-β expression and the ERK–JNK pathway in renal fibroblasts during kidney fibrosis. (A–C) Primary renal fibroblasts from WT mice were treated for 24 h with 100 ng/ml of various IFNs subtypes (IFN-α, IFN-β, IFN-γ, or IFN-λ2) or PBS. (A and B) Isg15 , Mx1 , Ifit1 , and Tgf-β mRNA levels were assessed by RT-qPCR ( n = 6), and (C) TGF-β protein in the culture supernatants was quantified by ELISA ( n = 4). (D–F) Primary skin fibroblasts from WT mice were treated for 24 h with 100 ng/ml of various IFNs subtypes (IFN-α, IFN-β, IFN-γ, or IFN-λ2) or PBS. (D and E) Isg15 , Mx1 , Ifit1 , and Tgf-β mRNA levels were detected by RT-qPCR ( n = 6), and (F) TGF-β protein in the culture supernatants was measured by ELISA ( n = 4). (G–J) WT, Ifnar –/– , and Ifngr1 −/− mice were subjected to sham or UUO surgery, and kidneys were collected on day 7. n = 6 per group. (G) Representative images and quantitative analysis of fibrotic areas with Masson’s trichrome and PSR staining (scale bars = 50 μm). (H) RT-qPCR analysis of Acta2 , fibronectin, and vimentin mRNA levels in kidneys. TGF-β mRNA and protein levels in kidneys were measured by RT-qPCR (I) and western blot (J). (K and L) Primary kidney fibroblasts were treated with 100 ng/ml IFN-α (K) or IFN-β (L) for the indicated times. (K and L) Western blot analysis of phosphorylated and total ERK and JNK protein levels. Data in A–I are pooled from two independent experiments. Data in J–L are representative of three independent experiments. Data are presented as mean ± SEM. *P < 0.05, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparison test (A–I). ns, no significant difference. Source data are available for this figure: .

Article Snippet: For pharmacological inhibition of ERK or JNK, WT mice undergoing UUO or sham surgery were treated with either ERK inhibitor SCH772984 (HY-50846; MedChemExpress) at 50 mg/kg in 100 μl PBS intraperitoneally or JNK inhibitor SP600125 (HY-12041; MedChemExpress) at 30 mg/kg in 100 μl PBS by gavage on days −1, 1, 3, and 5.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Interferon-λ drives renal fibrosis by coordinating epithelial–fibroblast crosstalk

doi: 10.1084/jem.20251858

Figure Lengend Snippet: IFN-λ induces TGF-β synthesis in renal fibroblasts through activation of the ERK and JNK signaling pathways. (A) Primary renal fibroblasts from WT mice were treated IFN-λ2 (100 ng/ml) for 0, 30, 60, and 120 min. Phosphorylated and total ERK, JNK, p38, PI3K, and mTOR were assessed by western blot. (B) Primary renal fibroblasts from WT and Ifnlr1 −/− mice were treated with IFN-λ2 (100 ng/ml) or PBS for 120 min p-ERK, ERK, p-JNK, and JNK were analyzed by western blot. (C and D) Primary renal fibroblasts were pretreated with ERK inhibitor SCH772984 (ERKi, 1 μM) for 1 h, then stimulated with IFN-λ2 (100 ng/ml) for 2 h (to assess ERK activation) or 24 h (to evaluate TGF-β expression) ( n = 6). (C) p-ERK and ERK were assessed by western blot; TGF-β expression were analyzed by western blot (C), RT-qPCR (D, left panel), and ELISA (D, right panel). (E and F) Primary renal fibroblasts were pretreated with JNK inhibitor SP600125 (JNKi, 10 μM) for 1 h, then stimulated with IFN-λ2 (100 ng/ml) for 2 h (to assess JNK activation) or 24 h (to evaluate TGF-β expression) ( n = 6). (E) p-JNK and JNK were assessed by western blot; TGF-β expression were analyzed by western blot (E), RT-qPCR (F, left panel), and ELISA (F, right panel). (G) Primary renal fibroblasts were treated with IFN-λ2 (100 ng/ml) for 24 h in the presence or absence of ERKi or JNKi. Acta2, fibronectin , and vimentin mRNA levels were measured by RT-qPCR ( n = 6). (H) WT mice underwent sham or UUO surgery for 7 days. Renal fibroblasts were isolated and analyzed for p-ERK, ERK, p-JNK, and JNK by western blot. (I and J) WT UUO mice were subcutaneously injected with 1 μg of IFN-λ2 on days −1, 1, 3, and 5, combined with intraperitoneal administration of ERKi (50 mg/kg) (I) or JNKi (50 mg/kg) (J). Kidneys were collected at day 7 after UUO. TGF-β protein and mRNA levels in renal fibroblasts were assessed by western blot and RT-qPCR ( n = 5). (K) Representative images and quantitative analysis of fibrotic area and p-SMAD2/3 MOD in kidneys using Masson’s trichrome, PSR, and IHC (scale bars = 50 μm) ( n = 5). (L) Renal Acta2, fibronectin , and vimentin mRNA levels were measured by RT-qPCR ( n = 5). Data are representative of three (A–C, E, and H–J) independent experiments. Data in D, F–G, and K–L are pooled from two independent experiments. Data are shown as mean ± SEM. ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparison test. MOD, mean OD. Source data are available for this figure: .

