Journal: The EMBO Journal

Article Title: The WNK-OXSR1 osmosensing pathway mediates intestinal regeneration via Hippo-YAP signaling

doi: 10.1038/s44318-026-00738-8

Figure Lengend Snippet: ( A ) Schematic representation of the DSS treatment regimen in mice. ( B ) OXSR1 protein levels in the crypts of the small intestine (SI) and colon from VilCre ERT2 ; Oxsr1 f/f mice, with or without Tamoxifen (TAM) treatment, were assessed via western blot. ( C ) VilCre ERT2 ; Oxsr1 f/f mice, with or without TAM treatment, were administered 2.5% DSS for 5 days, and their survival was subsequently monitored daily. Survival curves were analyzed using log-rank (Mantel–Cox) test. n = 7 mice. Note the increased mortality of Oxsr1 conditional knockout (cKO) mice after DSS treatment. ( D – G ) Colons from TAM-untreated or treated VilCre ERT2 ; Oxsr1 f/f mice were collected on day 4 of the regeneration phase following DSS treatment and subjected to further analyses. Data shown are representative results of the colon gross morphology ( D , left panel), quantification of colon length ( D , right panel), hematoxylin and eosin (H&E) staining of colon Swiss roll ( E ), immunohistochemical staining for Ki67 ( F ), and immunostaining for Lrig1 ( G ) in colonic crypts. For ( D ), data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d. ( n = 5 colons). Scale bars in ( E ): 500 μm (upper panel) and 100 μm (lower panel). Scale bar in ( F ): 100 μm. Scar bar in ( G ): 20 μm. ( H – J ) Phosphorylation levels of OXSR1 in the colonic crypts during the indicated stages of regeneration were assessed by immunohistochemistry ( H ), immunofluorescence ( I ), and immunoblotting ( J ). Note the elevated phosphorylation of OXSR1 at the early regeneration stage (Regen. d4), which returned to baseline levels at the late regeneration stage (Regen. d9). UI, uninjured. Scale bars in ( H , I ): 100 μm. ( K ) Dynamic changes in the Lrig1⁺ stem-cell population within colonic crypts at the indicated stages of injury and regeneration were assessed by immunostaining. Note that Lrig1 signals were reduced during injury and progressively reappeared during regeneration. Scale bar: 20 μm. ( L ) Representative immunohistochemistry images showing OXSR1 phosphorylation levels in colonic tissue from human IBD patients. Note the increased OXSR1 phosphorylation in the inflamed regions. Scale bar: 100 μm. The gel and microscopy images shown are representative of at least two independent experiments.

Article Snippet: To generate Oxsr1 conditional knockout mice, Villin-Cre ERT2 ; Oxsr1 fl/fl mice (8–12 weeks old) were intraperitoneal injected with Tamoxifen (120 mg/kg; dissolved in corn oil; MCE #HY-13757A) for 2 weeks.

Techniques: Western Blot, Knock-Out, Staining, Immunohistochemical staining, Immunostaining, Two Tailed Test, Phospho-proteomics, Immunohistochemistry, Immunofluorescence, Microscopy

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: FAM65A binds to Ras and activates the Ras/ERK signaling to mediate RSK activation (A) The volcano plot analysis results for the FAM65A high-expression and low-expression groups from the TCGA database were shown. (B) The KEGG and GO results were shown. (C) The GSEA results were shown. (D) GSEA on DEGs between the FAM65A high-expression group and low-expression group in the Reactome database were shown. (E) IP was performed to detect the binding of FAM65A and Ras/p-RSK. (F) IP was performed to detect the binding of Ras and FAM65A/p-RSK. (G) Immunofluorescence was performed to detect the co-localization of FAM65A and Ras. Scale bars, 20 μm. (H) Western blot analysis the Ras and p -ERK expression in FAM65A knockdown or overexpression cells. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Activation Assay, Expressing, Binding Assay, Immunofluorescence, Western Blot, Knockdown, Over Expression

