Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 enhances A. fumigatus –induced inflammatory response in HCECs. HCECs were stimulated with A. fumigatus hyphae (1 × 10 6 hyphal fragments/mL) for 0 (control), 1, 3, 6, 12, and 24 hours. ( A ) Protein levels of PIM1, PIM2, PIM3, and β-actin were analyzed by Western blot. ( B–D ) Quantification of PIM1 ( B ), PIM2 ( C ), and PIM3 ( D ) expression normalized to β-actin. For overexpression studies, HCECs were transfected with either empty vector pcDNA3.1 (Vector) or PIM1-expressing plasmid pcDNA3.1-PIM1 (PIM1) for 24 hours, followed by stimulation with A. fumigatus hyphae for 12 hours. ( E ) Western blot was used to detect the protein levels of PIM1 and β-actin. ( F ) Quantification of PIM1 protein levels in ( E ). ( G ) mRNA expression levels of TNF-α , IL-6 , IL-1β , and IFN-β were measured using qRT-PCR. ( H ) Secreted levels of TNF-α, IL-6, IL-1β, and IFN-β in culture supernatants were assessed by ELISA. Data are presented as the mean ± SD; # P > 0.05, * P < 0.05, ** P < 0.01; n = 3.

Article Snippet: The Flag-PIM1 and Myc-DDX41 expression vectors were generated by cloning the coding sequences of PIM1 and DDX41 into pcDNA3.1 plasmids containing Flag or Myc tags, respectively (Addgene, Watertown, MA, USA).

Techniques: Control, Western Blot, Expressing, Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 inhibition mitigates A. fumigatus –induced inflammatory response. HCECs were transfected with either negative control siRNA (siNC) or two specific PIM1-targeting siRNAs (siPIM1-1 and siPIM1-2) for 24 hours and then stimulated with A. fumigatus hyphae (1 × 10 6 hyphal fragments/mL) for an additional 12 hours. ( A ) Western blot analysis of PIM1 and β-actin protein levels. Quantitative analysis of PIM1 is provided in A. ( B ) mRNA expression levels of TNF-α , IL-6 , IL-1β , and IFN-β were evaluated by qRT-PCR. ( C ) Secreted levels of TNF-α, IL-6, IL-1β, and IFN-β in culture supernatants were measured using ELISA. HCECs were pretreated with the PIM1 inhibitor SGI-1776 (0, 2, and 4 µM) for 12 hours prior to 12-hour stimulation with A. fumigatus hyphae. ( D ) Western blot analysis of PIM1 and β-actin protein levels. Quantitative data are presented in B. ( E ) qRT-PCR analysis of TNF-α , IL-6 , IL-1β , and IFN-β mRNA expression. ( F ) Secreted levels of TNF-α, IL-6, IL-1β, and IFN-β in culture supernatants were assessed by ELISA. Data are presented as the mean ± SD; * P < 0.05, ** P < 0.01; n = 3.

Article Snippet: The Flag-PIM1 and Myc-DDX41 expression vectors were generated by cloning the coding sequences of PIM1 and DDX41 into pcDNA3.1 plasmids containing Flag or Myc tags, respectively (Addgene, Watertown, MA, USA).

Techniques: Inhibition, Transfection, Negative Control, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 directly interacts with DDX41 and regulates its phosphorylation. ( A ) HCECs transfected with either pcDNA3.1 (Flag) or pcDNA3.1 PIM1 (Flag-PIM1) were stimulated with A. fumigatus hyphae (1 × 10 6 hyphal fragments/mL) for 12 hours. Cell lysates were immunoprecipitated using anti-Flag beads, and bound proteins were separated by SDS-PAGE and visualized by silver staining. ( B ) A GST pull-down assay was performed to examine direct binding between PIM1 and DDX41. ( C ) Endogenous interaction between PIM1 and DDX41 in HCECs was assessed by co-IP. HEK293T cells were cotransfected with Flag-PIM1 and Myc-DDX41 for 48 hours. (D) Co-IP was carried out with anti-Flag beads, and Myc-DDX41 was detected with anti-Myc antibody. ( E ) Reciprocal co-IP was performed using anti-Myc beads, and Flag-PIM1 was probed with anti-Flag antibody. ( F ) HCECs were transfected with NC siRNA (siNC) or PIM1 siRNAs (siPIM1-1 and siPIM1-2) for 24 hours, then exposed to A. fumigatus hyphae for 12 hours. DDX41 was immunoprecipitated and immunoblotted with anti–p-Ser and DDX41 antibodies. ( G ) HCECs were transfected with pcDNA3.1 (Vector) or pcDNA3.1-PIM1 (PIM1) for 24 hours, then exposed to A. fumigatus hyphae for 12 hours, followed by DDX41 immunoprecipitation and immunoblotting with indicated antibodies. ( H ) HCECs were transfected with NC siRNA (siNC), PIM1 siRNA (siPIM1-2), and/or pcDNA3.1-PIM1 (PIM1) for 24 hours, then treated with A. fumigatus hyphae for 12 hours. Co-IP of DDX41 was performed, and associated proteins were analyzed by Western blot. ( I ) Quantification of protein levels in ( H ). ( J ) HCECs were pretreated with SGI-1776 (0 and 4 µM) for 12 hours prior to 12-hour stimulation with A. fumigatus hyphae. Co-IP assay was performed using anti-DDX41 antibody and immunoblotted with the antibodies indicated. ( K ) Quantification of protein levels in ( J ). ( L ) Recombinant His-PIM1 and GST-DDX41 were incubated in reaction buffer with or without phosphatase for 30 minutes. The protein levels of p-Ser, His-PIM1, and GST-DDX41 were detected by Western blot. Quantification of protein levels in F, G, and L is shown in A–C. Data are presented as the mean ± SD; ** P < 0.01; n = 3.

