Journal: Nature Communications

Article Title: Targeting leucine-rich repeat kinase 2 overcomes resistance to oncolytic herpes simplex virus-based therapies in glioblastoma

doi: 10.1038/s41467-026-70132-9

Figure Lengend Snippet: a A scatter plot showing differential gene expression in oHSV-GFP-infected U-251 MG cells treated with or without LRRK2-IN-1 (LRRK2i). U-251 MG cells were pretreated with LRRK2i (2.5 µM) or left untreated for 4 h, followed by infection with oHSV-GFP (MOI = 0.25 PFU/cell) for 4 h. RNA-Seq was used to assess differentially expressed genes among the treated cells. Blue dots represent genes significantly downregulated by LRRK2i during infection, and red dots represent genes significantly upregulated by LRRK2i during infection. b A heatmap showing the expression patterns of differentially expressed genes belonging to the “response to virus” category. The normalized expression values (Log 2 (TPM + 1)) in the heatmap depict the mean across three biological replicates. c GO enrichment analysis showing the top eight most enriched pathways downregulated by LRRK2i in oHSV-GFP-infected U-251 MG cells. d U-251 MG and U-87 MG cell lines were treated with LRRK2-IN-1 (2.5 μM) for 4 h, followed by IFNα and anti-IFNR1 (α-IFNR1) for an additional 4 h, and then infected with oHSV-GFP (MOI = 0.25 PFU/cell). Cell viability was determined 48 h post-infection. e Western blot analysis of p-STAT1, STAT1, and β-actin in GBM cell lines infected with oHSV-GFP or mock-infected, either with or without LRRK2-IN-1 (2.5 µM), 48 h post-infection. IFNα served as a positive control. The samples derive from the same experiment but different gels for p-STAT1, another for STAT1 and another for actin were processed in parallel. f p-STAT1 and STAT1 expression levels were quantified by densitometric analysis of bands from three independent Western blot experiments corresponding to Fig. 4e. g RT‒qPCR analysis of the expression of the indicated IFN-stimulated gene transcript expression in GBM cell lines infected with oHSV-GFP or mock-infected, either with or without LRRK2-IN-1 (2.5 µM), 12 h post-infection. h Western blot analysis of the indicated IFN-stimulated proteins (IFI16 and IRF9) in GBM cell lines infected with oHSV-GFP or mock-infected, either with or without LRRK2-IN-1 (2.5 µM), 48 h post-infection. IFI16 and IRF9 expression levels were quantified by densitometric analysis of bands from three independent Western blot experiments. The samples derive from the same experiment but different gels for IFI16, another for IRF9 and another for actin were processed in parallel. The data are presented as the mean ± SD from two independent experiments ( d ) or three independent experiments ( d , f – h ), and P values were calculated using an ordinary one-way ANOVA ( d , f – h ).

Article Snippet: Recombinant human STAT1 protein (ABclonal, RP01251) was incubated with active, baculovirus-expressed LRRK2 protein (Thermo Fisher Scientific, A15197 ) in 1 × kinase buffer (Cell Signaling Technologies, 9802S) containing 200 μM ATP (Cell Signaling Technologies, 9804S) for 30 min at 30 °C.

Techniques: Gene Expression, Infection, RNA Sequencing, Expressing, Virus, Western Blot, Positive Control

Journal: Nature Communications

Article Title: Targeting leucine-rich repeat kinase 2 overcomes resistance to oncolytic herpes simplex virus-based therapies in glioblastoma