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: Ras/ERK signaling activation was indispensable for FAM65A-mediated RSK activation and CRC progression (A) Western blot analysis of Ras and p -ERK expression in HCT116-FAM65A cells treated with 10 μM Abd-7, or without treatment. (B) Results from the CCK8 cell proliferation assay conducted on HCT116-FAM65A cells with and without the application of Abd-7, n = 3, ∗∗∗ p < 0.001. (C) Colony formation assay performed on HCT116-FAM65A cells treated with Abd-7 or not. (D) Quantitative analysis of the colony formation assay results, n = 3, ∗∗∗ p < 0.001. (E) Results from the EdU assay conducted on HCT116-FAM65A cells with and without the application of Abd-7. Scale bars, 100 μm. (F) Quantitative analysis of the EdU assay results, n = 3, ∗∗∗ p < 0.001. (G) Western blot analysis of Ki-67, cleaved Caspase 3, Bcl-2, and Bax expression in HCT116-FAM65A cells treated with Abd-7 or not. (H) Results from the apoptosis assay conducted on HCT116-FAM65A cells treated with Abd-7 or not. Scale bars, 50 μm. (I) Quantitative analysis of the apoptosis experiments, n = 3, ∗∗∗ p < 0.001. (J) Results from the Transwell migration assay conducted on HCT116-FAM65A cells with and without the application of Abd-7. Scale bars, 50 μm. (K) Quantitative analysis of the Transwell migration assay results, n = 3, ∗∗∗ p < 0.001. (L) Results from the wound healing assay performed on HCT116-FAM65A cells treated with Abd-7 or not. Scale bars, 50 μm. (M) Quantitative analysis of the wound healing assay results, n = 3, ∗∗∗ p < 0.001. (N) Western blot analysis the expression of EMT markers in HCT116-FAM65A cells treated with Abd-7 or not. (O) Proposed model of FAM65A in CRC progression. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Activation Assay, Western Blot, Expressing, Proliferation Assay, Colony Assay, EdU Assay, Apoptosis Assay, Transwell Migration Assay, Wound Healing Assay

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: Knockdown of FAM65A inhibits tumor progression in vivo (A) LOVO-shCtrl and LOVO-shFAM65A cells were administered into the fourth fat pad of nude mice, and the resulting tumor growth curves were subsequently generated, n = 5, ∗ p < 0.05. (B) The tumors excised from mice across various experimental groups are presented. (C) Hematoxylin and Eosin (HE) staining results of lung tissue from the different groups is displayed. (D) A quantitative analysis of metastatic lung nodules is provided, n = 5, ∗∗ p < 0.01. (E) IHC results for FAM65A, Ki-67, p -RSK, p -ERK, Ras, N-cadherin, vimentin, cleaved Caspase 3, ZO-1, and E-cadherin in tumor tissues are illustrated. (F) A quantitative analysis of the IHC results is included. Data are presented as mean ± SEM of biologically independent experiments, n = 5, ∗∗∗ p < 0.001.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Knockdown, In Vivo, Generated, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: AIF-1 Drives Corneal Neovascularization by Promoting Inflammatory Macrophage Activation via the MAPK and PI3K/AKT/mTOR Signaling Pathways

doi: 10.1167/iovs.67.2.22

Figure Lengend Snippet: AIF-1 siRNA inhibited the phosphorylation of P38, ERK1/2, JNK, PI3K, AKT, and mTOR proteins in corneal tissues following alkali burn. ( A ) WB analysis of p-P38, p-ERK1/2, and p-JNK expression levels in CNV corneas. ( B – D ) Quantitative analysis of the WB bands ( n = 3). ( E ) The protein expression level of p-PI3K, p-AKT, and p-mTOR in CNV corneas. ( F-H ) Quantitative analysis of the WB bands ( n = 3). β-Actin served as an internal control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are presented as the mean ± SEM.