Article Snippet: The Flag-PIM1 and Myc-DDX41 expression vectors were generated by cloning the coding sequences of PIM1 and DDX41 into pcDNA3.1 plasmids containing Flag or Myc tags, respectively (Addgene, Watertown, MA, USA).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, SDS Page, Silver Staining, Pull Down Assay, Binding Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Western Blot, Recombinant, Incubation

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 regulates STING signaling through DDX41. ( A ) HCECs were transfected with pcDNA3.1 (Vector) or pcDNA3.1-PIM1 (PIM1) for 24 hours, followed by stimulation with A. fumigatus hyphae (1 × 10 6 hyphal fragments/mL) for 12 hours. Protein levels of p-STING, STING, p-TBK1, TBK1, p-IRF3, IRF3, PIM1, and β-actin were assessed by Western blot. ( B ) HCECs were transfected with NC siRNA (siNC) or PIM1 siRNAs (siPIM1-1 and siPIM1-2) for 24 hours, then treated with A. fumigatus hyphae for 12 hours. Protein expression was analyzed via Western blot. ( C ) Quantification of protein levels in ( A ). ( D ) Quantification of protein levels in ( B ). ( E ) HCECs were transfected with NC siRNA (siNC), PIM1 siRNA-2 (siPIM1-2), and/or pcDNA3.1-PIM1 (PIM1) for 24 hours, followed by A. fumigatus hyphae treatment for 12 hours. Protein levels were detected by Western blot. ( F ) HCECs were pretreated with SGI-1776 (0, 2, 4 µM) for 12 hours, then exposed to A. fumigatus hyphae for 12 hours. Protein levels were detected by Western blot. ( G ) HCECs were transfected with NC siRNA (siNC), PIM1 siRNA-2 (siPIM1-2), DDX41 siRNA (siDDX41), or pcDNA3.1-DDX41 (DDX41) for 24 hours, followed by A. fumigatus hyphae treatment for 12 hours. Protein levels were detected by Western blot. ( H ) Quantification of protein levels in ( E ). ( I ) Quantification of protein levels in ( F ). Quantification of protein levels in G is provided in D. Data are presented as the mean ± SD; ** P < 0.01; n = 3.

Article Snippet: The Flag-PIM1 and Myc-DDX41 expression vectors were generated by cloning the coding sequences of PIM1 and DDX41 into pcDNA3.1 plasmids containing Flag or Myc tags, respectively (Addgene, Watertown, MA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 regulates A. fumigatus –induced inflammatory response by interacting with DDX41. HCECs were transfected with NC siRNA (siNC), PIM1 siRNA-2 (siPIM1-2), DDX41 siRNA (siDDX41), and/or pcDNA3.1-DDX41 (DDX41) for 24 hours, followed by stimulation with A. fumigatus hyphae (1 × 10 6 hyphal fragments/mL) for 12 hours. ( A ) Protein levels of DDX41, PIM1, and β-actin were analyzed by Western blot. Quantitative data are shown in A. ( B ) mRNA expression levels of TNF-α , IL-6 , IL-1β , and IFN-β were evaluated by qRT-PCR. ( C ) Secreted levels of TNF-α, IL-6, IL-1β, and IFN-β in culture supernatants were measured using ELISA. HCECs were transfected with pcDNA3.1 (Vector) or pcDNA3.1-PIM1 (PIM1) for 24 hours and then treated with A. fumigatus hyphae for 12 hours. C176 (1 µM) was added to the medium for 24 hours before harvest. ( D ) Protein levels of PIM1 and β-actin were analyzed by Western blot. Quantitative analysis is shown in B. ( E ) mRNA expression levels of TNF-α , IL-6 , IL-1β , and IFN-β were evaluated by qRT-PCR. ( F ) Secreted levels of TNF-α, IL-6, IL-1β, and IFN-β in culture supernatants were measured using ELISA. Data are presented as the mean ± SD; * P < 0.05, ** P < 0.01; n = 3.