doi: 10.1038/s41467-026-70132-9

Figure Lengend Snippet: a Anti-myc immunoprecipitates prepared from 293 T cells transfected with the indicated plasmids were subjected to SDS‒PAGE and blotted with the indicated antibodies. The samples derive from the same experiment but different gels for Myc and another for STAT1 were processed in parallel. b Endogenous LRRK2 and STAT1 complexes in U-118 MG cells were isolated by co-immunoprecipitation (co-IP) and detected using anti-LRRK2 antibodies. The samples derive from the same experiment but different gels for LRRK2 and another for STAT1 were processed in parallel. c The interactions between STAT1-Flag and Myc-LRRK2 mutants were detected by a co-IP assay. FL, full-length. The samples derive from the same experiment but different gels for Myc and another for Flag were processed in parallel. d , e The interactions between Myc-LRRK2 and STAT1-HA truncations were detected by a co-IP assay. The samples derive from the same experiment but different gels for Myc and another for HA were processed in parallel. f STAT1-His proteins were incubated with human FL LRRK2 proteins in the presence of ATP. The reaction products were analyzed with the indicated antibodies. CD300A proteins served as a negative control. The samples derive from the same experiment but different gels for Flag and another for His were processed in parallel. g , h 293 T cells were cotransfected with Flag-STAT1 and LRRK2 in the presence or absence of LRRK2-IN-1, and the phosphorylation of STAT1 was analyzed by western blot analysis. The samples derive from the same experiment but different gels for STAT1, another for p-STAT1 701 and another for p-STAT1 727 were processed in parallel. i 293 T cells were cotransfected with Flag-STAT1 and each LRRK2 mutant, either in the presence or absence of LRRK2-IN-1, and STAT1 phosphorylation was assessed via western blot analysis. The samples derive from the same experiment but different gels for STAT1, another for p-STAT1 701 and another for GAPDH were processed in parallel. j Western blotting was performed to assess and quantify phosphorylated STAT1 and HSV-derived GFP in U-87 MG cells treated with fludarabine (10 µM) for 4 h, either in the presence or absence of LRRK2-IN-1, followed by infection with oHSV-GFP (MOI = 1 PFU/cell). The samples derive from the same experiment but different gels for STAT1 and GFP, another for p-STAT1 701 and another for actin were processed in parallel. k Western blotting was performed to assess and quantify phosphorylated STAT1 and HSV-derived GFP in GBM-1 cells. Cells transfected with EV, LRRK2 or LRRK2 D1994A were infected with oHSV-GFP (MOI = 0.5 PFU/cell), with or without LRRK2-IN-1. The samples derive from the same experiment but different gels for STAT1 and GFP, another for p-STAT1 701 and another for actin were processed in parallel. l Western blotting was performed to assess and quantify phosphorylated TBK1, phosphorylated LRRK2, phosphorylated Rab10, and phosphorylated STAT1 in BX795 (10 µM)-treated U-87 cells following infection with oHSV-GFP (MOI = 0.5 PFU/cell). The samples derive from the same experiment but different gels for p-TBK1, another for p-LRRK2, another for LRRK2, another for p-Rab10 and p-STAT1, another for STAT1 and another for actin were processed in parallel. Western blot analyses were independently repeated twice for panels ( a – d and f )and three times for panels ( e , g and i – l ), with similar results. The data are presented as the mean ± SD from three independent experiments ( h – l ), and P values were calculated using an ordinary one-way ANOVA ( h – l ).

Article Snippet: Recombinant human STAT1 protein (ABclonal, RP01251) was incubated with active, baculovirus-expressed LRRK2 protein (Thermo Fisher Scientific, A15197 ) in 1 × kinase buffer (Cell Signaling Technologies, 9802S) containing 200 μM ATP (Cell Signaling Technologies, 9804S) for 30 min at 30 °C.

Techniques: Transfection, Isolation, Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, Negative Control, Phospho-proteomics, Western Blot, Mutagenesis, Derivative Assay, Infection

Journal: Investigative Ophthalmology & Visual Science

Article Title: Ly93 Inhibits Sphingomyelin Synthesis and Attenuates Inflammation and Injury in Dry Eye Conjunctival Organoids