Article Snippet: The PVDF membrane was incubated overnight at 4°C with primary antibodies, including AIF-1 (1:1000; Abcam, Cambridge, UK), VEGFA (1:1000; CST, Danvers, MA, USA), CD86 (1:1000; Abcam), TNF-α (1:1000; ABclonal, Wuhan, China), IL-1β (1:2000; ABclonal), IL-6 (1:1000; CST), CD31 (1:1000; R&D Systems, Minneapolis, MN, USA), proliferating cell nuclear antigen (PCNA, 1:1000; ABclonal), p-ERK1/2 (1:1500; Proteintech, Wuhan, China), ERK1/2 (1:1500; Proteintech), P38 (1:1000; CST), p-P38 (1:1000; CST), p-JNK (1:1000; CST), JNK (1:1000; CST), p-PI3K (1:500; Affinity Biosciences, Nanjing, China), PI3K (1:1000; Proteintech), p-AKT (1:3000; Proteintech), AKT (1:1000; Proteintech), p-mTOR (1:5000; Proteintech), mTOR (1:1000; Proteintech), and β-actin (1:2000; Elabscience, Wuhan, China).

Techniques: Phospho-proteomics, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Platelet-rich plasma-derived microRNA let-7a-5p alleviates knee osteoarthritis by regulating macrophage polarization and improving inflammatory microenvironment

doi: 10.3389/fimmu.2026.1756467

Figure Lengend Snippet: Validation of the targeting relationship between let-7a-5p and MAPK8. (A) Schematic representation of the predicted complementary binding site between let-7a-5p and the 3′-UTR of MAPK8. (B) Relative mRNA expression level of MAPK8 in macrophages overexpressing let-7a-5p detected by RT-qPCR. (C) WB analysis of MAPK8 protein expression in macrophages overexpressing let-7a-5p. (D) Inhibitory effect of let-7a-5p on MAPK8 expression was assessed via a dual luciferase reporter assay. ( **P < 0.01, ***P < 0.001).

Article Snippet: Subsequently, the tissue sections and cells were incubated overnight at 4°C with primary antibodies against iNOS (Proteintech 22226-1-AP), CD206 (Proteintech 18704-1-AP) and MAPK8 (Proteintech 66210-1-Ig).

Techniques: Biomarker Discovery, Binding Assay, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay

Journal: Frontiers in Immunology

Article Title: Platelet-rich plasma-derived microRNA let-7a-5p alleviates knee osteoarthritis by regulating macrophage polarization and improving inflammatory microenvironment

doi: 10.3389/fimmu.2026.1756467

Figure Lengend Snippet: Effect of PRP on expression of let-7a-5p and MAPK8. (A) Relative mRNA expression levels of let-7a-5p in knee joint sections of each group detected by RT-qPCR. (B) iNOS and CD206 co-immunolabeld with MAPK8 and counter-stained with DAPI in synovial tissues (Scale bar: 50 μm). (C, D) Quantification of iNOS and CD206 expression co-localized with MAPK8. ( **P < 0.01).

Article Snippet: Subsequently, the tissue sections and cells were incubated overnight at 4°C with primary antibodies against iNOS (Proteintech 22226-1-AP), CD206 (Proteintech 18704-1-AP) and MAPK8 (Proteintech 66210-1-Ig).

Techniques: Expressing, Quantitative RT-PCR, Staining

Journal: Frontiers in Immunology

Article Title: Platelet-rich plasma-derived microRNA let-7a-5p alleviates knee osteoarthritis by regulating macrophage polarization and improving inflammatory microenvironment

doi: 10.3389/fimmu.2026.1756467

Figure Lengend Snippet: The let-7a-5p/MAPK8 axis regulates macrophage polarization and inflammatory cytokine release in vitro . (A) IF staining showing expression of iNOS and CD206 in macrophages (Scale bar: 50 μm). (B) Quantification of iNOS-positive cell rate in transfected macrophages. (C) Quantification of CD206-positive cell rate in transfected macrophages. (D-G) Relative mRNA expression levels of pro-inflammatory cytokine (IL-1β and TNF-α) and anti-inflammatory cytokine (IL-4 and IL-10) in transfected macrophages detected by RT-qPCR. ( *P < 0.05, **P < 0.01, ns no significance).