Article Snippet: The Flag-PIM1 and Myc-DDX41 expression vectors were generated by cloning the coding sequences of PIM1 and DDX41 into pcDNA3.1 plasmids containing Flag or Myc tags, respectively (Addgene, Watertown, MA, USA).

Techniques: Transfection, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: Knockdown of PIM1 ameliorates A. fumigatus keratitis in mice. ( A ) Mouse corneas were infected with 5 µL of live A. fumigatus hyphae (1 × 10 8 hyphal fragments/mL) for 0.5, 1, and 3 days, then excised for analysis. Protein levels of p-STING, STING, PIM1, and β-actin were assessed by Western blot. Quantitative data are provided in A. ( B ) DDX41 was immunoprecipitated from corneal lysates and immunoblotted with anti–p-Ser and anti-DDX41 antibodies. Quantification is shown in B. Mice received subconjunctival injection of 5 µL control siRNA (siNC, 10 µM) or PIM1 siRNAs (siPIM1-1/siPIM1-2, 10 µM). After 24 hours, corneas were infected with 5 µL of live hyphae for an additional 24 hours before harvesting. ( C ) Keratitis severity was evaluated by slit-lamp examination. ( D ) Clinical scores were calculated based on slit-lamp observations. ( E ) Fungal burden was quantified by colony-forming unit (CFU) assays. ( F ) Protein expression of p-STING, STING, PIM1, and β-actin in mouse corneas was detected by Western blot. Quantitative results are shown in C. ( G ) DDX41 was immunoprecipitated and immunoblotted with anti–p-Ser and anti-DDX41 antibodies. Quantification is presented in D. ( H ) mRNA expression of TNF-α , IL-6 , IL-1β , and IFN-β was measured by qRT-PCR. ( I ) Secreted levels of TNF-α, IL-6, IL-1β, and IFN-β in corneal homogenates were detected using ELISA. Data are presented as the mean ± SD; ** P < 0.01; n = 6.

Article Snippet: The Flag-PIM1 and Myc-DDX41 expression vectors were generated by cloning the coding sequences of PIM1 and DDX41 into pcDNA3.1 plasmids containing Flag or Myc tags, respectively (Addgene, Watertown, MA, USA).

Techniques: Knockdown, Infection, Western Blot, Immunoprecipitation, Injection, Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 inhibitor inhibits A. fumigatus keratitis in vivo. Mouse corneas were infected with 5 µL of live A. fumigatus hyphae (1 × 10 8 hyphal fragments/mL) for 24 hours. Two hours prior to infection, mice received a subconjunctival injection of 5 µL SGI-1776 (1 µg/µL or 2 µg/µL). Corneas were harvested 24 hours postinfection. ( A ) Keratitis severity was evaluated by slit-lamp examination. ( B ) Clinical scores were calculated based on slit-lamp observations. ( C ) Fungal burden was quantified by CFU assays. (D) Protein expression of p-STING, STING, PIM1, and β-actin in mouse corneas was analyzed by Western blot. Quantification is presented in A. ( E ) DDX41 was immunoprecipitated from corneal lysates and immunoblotted with anti–p-Ser and anti-DDX41 antibodies. Quantification is provided in B. ( F ) mRNA expression levels of TNF-α , IL-6 , IL-1β , and IFN-β were measured by qRT-PCR. ( G ) Protein levels of TNF-α, IL-6, IL-1β, and IFN-β in corneal homogenates were determined using ELISA. Data are presented as the mean ± SD; * P < 0.05, ** P < 0.01; n = 6.

Article Snippet: The Flag-PIM1 and Myc-DDX41 expression vectors were generated by cloning the coding sequences of PIM1 and DDX41 into pcDNA3.1 plasmids containing Flag or Myc tags, respectively (Addgene, Watertown, MA, USA).

