Journal: Nature Communications

Article Title: YBX1 Confers immunosuppressive bone metastatic traits in non-small cell lung cancer

doi: 10.1038/s41467-026-73931-2

Figure Lengend Snippet: A ELISA detection of IL6 and CCL5 concentrations in the serum of mice bearing A549 High-M tumors. Biological replicates n = 5, independent experiments, Unpaired t test (Two-tailed); Mean ± SD. B Dual-luciferase reporter assay analyzing the effect of YBX1 overexpression on the activity of the IL6 and CCL5 promoters. Biological replicates n = 6, independent experiments, Unpaired t test (Two-tailed); Mean ± SD. The schematic diagram was created by the author using Adobe Illustrator. C Representative immunofluorescence images of the number of YBX1 condensates in the nuclei of A549 High-M and A549 Low-M cells ( Scale bar: 10 μm ). Biological replicates n = 6, independent experiments, Unpaired t test (Two-tailed) . A549 Low-M : n = 6, minima = 3, maxima = 8, center = 6, Q1/Lower Quartile = 5.25, Q3/Upper Quartile = 6.75, Lower Whisker = 3, Upper Whisker = 8, IQR / Interquartile Range = 1.5; A549 High-M : n = 6, minima = 13, maxima = 26, center = 19.5, Q1/Lower Quartile = 18.25, Q3/Upper Quartile = 23, Lower Whisker = 13, Upper Whisker = 26, IQR / Interquartile Range = 4.75 . D Representative immunofluorescence images of the changes after treatment with 5% 1,6-Hex ( Scale bar: 10 μm ). Biological replicates n = 6, independent experiments, Unpaired t test (Two-tailed) . Ctrl: n = 6, minima = 15, maxima = 28, center = 22, Q1/Lower Quartile = 17.5, Q3/Upper Quartile = 25.75, Lower Whisker = 15, Upper Whisker = 28, IQR / Interquartile Range = 28.25; 1,6Hex: n = 6, minima = 2, maxima = 6, center = 3.5, Q1/Lower Quartile = 3, Q3/Upper Quartile = 4.75, Lower Whisker = 2, Upper Whisker = 6, IQR / Interquartile Range = 1.75 . E Representative images of the dynamics of YBX1 condensates in fluorescence bleaching recovery experiment ( Scale bar: 10 μm ). Biological replicates n = 6, independent experiments . F Representative images of nuclear co-localization of YBX1 condensates with transcriptional activity markers p-RNA Pol II (Ser5) and H3K27ac ( Scale bar: 10 μm ). YBX1+Pol II-S5P: n = 20, perspective, minima = 0.825, maxima = 0.987, center = 0.955, Q1/Lower Quartile = 0.90275, Q3/Upper Quartile = 0.97625, Lower Whisker = 0.825, Upper Whisker = 0.987, IQR/Interquartile Range = 0.0735; YBX1 + H3K27ac: n = 20, perspective, minima = 0.814, maxima = 0.985, center = 0.9185, Q1/Lower Quartile = 0.846, Q3/Upper Quartile = 0.95175, Lower Whisker = 0.814, Upper Whisker = 0.985, IQR / Interquartile Range = 0.10575 . G RT-qPCR analysis of IL6 and CCL5 mRNA expression levels in A549 High-M cells treated with 1,6-hexanediol. Biological replicates n = 5, independent experiments, Unpaired t test (Two-tailed); Mean ± SD. H Establish a conditional culture medium experiment: Representative images of TRAP assay analyzing the osteoclast differentiation capacity induced by conditioned medium of A549 cells pre-treated with 1,6-hexanediol ( n = 3, independent experiments), *p < 0.05 ) ( Scale bar: 10 μm ). Biological replicates n = 3, Unpaired t test (Two-tailed); Mean ± SD. The schematic diagram was created by the author using Adobe Illustrator. I Representative images of the activation effect of A549 High-M cell conditioned medium on osteoclasts under IL6 neutralizing antibody (Siltuximab) treatment ( Scale bar: 200 μm ). Biological replicates n = 3, independent experiments, Unpaired t test (Two-tailed) ; Mean ± SD. The schematic diagram was created by the author using Adobe Illustrator.

Article Snippet: For the immunocompetent bone metastasis mouse model, Luci-Lewis lung cancer cells were injected into 4-6-week-old Female C57BL/6 mice (RRID: MGI:7264769) via the tibial metaphysis, with corresponding treatment groups established (e.g., PD-1 mAb, IL6 neutralizing antibody, ICA (MCE, HY-N0678), O-GlcNAcase inhibitor (OGA-i, MCE, HY-12588, etc.).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Luciferase, Reporter Assay, Over Expression, Activity Assay, Immunofluorescence, Whisker Assay, Fluorescence, Quantitative RT-PCR, Expressing, TRAP Assay, Activation Assay