Article Snippet: Subsequently, the tissue sections and cells were incubated overnight at 4°C with primary antibodies against iNOS (Proteintech 22226-1-AP), CD206 (Proteintech 18704-1-AP) and MAPK8 (Proteintech 66210-1-Ig).

Techniques: In Vitro, Staining, Expressing, Transfection, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Constitutively Active Homo-oligomeric Angiotensin II Type 2 Receptor Induces Cell Signaling Independent of Receptor Conformation and Ligand Stimulation

doi: 10.1074/jbc.m500639200

Figure Lengend Snippet: FIG. 1. a, AT2 receptors form homo-oligomerization and up-regulate after serum-free conditions in PC12W cells. Cell membrane was prepared under serum or serum-free conditions, subjected to SDS-gel electrophoresis under non-reducing conditions, and immunoblotted with anti-AT2 receptor antibody. 30 g of protein was used in each lane. Bmax on the AT2 receptors was also analyzed by Scatchard plot analysis. b, AT2-WT-EGFP receptor localized in the cell membrane after 24 h under serum-free conditions. EGFP- and AT2-WT-EGFP receptor-expressing CHO cell lines were grown under serum or serum-free conditions and stained with DAPI to visualize nuclear morphology as assessed by a laser scanning confocal microscope. Arrows indicate AT2-WT-EGFP receptor translocation in the cell membrane. c, AT2-WT-EGFP receptor induced apoptosis after 48 h under serum-free conditions. AT2-WT-EGFP receptor- and AT2-N127G-EGFP receptor-expressing CHO cell lines were grown under serum conditions or for up to 48 h under serum-free conditions, stained with DAPI, and imaged by a digital fluorescent microscope. d, pharmacological intervention in apoptosis mediated by AT2-WT-EGFP receptor and AT2-N127G-EGFP receptor was analyzed by treating cells under serum conditions and with or without 10 M p38 MAPK inhibitor (SB203580) and 1 M caspase-3 inhibitor (DEVD-cmk) for 48 h under serum-free conditions. Data are shown as the percentage of apoptotic cells in three independent experiments as assessed by TUNEL as described under “Experimental Procedures.” Histograms show that AT2-WT-EGFP receptor transfected CHO cells induced apoptosis under serum (A) or after 48 h serum-free conditions (B). 65% of AT2-WT-EGFP receptor transfected CHO cells showed apoptosis after 48 h of serum-free conditions. *, p 0.05 versus serum conditions. t, p 0.05 versus serum-free conditions without treatment. Data are given as mean S.E. (n 4). e, the AT2 receptor formed a homo-oligomer in the cell membrane. Cell membranes were prepared in serum-free conditions for 24 h, subjected to SDS-gel electro- phoresis after being pretreated with () or without ( ) DTT under non-reducing conditions, and immunoblotted with anti-EGFP antibody. 20 mg of protein was used in each lane. Arrows indicate the monomer and dimer species of the receptor. f, AT2-WT-EGFP receptor formed only homo-oligomer, whereas AT2-N127G-EGFP receptor formed both homo-oligomer and monomer in the cell membrane. Cell membranes were prepared in serum () or serum-free ( ) conditions for up to 48 h, subjected to SDS-gel electrophoresis under non-reducing conditions, and immunoblotted with anti-EGFP antibody. 40 g of protein was used in each lane. Arrows indicate the monomer and dimer species of the receptor. Representative immunoblots (a, d, and f) and pictures (b and c) are shown. Three independent determinations were performed, and similar results were observed (a–d and f).

Article Snippet: Materials—The following antibodies and reagents were generously provided as indicated or purchased: AT2 receptor-selective non-peptide antagonist PD123319 (Research Biochemical International) and the p38 MAP kinase inhibitor SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)1H-imidazole (Upstate Biotechnology); the caspase-3 inhibitor Ac-DEVD-chloro methyl ketone (Calbiochem); antiAT2 receptor antibody catalog number sc-7420 (Santa Cruz Biotechnology).