Techniques: In Vivo, Infection, Injection, Expressing, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: Working model of the PIM1-DDX41-STING axis in A. fumigatus keratitis. A. fumigatus infection upregulates PIM1 expression. PIM1 then directly binds to and phosphorylates the innate immune sensor DDX41. Phosphorylated DDX41 activates the STING signaling pathway, leading to the phosphorylation of TBK1 and IRF3. This cascade ultimately promotes the transcription and secretion of proinflammatory cytokines (TNF-α, IL-6, IL-1β, IFN-β), contributing to the immunopathology of fungal keratitis. Pharmacologic inhibition of PIM1 by SGI-1776 disrupts this axis, attenuating DDX41/STING activation and the subsequent inflammatory response.

Article Snippet: The Flag-PIM1 and Myc-DDX41 expression vectors were generated by cloning the coding sequences of PIM1 and DDX41 into pcDNA3.1 plasmids containing Flag or Myc tags, respectively (Addgene, Watertown, MA, USA).

Techniques: Infection, Expressing, Phospho-proteomics, Inhibition, Activation Assay

Journal: Inflammation

Article Title: Keratinocyte-derived Exosomal LKB1 Drives Vitiligo Progression by Activating CD8 + T Cells

doi: 10.1007/s10753-026-02479-6

Figure Lengend Snippet: Exosomal LKB1 serves as a key mediator of vitiligo progression. Proteomic profiling of differentially expressed proteins between Vexo versus Hexo ( A ) and H₂O₂/exo versus Con/exo ( B ), screened using p < 0.05 and fold change > 2. Red indicates upregulated proteins; blue indicates downregulated proteins. ( C ) Venn diagram showing the intersection of proteins upregulated in both Vexo versus Hexo and H₂O₂/exo versus Con/exo datasets, identifying LKB1 as the sole common candidate. ( D ) Western blot analysis of LKB1 expression in keratinocytes isolated from lesional skin of three vitiligo patients and keratinocytes from three healthy individuals. ( E ) Multiplex immunofluorescence staining of CD8 (red) and CD69 (green) expression in lesional skin from vitiligo patients with nuclear counterstaining by DAPI (blue). ( F ) Multiplex immunofluorescence staining of LKB1 (red) and CD69 (yellow) expression in lesional skin from vitiligo patients and healthy control skin, with nuclear counterstaining by DAPI (blue). ( G ) Quantification of LKB1 expression in skin biopsies from 12 vitiligo patients and 12 healthy controls. ( H ) Correlation analysis between LKB1 expression and CD69 expression in the lesional skin of vitiligo patients. Vexo, exosomes derived from keratinocytes of vitiligo patients; Hexo, exosomes derived from healthy donors; H 2 O 2 /exo, exosomes derived from H 2 O 2 -treated HaCaT cells; Con/exo, exosomes derived from untreated HaCaT cells; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant

Article Snippet: Vitiligo mice treated with 40 mg/kg LKB1 inhibitor (HY-10371, MCE, USA) administered intraperitoneally.

Techniques: Western Blot, Expressing, Isolation, Multiplex Assay, Immunofluorescence, Staining, Control, Derivative Assay

Journal: Inflammation

Article Title: Keratinocyte-derived Exosomal LKB1 Drives Vitiligo Progression by Activating CD8 + T Cells

doi: 10.1007/s10753-026-02479-6

Figure Lengend Snippet: Exosomal LKB1 drives CD8⁺ T cells activation and vitiligo progression. Western blot analysis of LKB1 expression in CD8⁺ T cells treated with Hexo or Vexo ( A ) and Con/exo or H₂O₂/exo ( B ). ELISA quantification of granzyme B ( C ) and IFN-γ ( D ) secretion by CD8⁺ T cells treated with exosomes from LKB1-overexpressing HaCaT cells (HaCaT/exo LKB1 ) or vector control exosomes (HaCaT/exo vector ). ELISA quantification of granzyme B ( E ) and IFN-γ ( F ) secretion by CD8⁺ T cells treated with exosomes from H₂O₂-treated HaCaT cells with LKB1 knockdown (H₂O₂/exo shLKB1 ) or negative control shRNA (H₂O₂/exo shNC ). Flow cytometric analysis of melanocyte apoptosis following co-culture with CD8⁺ T cells treated with HaCaT/exo LKB1 or HaCaT/exo vector , shown as representative plots ( G ) and quantitative summary ( H ). ( I ) Representative tail skin photographs of vitiligo mice from the untreated control group and the group treated with 40 mg/kg LKB1 inhibitor administered intraperitoneally. Immunofluorescence staining of CD69 expression in tail skin from control and LKB1 inhibitor–treated vitiligo mice, shown as representative images ( J ) and quantification ( K ). Vexo, exosomes derived from keratinocytes of vitiligo patients; Hexo, exosomes derived from healthy donors; HaCat/exoLKB1, exosomes derived from LKB1-overexpressing HaCat keratinocytes; H 2 O 2 /exo shLKB1 , exosomes derived from LKB1-knockdown keratinocytes under H 2 O 2 treat; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant

Article Snippet: Vitiligo mice treated with 40 mg/kg LKB1 inhibitor (HY-10371, MCE, USA) administered intraperitoneally.

