Journal: The FASEB Journal

Article Title: Distinct cAMP Regulation in Scleroderma Lung and Skin Myofibroblasts Governs Their Dedifferentiation via p38α Inhibition

doi: 10.1096/fj.202500694RR

Figure Lengend Snippet: cAMP‐mediated p38 inhibition promotes dedifferentiation of lung and skin MFs. (A, B) Western blot and densitometric analysis of the phosphorylated proteins p‐p38, p‐ERK, and p‐JNK following SSc lung (A) and skin (B) MF treatment with forskolin (20 μM) for 6 h. Phosphorylated proteins were normalized to total levels of their respective proteins. (C) Western blot and densitometric analysis of the fibrosis‐associated genes Col1A1 and αSMA following SSc lung and skin MF treatment with SB203580 (20 μM) for 96 h. (D) αSMA stress fibers were identified by immunofluorescence microscopy using an anti‐αSMA‐FITC‐conjugated antibody (using the same protocol in C). Nuclei were stained with DAPI. Data points represent distinct patient‐derived cell lines. Significance for densitometric data ( n = 5–7) in (A and B) was determined by a 2‐tailed paired t‐test and by one‐way ANOVA in (C). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The pan‐p38 inhibitor SB203580 (20 μM) and p38α inhibitor VX‐702 (50 μM) were purchased from Cayman Chemicals (13067) and Selleckchem (HY‐10401), respectively.

Techniques: Inhibition, Western Blot, Immunofluorescence, Microscopy, Staining, Derivative Assay

Journal: The FASEB Journal

Article Title: Distinct cAMP Regulation in Scleroderma Lung and Skin Myofibroblasts Governs Their Dedifferentiation via p38α Inhibition

doi: 10.1096/fj.202500694RR

Figure Lengend Snippet: p38α inhibition promotes SSc lung and skin MF dedifferentiation. (A) qPCR data representing the relative expression of the genes encoding p38α, p38β, p38γ and p38δ in SSc lung and skin MFs. (B) Western blot and densitometric analysis of the fibrosis‐associated genes Col1A1 and αSMA following SSc lung and skin MF treatment with the isoform‐specific p38α inhibitor VX‐702 (50 μM) for 96 h. (C) αSMA stress fibers were identified by immunofluorescence microscopy using an anti‐αSMA‐FITC‐conjugated antibody (using the same protocol in B). Nuclei were stained with DAPI. Data points represent distinct patient‐derived cell lines. Significance for data in (A) ( n = 8) was determined by two‐tailed unpaired or paired t ‐test where appropriate and by one‐way ANOVA in (B) ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The pan‐p38 inhibitor SB203580 (20 μM) and p38α inhibitor VX‐702 (50 μM) were purchased from Cayman Chemicals (13067) and Selleckchem (HY‐10401), respectively.

Techniques: Inhibition, Expressing, Western Blot, Immunofluorescence, Microscopy, Staining, Derivative Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Mitochondrial damage and IL-1β production in monocytes caused by Neospora caninum infection are mediated by dense granule protein 7 and prohibitins

doi: 10.3389/fimmu.2025.1408992

Figure Lengend Snippet: IL-1β and TNF-α production in THP-1 cells and inflammasome-reconstructed 293T cells. (A) THP-1 cells were infected with the parental strain Nc1 or the NcGRA6-, NcGRA7-, or NcGRA14-deficient (KO) parasites at a multiplicity of infection (MOI) of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. (B) 293T cells were transfected with inflammasome-reconstruction plasmids encoding NLRP3, ASC, procaspase-1, and pro-IL-1β, together with NcGRA7 cDNA or an empty vector. Untransfected 293T cells were used as a negative control (no plasmid). At 20 h posttransfection, the culture supernatants were collected for analysis. (C–H) THP-1 cells were pretreated with 10 μM MCC950 (an NLARP3 inhibitor), 100 μM VX765 (a CASP1 inhibitor), and 18 μM SN50 (an NF-κB inhibitor) for 2 hr and then infected with the Nc1 strain of N. caninum at a MOI of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. Each value represents the mean ± SD of 4 replicates (technical replicates) in one representative experiment. Each experiment (biological replicate) was repeated two (G, H) , three (A, D, E, F) and four times (B, C) . Statistically significant differences according to one-way ANOVA or two-way ANOVA and a Tukey–Kramer post hoc analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MCC950 (an inhibitor of NLRP3 inflammasome activation by the inhibition of IL-1β release; Cayman Chemical), VX765 (an inhibitor of NLRP3 inflammasome activation by the inhibition of caspase 1 and IL-1β cleavage and release; LKT Laboratories), SN50 (an inhibitor of the nuclear transcription factor κB; Selleck, Houston, TX, USA), a carbonyl cyanide m-chlorophenyl hydrazone (CCCP, a uncoupling agent for oxidative phosphorylation that inhibits mitochondrial function; FUJFILM Wako Pure Chemical Corporation), rocaglamid A (Cayman, Ann Arbor, MI, USA) with biochemical research grades were used in this study.