doi: 10.1167/iovs.67.2.58

Figure Lengend Snippet: Hyperosmolarity induced DED-associated injury and inflammation of conjunctival organoids. ( A ) Representative images showing hyperosmolarity-induced organoid disintegration ( scale bar , 100 µm). ( B ) Quantification of the percentage of intact organoids. ( C ) Western blotting analysis of proliferation markers (p63 and Ki67), inflammatory mediators (MMP-9 and IL-1β), signaling molecules (NF-κB p65, STAT1, and PI3K), ferroptosis markers (TFR and GPX4), and apoptosis marker (caspase-3). ( D – F ) Immunofluorescence staining of p63 + and Ki67 + cells in organoids (scale bar , 50 µm). ( G ) Immunofluorescence staining of caspase-9, NF-κB p65, STAT1, and IL-1β in organoids ( scale bar , 50 µm). * P < 0.05; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Immunofluorescence staining was performed to evaluate the expression of K19 (1:200, Cell Signaling Technology, Danvers, MA, USA), K13 (1:200, Cell Signaling Technology), IL-1β (1:200, Abcam, Cambridge, UK), STAT1 (1:200, Proteintech, Rosemont, IL, USA), p63 (1:250, Abcam), Ki67 (1:200, Abcam), NF-κB p65 (1:200, Abcam), MUC1 (1:200, Abcam), MUC5AC(1:250, Abcam), caspase-9 (1:200, Cell Signaling Technology), ZO-1 (1:200, Abcam), and 4-hydroxynonenal (4-HNE) (1:100, Invitrogen, Carlsbad, CA, USA) in frozen sections of the conjunctival organoids.

Techniques: Western Blot, Marker, Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Ly93 Inhibits Sphingomyelin Synthesis and Attenuates Inflammation and Injury in Dry Eye Conjunctival Organoids

doi: 10.1167/iovs.67.2.58

Figure Lengend Snippet: Exogenous SM induced inflammation and injury of conjunctival organoids. ( A ) Effects of SM treatment on the viability of conjunctival cells and morphology of organoids ( scale bar , 100 µm for cells and 200 µm for organoids). ( B , C ) Western blotting analysis of inflammatory markers (phospho-STAT1, STAT1, IL-1β, and iNOS) in SM-treated organoids. ( D – F ) Relative mRNA expression of IL-1β, IL-6, and STAT1 after SM treatment. ( G ) Immunofluorescence staining of K13, K19, Ki67, MUC1, and MUC5AC in SM-treated organoids ( scale bar , 50 µm). ( H – L ) Comparative analysis of immunofluorescent results: K13 ( H ), K19 ( I ), Ki67 ( J ), MUC1 ( K ), and MUC5AC ( L ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Immunofluorescence staining was performed to evaluate the expression of K19 (1:200, Cell Signaling Technology, Danvers, MA, USA), K13 (1:200, Cell Signaling Technology), IL-1β (1:200, Abcam, Cambridge, UK), STAT1 (1:200, Proteintech, Rosemont, IL, USA), p63 (1:250, Abcam), Ki67 (1:200, Abcam), NF-κB p65 (1:200, Abcam), MUC1 (1:200, Abcam), MUC5AC(1:250, Abcam), caspase-9 (1:200, Cell Signaling Technology), ZO-1 (1:200, Abcam), and 4-hydroxynonenal (4-HNE) (1:100, Invitrogen, Carlsbad, CA, USA) in frozen sections of the conjunctival organoids.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Ly93 Inhibits Sphingomyelin Synthesis and Attenuates Inflammation and Injury in Dry Eye Conjunctival Organoids

doi: 10.1167/iovs.67.2.58

Figure Lengend Snippet: Ly93 attenuated DED-associated inflammation in conjunctival organoids. ( A ) Immunofluorescence staining of caspase-9, ZO-1, IL-1β, STAT1, and NF-κB p65 between vehicle control, DED, and DED + Ly93 groups ( scale bar , 50 µm). ( B ) Intracellular Fe 2+ analysis ( scale bar , 50 µm). ( C ) Immunofluorescence staining of 4-HNE ( scale bar , 50 µm). ( D – H ) Western blotting analysis of GPX4 ( D ), TFR ( E ), AKT pathway ( F ), caspase-9 and NF-κB p65 pathway ( G ), and IL-1β and STAT1 pathway ( H ) protein expression.