Techniques: Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Control, Knockdown, Negative Control, shRNA, Co-Culture Assay, Immunofluorescence, Staining, Derivative Assay

Journal: Inflammation

Article Title: Keratinocyte-derived Exosomal LKB1 Drives Vitiligo Progression by Activating CD8 + T Cells

doi: 10.1007/s10753-026-02479-6

Figure Lengend Snippet: Exosomal LKB1 mediates CD8 + T cells activation via the AMPK pathway. ( A ) Heatmap of RNA-seq analysis showing differentially expressed genes in CD8⁺ T cells treated with Hexo or Vexo. ( B ) KEGG pathway enrichment analysis of differentially expressed genes in Vexo-treated versus Hexo-treated CD8⁺ T cells, highlighting significant activation of the AMPK signaling pathway. ( C–D ) Western blot analysis of LKB1 and p-AMPK expression in CD8⁺ T cells treated with different exosome preparations (Hexo, Vexo, H₂O₂/exo, or Con/exo) in the presence or absence of LKB1 inhibitor (10 µM). ( E ) Western blot analysis of p-AMPK expression in CD8⁺ T cells after LKB1 overexpression. ELISA quantification of granzyme B ( F ) and IFN-γ ( G ) secretion by CD8⁺ T cells treated with indicated exosome groups with or without AMPK inhibition (20 µM). ( H ) Flow cytometric analysis of IFN-γ⁺ CD8⁺ T cells following treatment with indicated exosome preparations. Vexo, exosomes derived from keratinocytes of vitiligo patients; Hexo, exosomes derived from healthy donors; LBK1in, LKB1 inhibitor; H 2 O 2 /exo, exosomes derived from H 2 O 2 -treated HaCaT cells; Con/exo, exosomes derived from untreated HaCaT cells; LKB1-oe, LBK1 overexpression; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant

Article Snippet: Vitiligo mice treated with 40 mg/kg LKB1 inhibitor (HY-10371, MCE, USA) administered intraperitoneally.

Techniques: Activation Assay, RNA Sequencing, Western Blot, Expressing, Over Expression, Enzyme-linked Immunosorbent Assay, Inhibition, Derivative Assay

Journal: Inflammation

Article Title: Keratinocyte-derived Exosomal LKB1 Drives Vitiligo Progression by Activating CD8 + T Cells

doi: 10.1007/s10753-026-02479-6

Figure Lengend Snippet: IFN-γ secreted by CD8 + T cells positively feeds back on the excessive production of LKB1 in keratinocytes. ( A ) qRT-PCR analysis of LKB1 expression in HaCaT cells at various time points following IFN-γ treatment. qRT-PCR ( B ) and Western blot ( C ) analyses of LKB1 expression in HaCaT cells treated with increasing concentrations of IFN-γ. ( D ) Schematic representation of the co-culture system between keratinocytes and CD8⁺ T cells pretreated with Hexo or Vexo. qRT-PCR analysis of LKB1 expression in HaCaT cells co-cultured with CD8⁺ T cells treated with Vexo ( E ) or H₂O₂/exo ( F ). ( G ) Western blot analysis of p-STAT1 levels in HaCaT cells following IFN-γ stimulation. ( H ) Chromatin immunoprecipitation–qRT-PCR analysis showing recruitment of STAT1 to the LKB1 promoter region. ( I ) Predicted STAT1-binding motifs within the LKB1 promoter identified from the JASPAR database. A dual luciferase reporter plasmid containing the wild-type or mutant binding sites (#1–3) was constructed. ( J ) Luciferase activity in HEK293T cells co-transfected with the indicated LKB1 promoter constructs and STAT1 expression plasmid. qRT-PCR ( K ) and Western blot ( L ) analyses of LKB1 expression in HaCaT cells treated with IFN-γ in the presence or absence of a STAT1 inhibitor (5 µM). * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant

Article Snippet: Vitiligo mice treated with 40 mg/kg LKB1 inhibitor (HY-10371, MCE, USA) administered intraperitoneally.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Co-Culture Assay, Cell Culture, Chromatin Immunoprecipitation, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Construct, Activity Assay, Transfection