Techniques: Membrane, SDS-Gel, Electrophoresis, Expressing, Staining, Microscopy, Translocation Assay, TUNEL Assay, Transfection, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing Ras-Related C3 Botulinum Toxin Substrate 1 Inhibits Growth and Migration of Hypopharyngeal Squamous Cell Carcinoma via the P38 Mitogen-Activated Protein Kinase Signaling Pathway

doi: 10.12659/MSM.907468

Figure Lengend Snippet: Silencing Rac1 inhibits the activation of P38 MAPK signal in vitro . The levels of MKK3 and p-MKK3 ( A, B ) and P38 and p-P38 ( C, D ) were detected by Western blot analysis. GAPDH served as the internal reference. All experiments were repeated 3 times and the results are presented as mean ±SD. *** P<0.001 compared with the negative control group.

Article Snippet: The membranes were blocked with 5% skim milk or 1% bovine serum albumin, and then incubated with Rac1 antibody, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1: 1000; Proteintech, Wuhan, China), Cyclin D1 antibody (1: 1000; BOSTER, Wuhan, China), Cyclin E antibody, Cyclin B antibody (1: 500; Bioss, Beijing, China), metal matrix proteinase (MMP)-2 antibody, MMP-9 antibody, B cell lymphoma-2 (Bcl-2) antibody, Bcl-2-associated X protein (Bax) antibody, phosphorylated-MAP kinase (p-MKK3) antibody (1: 500; Sangon Biotech, Shanghai, China), caspase-3 antibody, caspase-9 antibody, poly ADP-ribose polymerase (PARP) antibody (1: 1000; Cell Signaling Technology, Beverly, MA, USA), p38 mitogen-activated protein kinase (MAPK) antibody, p-p38 MAPK antibody (1: 500; KeyGen, Nanjing, China), or MKK3 antibody (1: 300; BOSTER) overnight at 4°C.

Techniques: Activation Assay, In Vitro, Western Blot, Negative Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing Ras-Related C3 Botulinum Toxin Substrate 1 Inhibits Growth and Migration of Hypopharyngeal Squamous Cell Carcinoma via the P38 Mitogen-Activated Protein Kinase Signaling Pathway

doi: 10.12659/MSM.907468

Figure Lengend Snippet: Rac1 silencing inhibits the activation of P38 MAPK signal in vivo . The levels of MKK3 and p-MKK3 ( A, B ) and P38 and p-P38 ( C, D ) in each group were detected by Western blot analysis with GAPDH as the internal reference. All experiments were repeated 3 times. The results are presented as mean ±SD. N=6. *** P<0.001 compared with the negative control group.

Article Snippet: The membranes were blocked with 5% skim milk or 1% bovine serum albumin, and then incubated with Rac1 antibody, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1: 1000; Proteintech, Wuhan, China), Cyclin D1 antibody (1: 1000; BOSTER, Wuhan, China), Cyclin E antibody, Cyclin B antibody (1: 500; Bioss, Beijing, China), metal matrix proteinase (MMP)-2 antibody, MMP-9 antibody, B cell lymphoma-2 (Bcl-2) antibody, Bcl-2-associated X protein (Bax) antibody, phosphorylated-MAP kinase (p-MKK3) antibody (1: 500; Sangon Biotech, Shanghai, China), caspase-3 antibody, caspase-9 antibody, poly ADP-ribose polymerase (PARP) antibody (1: 1000; Cell Signaling Technology, Beverly, MA, USA), p38 mitogen-activated protein kinase (MAPK) antibody, p-p38 MAPK antibody (1: 500; KeyGen, Nanjing, China), or MKK3 antibody (1: 300; BOSTER) overnight at 4°C.

Techniques: Activation Assay, In Vivo, Western Blot, Negative Control