Article Snippet: Immunofluorescence staining was performed to evaluate the expression of K19 (1:200, Cell Signaling Technology, Danvers, MA, USA), K13 (1:200, Cell Signaling Technology), IL-1β (1:200, Abcam, Cambridge, UK), STAT1 (1:200, Proteintech, Rosemont, IL, USA), p63 (1:250, Abcam), Ki67 (1:200, Abcam), NF-κB p65 (1:200, Abcam), MUC1 (1:200, Abcam), MUC5AC(1:250, Abcam), caspase-9 (1:200, Cell Signaling Technology), ZO-1 (1:200, Abcam), and 4-hydroxynonenal (4-HNE) (1:100, Invitrogen, Carlsbad, CA, USA) in frozen sections of the conjunctival organoids.

Techniques: Immunofluorescence, Staining, Control, Western Blot, Expressing

Journal: Aging Cell

Article Title: Rapamycin Reverses the Hepatic Response to Diet‐Induced Metabolic Stress That Is Amplified by Aging

doi: 10.1111/acel.70395

Figure Lengend Snippet: Rapamycin co‐treatment mitigates proinflammatory transcriptional hyperactivation in aged mice exposed to HFD. (A) Protocol: Mice received ND or eRapamycin‐containing diet from 4 to 18 months, followed by 9 weeks on HFD or ND. (B) Western blot of liver tissue. (C) Fold change of p‐S6/S6 compared to control diet. (D) PCA of RNA‐seq from isolated hepatocytes. (E) Number of DEGs across treatment groups. (F) Scatter plot comparing HFD‐induced gene expression changes (HFD + veh vs. ND + veh) to all gene expression changes with eRapamycin (eRapa+HFD vs. HFD + veh). (G) Heatmap of genes upregulated by HFD + vehicle treatment. (H) Venn diagram showing overlap between transcripts elevated by HFD versus ND and those decreased by rapamycin. (I) KEGG enrichment analysis of overlapping genes. (J) Heatmap of innate immune response genes. (K) Top transcription factor pathways predicted by IPA in the overlapping gene set. (L) Heatmap of Stat1 target genes. (M) Tumorigenic index score calculated per mouse. Statistical analyses for p‐S6/S6 ratios and tumorigenic index scores were performed using one‐way ANOVA with post hoc Tukey's test; p < 0.05 (*), p < 0.1 (**).

Article Snippet: Antibodies used: total Stat1 (Cell Signaling Technologies 9172S), S6 Ribosomal protein (Cell Signaling Technologies 2317), phospho‐S6 Ribosomal protein (Cell Signaling Technologies 5364).

Techniques: Western Blot, Control, RNA Sequencing, Isolation, Gene Expression

Journal: Cell Death Discovery

Article Title: Fexofenadine protects against osteoarthritis by targeting Smad2 and STAT1 to enhance anabolism and binding cPLA2 to inhibit catabolism

doi: 10.1038/s41420-025-02754-9

Figure Lengend Snippet: A Coomassie Brilliant Blue staining of SDS-PAGE gels after the DARTS experiment; bands with molecular weights around 80 kDa were protected by FFD. Mass spectrum of STAT1. B Western blot analysis of STAT1 protein levels in Human normal chondrocytes treated with increasing concentrations of FFD (0, 10, 50, 100 μM) under proteolytic digestion. C Human normal chondrocytes were digested with different concentrations of proteases with or without FFD, after which STAT1 protein levels were determined using Western blotting. D , F Human normal chondrocyte lysates were denatured at different temperatures with or without FFD, after which STAT1 protein levels were assessed by Western blotting and Image J software (n = 3). E , G Isothermal dose-response curve (n = 3). H MST assay showing affinity between FFD and purified His-tagged STAT1 protein. I Molecular docking simulation of the interaction of FFD with human STAT1. STAT1 is shown in cyan. Hydrogen bonding is indicated by green dashed lines in 3D plots, with values indicating the bonding distances. Hydrogen bonding is indicated by purple arrows in 2D plots. Hydrophobic amino acids are shown in green, acidic amino acids in red, basic amino acids in blue, and uncharged polar amino acids in cyan. J Human normal chondrocytes were transfected using a myc-tagged STAT1 point mutant plasmid. Western blotting was used to assess MYC signalling after the DARTS experiment. K , L STAT1 protein expression in chondrocytes from healthy individuals and OA patients shown by Western blotting, and quantified using Image J (n = 6). M , N Protein expression of p-STAT1 (S727) in the ACLT animal model was assessed by immunohistochemical staining. Scale bars = 200 µm (left panel) and 100 µm (right panel). Quantitative analysis of immunohistochemical staining using Image J (n = 6). O , P OA chondrocytes were transiently transfected with pcDNA4, STAT1, or STAT1β plasmids for 24 h, after which they were treated for 30 min with or without FFD (10 µM). Western blotting and quantification of phosphorylated Smad2 (n = 3). Q OA chondrocytes were transiently transfected with pcDNA4 STAT1α plasmids for 24 h, after which they were treated for 24 h with or without FFD (10 µM). mRNA levels of ACAN and COL2A1 were measured using qRT-PCR (n = 3). R , S Knockdown of STAT1 in OA chondrocytes by siRNA, after which the cells were treated for 30 min with or without FFD (10 µM). Smad2 phosphorylation was assessed by immunoblotting. Densitometric analysis of the gels by Image J (n = 3). T Co-immunoprecipitation (Co-IP) was used to assess STAT1 interaction with ALK5 with or without FFD treatment. Significant differences are indicated as follows: * P < 0:05, ** P < 0:01, and *** P < 0:001; ns: not statistically significant.