Journal: Inflammation

Article Title: Keratinocyte-derived Exosomal LKB1 Drives Vitiligo Progression by Activating CD8 + T Cells

doi: 10.1007/s10753-026-02479-6

Figure Lengend Snippet: Proposed mechanistic model of keratinocyte-derived exosomal LKB1 in vitiligo pathogenesis. Schematic illustration of the proposed pathogenic feedback loop in vitiligo. Vitiligo lesional keratinocytes secrete exosomes enriched in LKB1 (Vexo), which are internalized by CD8⁺ T cells. Exosomal LKB1 activates the AMPK signaling pathway in CD8⁺ T cells, leading to increased secretion of IFN-γ and granzyme B, thereby enhancing melanocyte-directed cytotoxicity. IFN-γ released by activated CD8⁺ T cells further stimulates keratinocytes via the JAK/STAT1 pathway, resulting in increased LKB1 expression and further exosome release. This establishes a self-amplifying loop between keratinocytes and CD8⁺ T cells that sustains autoimmune melanocyte destruction and promotes vitiligo progression

Article Snippet: Vitiligo mice treated with 40 mg/kg LKB1 inhibitor (HY-10371, MCE, USA) administered intraperitoneally.

Techniques: Derivative Assay, Expressing

Journal: Research

Article Title: Pim1 Serves as a Therapeutic Target for Inflammatory Arthritis via Mitochondrial Metabolism and Th17 Cell Differentiation

doi: 10.34133/research.1137

Figure Lengend Snippet: Pim1 expression is increased in CD4 + T cells in inflammatory arthritis. (A) Representative immunofluorescence (IF) images showing Pim1 expression in CD4 + cells in the OA and RA synovium and the LDH and AS entheses. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (B) Representative immunofluorescence images showing Pim1 expression in CD4 + cells in ankle tissues from CIA and SKG arthritis model mice and the corresponding normal controls. Scale bars, 50 μm. (C and D) Relative protein levels of Pim1 in CD4 + T cells in ankle tissues (C) and spleens (D) of CIA and SKG arthritis mice over time following arthritis induction ( n = 5). (E) Relative protein levels of Pim1 in peripheral blood CD4 + T cells from patients with RA and AS and HCs ( n = 12). The data were statistically analyzed via one-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc comparisons test (C and D) and 2-tailed Student’s t test (E).

Article Snippet: Pim1 flox and Pim1 cKO mice were purchased from Shanghai Model Organism.

Techniques: Expressing, Immunofluorescence

Journal: Research

Article Title: Pim1 Serves as a Therapeutic Target for Inflammatory Arthritis via Mitochondrial Metabolism and Th17 Cell Differentiation

doi: 10.34133/research.1137

Figure Lengend Snippet: Specifically intercepting Pim1 alleviates the development of inflammatory arthritis by impeding Th17 cell differentiation. (A) Identification of Pim1 flox and CD4-cre gene. WT, wild type. (B) Relative protein levels of Pim1 in CD4 + T cells isolated from Pim1 flox and Pim1 cKO mice. (C) Macroscopic images of the ankles, arthritis scores, and hind paw thicknesses of Pim1 flox and Pim1 cKO CIA mice ( n = 5). (D and E) Representative histological images with H&E staining (D) and safranin O-fast green staining (E) of ankles from Pim1 flox and Pim1 cKO CIA mice ( n = 10). Scale bars, 200 μm. (F) Representative micro-CT images of the ankles of Pim1 flox and Pim1 cKO CIA mice. (G) Frequencies of IFN-γ + , IL-4 + , IL-17A + , and Foxp3 + cells among CD4 + cells in the spleens of Pim1 flox and Pim1 cKO CIA mice ( n = 5). (H) Relative mRNA expression of RORγt, T-bet, GATA3, and Foxp3 in the ankles of Pim1 flox or Pim1 cKO CIA mice ( n = 5). (I) Frequencies of IL-17A + cells among CD4 + cells in the ankles of Pim1 flox and Pim1 cKO CIA mice ( n = 5). (J) Relative mRNA expression of IL-17A in the ankles of Pim1 flox or Pim1 cKO CIA mice ( n = 5). (K) Representative immunohistochemical (IHC) images showing IL-17A expression in the ankle tissues of Pim1 flox and Pim1 cKO CIA mice. Scale bars, 50 μm. The data were statistically analyzed via 2-tailed Student’s t test (B, D, E, and G to J) and 2-way repeated-measures ANOVA (C).

Article Snippet: Pim1 flox and Pim1 cKO mice were purchased from Shanghai Model Organism.