Techniques: Infection, Transfection, Plasmid Preparation, Negative Control

Journal: Cell Death & Disease

Article Title: EIF4B Ser93 phosphorylation by ERK2 promotes epithelial-mesenchymal transition to drive colorectal cancer metastasis

doi: 10.1038/s41419-025-08375-5

Figure Lengend Snippet: A The top six kinase which might regulate eIF4B Ser93 phosphorylation predicted by the “Kinase Prediction” website ( https://www.phosphosite.org/kinaseLibraryAction ) were shown. B The conserved amino acid sequence surrounding eIF4B S93 matches the consensus MAPK phosphorylation motif. C ERK2 was predicted as the kinase that regulated eIF4B Ser93 phosphorylation. D PPI network diagram of ERK2 (MAPK1) and eIF4B. E HT29 cell lysates were prepared by RIPA lysis and co-immunoprecipitated by ERK2 or eIF4B antibody with IgG as negative control. The immunoprecipitated protein was detected by western blotting. F GST-pull down assay showed the interaction between His-ERK2 and GST-eIF4B (T53-E145) protein in vitro. In vitro kinase assay and western blotting analysis of p-eIF4B (T53-E145) Ser93 . The following assays were conducted in eIF4B WT and eIF4B S93D CRC cells following Vx-11e treatment (1 μM) or the control (DMSO) for 24 h. G The phosphorylation of eIF4B Ser93, eIF4B and EMT markers (Snai1, E-cad) protein were detected by western blotting. H The levels of mesenchymal makers mRNA were measured by RT‒qPCR. I RT-qPCR analysis of the mRNA abundance of mesenchymal markers in polyribosome fractions. All the data are presented as mean ± SEM from at least three independent experiments. P values were determined by two-sided Student t test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: ERK2 inhibitor Vx-11e (Selleck, S7709) were used in a final concentration as 1 μM in specific assays.

Techniques: Phospho-proteomics, Sequencing, Lysis, Immunoprecipitation, Negative Control, Western Blot, Pull Down Assay, In Vitro, Kinase Assay, Control, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: EIF4B Ser93 phosphorylation by ERK2 promotes epithelial-mesenchymal transition to drive colorectal cancer metastasis

doi: 10.1038/s41419-025-08375-5

Figure Lengend Snippet: The following assays were performed in eIF4B WT and eIF4B S93D CRC cells treated with Vx-11e (1 μM) or the control (DMSO). A CCK-8 assays were conducted to evaluate cells viabilities. B , C Colony formation assays were used to assess the cells proliferation capacities. Representative images and quantification of colonies were presented. D , E The cell migratory abilities were evaluated by wound‑healing assays. Representative images and quantification of wound closure are presented. Scale bar, 200 μm. F , G Transwell migration and invasion assays were conducted to measure cell migratory and invasive abilities. The number of migrated cells and invaded cells were presented as normalized values. Representative images were presented with scale bar as 100 μm. All the data are presented as mean±SEM from at least three independent experiments. H Schematic diagram of the xenograft tumor model following Vx-11e (50 mg/kg, twice a day) or the control (drug solvent without Vx-11e) treatment for 14 days established in nude mice with eIF4B WT or eIF4B S93D cells. I , J At day 14 after treatments, all mice were sacrificed, and tumors were dissected and photographed. Tumor weights and volumes were recorded and analyzed. K Immunohistochemistry was used to visualize and compare the level of p-eIF4B Ser93 , and the protein levels of eIF4B, Ki67, Snai1, Vim and N-cad in tumors. Scale bar: 100 μm. All the data are presented as mean±SEM ( n = 5). P values were determined by two-sided Student t test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: ERK2 inhibitor Vx-11e (Selleck, S7709) were used in a final concentration as 1 μM in specific assays.

Techniques: Control, CCK-8 Assay, Migration, Solvent, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: EIF4B Ser93 phosphorylation by ERK2 promotes epithelial-mesenchymal transition to drive colorectal cancer metastasis

doi: 10.1038/s41419-025-08375-5

Figure Lengend Snippet: EIF4B Ser93 phosphorylation by ERK2 promotes mesenchymal markers translation to drive CRC metastasis, while inhibition of ERK2 by Vx-11e suppresses eIF4B phosphorylation at Ser93 site and facilitates eIF4B degradation by ubiquitination, and thus inhibiting EMT and tumor metastasis (Created with BioRender. com).

Article Snippet: ERK2 inhibitor Vx-11e (Selleck, S7709) were used in a final concentration as 1 μM in specific assays.

Techniques: Phospho-proteomics, Inhibition, Ubiquitin Proteomics