Article Snippet: Purified recombinant STAT1 protein (RP01251; Abclonal) was labelled using the Protein Labelling Kit RED-NHS (NanoTemper, Beijing, China).

Techniques: Staining, SDS Page, Western Blot, Software, Purification, Transfection, Mutagenesis, Plasmid Preparation, Expressing, Animal Model, Immunohistochemical staining, Quantitative RT-PCR, Knockdown, Phospho-proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cell Death Discovery

Article Title: Fexofenadine protects against osteoarthritis by targeting Smad2 and STAT1 to enhance anabolism and binding cPLA2 to inhibit catabolism

doi: 10.1038/s41420-025-02754-9

Figure Lengend Snippet: A , B STAT1 knockdown via siRNA in Human normal chondrocytes, after which the cells were treated with or without (10 µM, 30 min); Smad2 phosphorylation was assessed via Western blot and quantified using Image J (n = 3). C qRT-PCR analysis of ACAN and COL2A1 mRNA levels in STAT1-silenced chondrocytes treated with or without FFD (10 µM, 24 h) (n = 3). D Pull-down of target proteins from human chondrocytes using FFD-conjugated Fe 3 O 4 beads, followed by mass spectrometry. E , F DARTS assay confirming FFD–Smad2 interaction. G , H CETSA demonstrating thermal stabilisation of Smad2 by FFD. I , J Isothermal dose-response assay showing Smad2 stabilisation by FFD, with Western blot and associated quantitative curve. K Surface plasmon resonance (SPR) assay assessing affinity between FFD and purified His-tagged Smad2 protein. L Molecular docking of FFD to Smad2 illustrating surface and 3D binding modes. M Western blot validation of siRNA-mediated Smad2 knockdown in human chondrocytes and C3H10T1/2 MSCs. N mRNA levels of ACAN , COL2A1 , and COMP in Human normal chondrocytes assessed via qRT-PCR (n = 3). O COMP protein levels in Human normal chondrocyte culture supernatants measured by ELISA (n = 3). P qRT-PCR analysis of ACAN , COL2A1 , and COMP mRNA levels in C3H10T1/2 MSCs (n = 3). Q , R Western blot and Image J quantification of SOX-9 protein levels in C3H10T1/2 MSCs (n = 3). S COMP protein levels in culture supernatants of C3H10T1/2 MSCs measured by ELISA (n = 3). Statistical significance: * P < 0:05, ** P < 0:01, and *** P < 0:001; ns not statistically significant.

Article Snippet: Purified recombinant STAT1 protein (RP01251; Abclonal) was labelled using the Protein Labelling Kit RED-NHS (NanoTemper, Beijing, China).

Techniques: Knockdown, Phospho-proteomics, Western Blot, Quantitative RT-PCR, Mass Spectrometry, SPR Assay, Purification, Binding Assay, Biomarker Discovery, Enzyme-linked Immunosorbent Assay