Techniques: Cell Differentiation, Isolation, Staining, Micro-CT, Expressing, Immunohistochemical staining

Journal: Research

Article Title: Pim1 Serves as a Therapeutic Target for Inflammatory Arthritis via Mitochondrial Metabolism and Th17 Cell Differentiation

doi: 10.34133/research.1137

Figure Lengend Snippet: Pim1 promotes Th17 cell differentiation in vitro. (A) Relative mRNA levels of Pim1 during Th17 cell differentiation ( n = 9). (B) Relative protein levels of pSTAT3, RORγt, and Pim1 during Th17 cell differentiation ( n = 9). (C and D) Frequency of Th17 cells among CD4 + cells after AZD1208 treatment ( n = 9). (E) Relative protein levels of Pim1 among CD4 + T cells overexpressing (OE) vector or Pim1 ( n = 9). (F) Frequency of Th17 cells in CD4 + cells after overexpressing vector or Pim1 ( n = 9). (G) Relative protein levels of RORγt and pSTAT3 after AZD1208 treatment or Pim1 overexpression ( n = 9). (H and I) Relative mRNA levels of Th17-cell-associated pathogenic genes after AZD1208 treatment (H) or Pim1 overexpression (I) ( n = 9). (J and K) Concentration of IL-17A, IL-17F, IL-22, and GM-CSF in the cell supernatant after AZD1208 treatment (J) or Pim1 overexpression (K). (L) Frequencies of Th1, Th2, and Treg cells among CD4 + T cells after treatment with AZD1208 ( n = 9). The data were statistically analyzed via one-way ANOVA, followed by Bonferroni’s post hoc comparisons (A to D) and paired t test (E to L).

Article Snippet: Pim1 flox and Pim1 cKO mice were purchased from Shanghai Model Organism.

Techniques: Cell Differentiation, In Vitro, Plasmid Preparation, Over Expression, Concentration Assay

Journal: Research

Article Title: Pim1 Serves as a Therapeutic Target for Inflammatory Arthritis via Mitochondrial Metabolism and Th17 Cell Differentiation

doi: 10.34133/research.1137

Figure Lengend Snippet: Pim1 regulates Th17 cell differentiation through OXPHOS. (A to C) The MFI of TMRE (A) and the relative levels of NADH (B) and ATP (C) in cells at different stages of Th17 cell differentiation and in cells treated with different concentrations of AZD1208 and overexpressing Pim1 ( n = 9). (D to F) OCR of cells at different stages of Th17 cell differentiation (D), cells treated with different concentrations of AZD1208 (E), and cells overexpressing Pim1 (F) ( n = 9). OLI, oligomycin. (G and H) Mitochondrial cristae frequency in cells treated with AZD1208 (G) and cells overexpressing Pim1 (H) ( n =15). Scale bars, 200 nm. (I) ROS levels of cells in naïve, Th17, and AZD1208 groups ( n = 9). (J) ECAR of cells in the naïve, Th17, and AZD1208 groups ( n = 9). (K) Relative mRNA levels of the key enzymes involved in fatty acid synthesis of cells in naïve, Th17, and AZD1208 groups ( n = 9). (L) Frequency of Th17 cells among CD4 + cells after CCCP treatment in the presence or absence of Pim1 overexpression ( n = 9). (M and N) Relative protein levels of RORγt and pSTAT3 in cells treated with CCCP in the presence or absence of Pim1 overexpression ( n = 9). (O) Relative mRNA levels of Th17-cell-associated pathogenic genes in cells treated with CCCP in the presence or absence of Pim1 overexpression ( n = 9). The data were statistically analyzed via one-way ANOVA, followed by Bonferroni’s post hoc comparisons (A to E and I to O), paired t test (A to C and F), and 2-tailed Student’s t test (G and H)

Article Snippet: Pim1 flox and Pim1 cKO mice were purchased from Shanghai Model Organism.

Techniques: Cell Differentiation, Over Expression

Journal: Research

Article Title: Pim1 Serves as a Therapeutic Target for Inflammatory Arthritis via Mitochondrial Metabolism and Th17 Cell Differentiation