Article Snippet: For pharmacological inhibition of ERK or JNK, WT mice undergoing UUO or sham surgery were treated with either ERK inhibitor SCH772984 (HY-50846; MedChemExpress) at 50 mg/kg in 100 μl PBS intraperitoneally or JNK inhibitor SP600125 (HY-12041; MedChemExpress) at 30 mg/kg in 100 μl PBS by gavage on days −1, 1, 3, and 5.

Techniques: Activation Assay, Protein-Protein interactions, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Isolation, Injection, Comparison

Journal: PLOS Biology

Article Title: Angptl5 restricts primitive hematopoiesis by promoting retinoic acid signaling in zebrafish

doi: 10.1371/journal.pbio.3003858

Figure Lengend Snippet: (A) WISH of cyp26a1 in WT embryos. Embryos were treated with ERK activator (C16-PAF, ERKa) or ERK inhibitor (Mirdametinib, ERKi) from the shield stage, or injected with angptl5 + itga6l + itgb5 mRNA at the 1-cell stage. Untreated embryos were used as control. (B–D) Schematic diagram of experimental setup (B) for WISH of dhrs9 and aldh1a2 in WT embryos. caERK2 (C) or angptl5 + itga6l + itgb5 (D) mRNA injected into one blastomere on the animal pole at the 128-cell stage and then detected at 6 hpf. (E–G) Schematic diagram of experimental setup (E) for WISH of dhrs9 . WT embryos were first injected with angptl5 mRNA at the 1-cell stage. Subsequently, itga6l ± itgb5 mRNA was injected into one blastomere on the animal pole at the 128-cell stage. Embryos were then continuously treated with or without ERK inhibitor until the shield stage (G) . Uninjected embryos and only angptl5 mRNA injected embryos (F) were used as control. (H) WISH of dhrs9 in WT embryos. caERK2 mRNA ± junba/bb MO injected into one blastomere on the animal pole at the 128-cell stage and then detected at 6 hpf. (I) ChIP-qPCR analysis of Junb binding to upstream regions of dhrs9. Embryos injected with HA-Junba mRNA were subjected to chromatin immunoprecipitation with HA-agarose, using IgG-agarose as a control. Enrichment at three predicted binding sites upstream of the dhrs9 gene was quantified by qPCR. Data are presented as the mean ± SD from three independent biological replicates. *** P < 0.001 (Unpaired t t est). (J and K) WISH of spi1b in angptl5 Δ10/Δ10 embryos. Embryos were injected with itga6l + itgb5 mRNA at the 1-cell stage and treated with or without ERK inhibitor from the shield stage to the 18-somite stage (J), or injected with caERK 2 mRNA at the 1-cell stage and treated with or without RA receptor antagonist AGN 193109 (K). Uninjected embryos were used as control. Statistics are shown below. Data presented as the mean ± SD, n( angptl5 Δ10/Δ10 ) = 22, n( angptl5 Δ10/Δ10 + itga6l + itgb5 ) = 26, n( angptl5 Δ10/Δ10 + itga6l + itgb5 + ERKi) = 28 (J); n( angptl5 Δ10/Δ10 ) = 22, n( angptl5 Δ10/Δ10 + caERK ) = 22, and n( angptl5 Δ10/Δ10 + caERKi + AGN) = 26 (K). Statistical significance: ** P < 0.01, *** P < 0.001 (One-way ANOVA). LV, lateral view; AV, animal view (C–H), anterior view (J and K). The data for this figure can be found in .