doi: 10.34133/research.1137

Figure Lengend Snippet: Pim1 promotes OXPHOS by facilitating mito-Ca 2+ influx. (A) MFI of Rhod-2AM of cells in the naïve, Th17, and AZD1208 groups ( n = 9). (B) MFI of Rhod-2AM of cells overexpressing the vector or Pim1 ( n = 9). (C to E) The MFI of TMRE (C) and the relative levels of NADH (D) and ATP (E) in cells treated with RR or Ru360 in the presence or absence of Pim1 overexpression ( n = 9). (F) OCR of cells treated with RR or Ru360 in the presence or absence of Pim1 overexpression ( n = 9). (G) Frequency of Th17 cells among CD4 + cells after treatment with RR or Ru360 in the presence or absence of Pim1 overexpression ( n = 9). (H) Relative protein levels of RORγt and pSTAT3 in cells treated with RR or Ru360 in the presence or absence of Pim1 overexpression ( n = 9). (I) Relative mRNA levels of Th17-cell-associated pathogenic genes in cells treated with RR or Ru360 in the presence or absence of Pim1 overexpression ( n = 9). (J) IP showing the interaction of Pim1 and MICU1 in Th17 cells (top) and HEK-293T cells (bottom). (K) Phosphorylation level of MICU1 in cells after AZD1208 treatment or Pim1 overexpression ( n = 6). The data were statistically analyzed via one-way ANOVA, followed by Bonferroni’s post hoc comparisons (A and C to I) and paired t test (B and K).

Article Snippet: Pim1 flox and Pim1 cKO mice were purchased from Shanghai Model Organism.

Techniques: Plasmid Preparation, Over Expression, Phospho-proteomics

Journal: Research

Article Title: Pim1 Serves as a Therapeutic Target for Inflammatory Arthritis via Mitochondrial Metabolism and Th17 Cell Differentiation

doi: 10.34133/research.1137

Figure Lengend Snippet: Molecular docking and molecular dynamics simulations of Pim1. (A) Molecular docking showing that drospirenone, olaparib, nilotinib, and dutasteride dock to Pim1 (PDB 1XWS) in the same drug-binding pocket as AZD1208 and that the bonds between the 4 drugs and Pim1. The blue dashed line represents hydrogen bonds, gray represents hydrophobic bonds, green cyan represents halogen bonds, and green represents π-stacking. (B) RMSD of 50-ns molecular dynamics simulations between Pim1 and drospirenone, olaparib, nilotinib, and dutasteride, respectively. (C) RMSFs of 50-ns molecular dynamics simulations between Pim1 and drospirenone, olaparib, nilotinib, and dutasteride, respectively. (D) The original and terminal structures of Pim1 interacting with drospirenone, olaparib, nilotinib, and dutasteride in 50-ns molecular dynamics simulations.

Article Snippet: Pim1 flox and Pim1 cKO mice were purchased from Shanghai Model Organism.

Techniques: Binding Assay

Journal: Research

Article Title: Pim1 Serves as a Therapeutic Target for Inflammatory Arthritis via Mitochondrial Metabolism and Th17 Cell Differentiation

doi: 10.34133/research.1137

Figure Lengend Snippet: Nilotinib inhibits Th17 cell differentiation and alleviates inflammatory arthritis by targeting Pim1. (A) Enzyme activity of Pim1 after incubation with AZD1208, drospirenone, olaparib, nilotinib, or dutasteride ( n = 9). (B) Frequency of Th17 cells among CD4 + cells after treatment with nilotinib in the presence or absence of Pim1 overexpression ( n = 9). (C) MFI of Rhod-2AM in cells treated with nilotinib in the presence or absence of Pim1 overexpression ( n = 9). (D) OCR of cells treated with nilotinib in the presence or absence of Pim1 overexpression ( n = 9). (E) Macroscopic images of the ankles of Pim1 flox and Pim1 cKO CIA mice treated with vehicle or nilotinib. (F) Arthritis scores and hind paw thickness of Pim1 flox and Pim1 cKO CIA mice treated with vehicle or nilotinib ( n = 5). (G and H) Representative histological images with H&E staining (G) and safranin O-fast green staining (H) of the ankles of Pim1 flox and Pim1 cKO CIA mice treated with vehicle or nilotinib ( n = 10). Scale bars, 200 μm. (I) Representative micro-CT images of ankles of Pim1 flox and Pim1 cKO CIA mice treated with vehicle or nilotinib. (J and K) Frequency of Th17 cells among CD4 + cells in spleens (J) and ankles (K) of Pim1 flox and Pim1 cKO CIA mice treated with vehicle or nilotinib ( n = 5). (L) Representative immunohistochemical images showing IL-17A expression in the ankle tissues of Pim1 flox and Pim1 cKO CIA mice treated with vehicle or nilotinib. Scale bars, 50 μm. The data were statistically analyzed via one-way ANOVA, followed by Bonferroni’s post hoc comparisons (B to D, G, H, J, and K) and 2-way repeated-measures ANOVA (F).

Article Snippet: Pim1 flox and Pim1 cKO mice were purchased from Shanghai Model Organism.

Techniques: Cell Differentiation, Activity Assay, Incubation, Over Expression, Staining, Micro-CT, Immunohistochemical staining, Expressing