Article Snippet: Detailed pharmacological parameters and experimental concentrations are tabulated below: RA (sigma), Aldehyde dehydrogenase inhibitors 4-diethylaminobenzaldehyde (DEAB) (MCE, 10 μM), RA receptor (RARs) antagonists AGN 193109 (MCE, 20 μM), FAK inhibitor Defactinib (MCE, 2 μM/8 μM), MAPK inhibitor Adezmapimod (MCE, 10 μM), ERK inhibitor Mirdametinib (MCE, 10 μM), ERK activator C16-PAF (MCE, 1 μM/5 μM), NF-κB inhibitor BAY 11-7082 (MCE, 0.1 μg/mL, 0.4 μg/mL).

Techniques: Injection, Control, ChIP-qPCR, Binding Assay, Chromatin Immunoprecipitation

Journal: PLOS Biology

Article Title: Angptl5 restricts primitive hematopoiesis by promoting retinoic acid signaling in zebrafish

doi: 10.1371/journal.pbio.3003858

Figure Lengend Snippet: (A) WISH of cyp26a1 in WT embryos. Embryos were treated with ERK activator (C16-PAF, ERKa) or ERK inhibitor (Mirdametinib, ERKi) from the shield stage, or injected with angptl5 + itga6l + itgb5 mRNA at the 1-cell stage. Untreated embryos were used as control. (B–D) Schematic diagram of experimental setup (B) for WISH of dhrs9 and aldh1a2 in WT embryos. caERK2 (C) or angptl5 + itga6l + itgb5 (D) mRNA injected into one blastomere on the animal pole at the 128-cell stage and then detected at 6 hpf. (E–G) Schematic diagram of experimental setup (E) for WISH of dhrs9 . WT embryos were first injected with angptl5 mRNA at the 1-cell stage. Subsequently, itga6l ± itgb5 mRNA was injected into one blastomere on the animal pole at the 128-cell stage. Embryos were then continuously treated with or without ERK inhibitor until the shield stage (G) . Uninjected embryos and only angptl5 mRNA injected embryos (F) were used as control. (H) WISH of dhrs9 in WT embryos. caERK2 mRNA ± junba/bb MO injected into one blastomere on the animal pole at the 128-cell stage and then detected at 6 hpf. (I) ChIP-qPCR analysis of Junb binding to upstream regions of dhrs9. Embryos injected with HA-Junba mRNA were subjected to chromatin immunoprecipitation with HA-agarose, using IgG-agarose as a control. Enrichment at three predicted binding sites upstream of the dhrs9 gene was quantified by qPCR. Data are presented as the mean ± SD from three independent biological replicates. *** P < 0.001 (Unpaired t t est). (J and K) WISH of spi1b in angptl5 Δ10/Δ10 embryos. Embryos were injected with itga6l + itgb5 mRNA at the 1-cell stage and treated with or without ERK inhibitor from the shield stage to the 18-somite stage (J), or injected with caERK 2 mRNA at the 1-cell stage and treated with or without RA receptor antagonist AGN 193109 (K). Uninjected embryos were used as control. Statistics are shown below. Data presented as the mean ± SD, n( angptl5 Δ10/Δ10 ) = 22, n( angptl5 Δ10/Δ10 + itga6l + itgb5 ) = 26, n( angptl5 Δ10/Δ10 + itga6l + itgb5 + ERKi) = 28 (J); n( angptl5 Δ10/Δ10 ) = 22, n( angptl5 Δ10/Δ10 + caERK ) = 22, and n( angptl5 Δ10/Δ10 + caERKi + AGN) = 26 (K). Statistical significance: ** P < 0.01, *** P < 0.001 (One-way ANOVA). LV, lateral view; AV, animal view (C–H), anterior view (J and K). The data for this figure can be found in .

Article Snippet: Detailed pharmacological parameters and experimental concentrations are tabulated below: RA (sigma), Aldehyde dehydrogenase inhibitors 4-diethylaminobenzaldehyde (DEAB) (MCE, 10 μM), RA receptor (RARs) antagonists AGN 193109 (MCE, 20 μM), FAK inhibitor Defactinib (MCE, 2 μM/8 μM), MAPK inhibitor Adezmapimod (MCE, 10 μM), ERK inhibitor Mirdametinib (MCE, 10 μM), ERK activator C16-PAF (MCE, 1 μM/5 μM), NF-κB inhibitor BAY 11-7082 (MCE, 0.1 μg/mL, 0.4 μg/mL).

Techniques: Injection, Control, ChIP-qPCR, Binding Assay, Chromatin Immunoprecipitation