Journal: Nature Communications

Article Title: YBX1 Confers immunosuppressive bone metastatic traits in non-small cell lung cancer

doi: 10.1038/s41467-026-73931-2

Figure Lengend Snippet: A Experimental flowchart: includes tissue collection, multiplex immunofluorescence staining, and automated scanning analysis. The schematic diagram was created by the author using Adobe Illustrator. B Representative multiplex immunofluorescence images showing the infiltration of immune cells in metastatic and non-metastatic tissues ( Scale bar: 2000 μm ). Biological replicates n = 38, patient samples . C Statistical analysis shows the infiltration levels of Treg cells (CD4 + CD25 + FoxP3 + ) and IFN-γ + CD8 + T cells, as well as YBX1 expression levels, in primary tissues from patients without metastasis ( n = 38 ) and with bone metastasis ( n = 38 ). Biological replicates, Unpaired t test (Two-tailed) . For Treg cells: Non-metastasis: n = 38, patient samples, minima = 2.6, maxima = 22.3, center = 11.1, Q1/Lower Quartile = 9.225, Q3/Upper Quartile = 14.45, Lower Whisker = 2.6, Upper Whisker = 22.2875, IQR/Interquartile Range = 5.225; Metastasis: n = 38, patient samples, minima = 4.7, maxima = 29.1, center = 15.85, Q1/Lower Quartile = 10.7, Q3/Upper Quartile = 18.1, Lower Whisker = 4.7, Upper Whisker = 29.1, IQR / Interquartile Range = 7.4; For IFN-γ + CD8 + T cells: Non-metastasis: n = 38, patient samples, minima = 11.8, maxima = 56.5, center = 34, Q1/Lower Quartile = 26.825, Q3/Upper Quartile = 37.575, Lower Whisker = 11.8, Upper Whisker = 53.7, IQR/Interquartile Range = 10.75; Metastasis: n = 38, patient samples, minima = 11.3, maxima = 51.2, center = 21.85, Q1/Lower Quartile = 17.225, Q3/Upper Quartile = 28.975, Lower Whisker = 11.3, Upper Whisker = 46.6, IQR/Interquartile Range = 11.75; For YBX1: Non-metastasis: n = 38,patient samples, minima = 10.8, maxima = 65.2, center = 33.5, Q1/Lower Quartile = 26.95, Q3/Upper Quartile = 39.75, Lower Whisker = 10.8, Upper Whisker = 58.95, IQR/Interquartile Range = 12.8; Metastasis: n = 38, patient samples, minima = 15.3, maxima = 69.2, center = 45.95, Q1/Lower Quartile = 36.15, Q3/Upper Quartile = 53.075, Lower Whisker = 15.3, Upper Whisker = 69.2, IQR/Interquartile Range = 16.925 . The correlation between YBX1 expression and Treg cell infiltration, as well as IFN-γ + CD8⁺ T cell infiltration level. Biological replicates n = 38, patient samples, Pearson’s correlation test (Two-tailed) . D Schematic diagram of mouse model construction and experimental treatments (including sgRNA-mediated YBX1 knockout and immune checkpoint inhibitor therapy). Treatment: IgG/PD-1 mAb 200 µg/mouse (Intraperitoneal injection), ICA 35 mg/kg/day (Intragastric administration). The schematic diagram was created by the author using Adobe Illustrator. E , F Representative flow cytometry plots ( E ) and quantitative analysis ( F ) of the infiltration of IFN-γ + CD8 + T cells and Treg in the tumor microenvironment of bone metastatic lesions. Biological replicates n = 3, mice; One-way ANOVA (multivariate comparison: Tukey) . G Relative levels of IL6 and CCL5 in the serum of mice from different treatment groups. ( n = 5, mice ). Biological replicates n = 5, mice; One-way ANOVA (multivariate comparison: Tukey); Mean ± SD.

Article Snippet: For the immunocompetent bone metastasis mouse model, Luci-Lewis lung cancer cells were injected into 4-6-week-old Female C57BL/6 mice (RRID: MGI:7264769) via the tibial metaphysis, with corresponding treatment groups established (e.g., PD-1 mAb, IL6 neutralizing antibody, ICA (MCE, HY-N0678), O-GlcNAcase inhibitor (OGA-i, MCE, HY-12588, etc.).

Techniques: Multiplex Assay, Immunofluorescence, Staining, Expressing, Two Tailed Test, Whisker Assay, Knock-Out, Injection, Flow Cytometry, Comparison

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: Differential Effects of vIL6 and hIL6 on the frequency and distribution of KSHV infection in B cells. Naïve B cells were isolated from 15 unique tonsils and Mock infected or infected with BAC16‐derived KSHV‐WT or KSHV‐ΔK2. Cultures were treated with IL6 neutralizing antibodies or left untreated and analyzed at 3 dpi for the distribution of KSHV infection within B cell lineages (Table ) using the GFP reporter by flow cytometry. (A) percent of viable CD19 + B lymphocytes that were GFP+ in each condition. Two‐way repeated measures ANOVA p = 0.006, F = 10.6 for Nab treatment, post hoc paired T ‐test p = 0.04 comparing no treatment to IL6 neutralization in KSHV‐WT infection. (B) Percent of GFP+ plasmablasts; paired T ‐test p = 0.05 comparing WT to ∆K2 in untreated cultures (C) percent of GFP+ classical memory cells; Two‐way repeated measures ANOVA p = 0.03, F = 5.7 for Nab treatment, p = 0.03, F = 5.9 for virus strain; paired T ‐test p = 0.04 comparing WT to ∆K2 in untreated cultures. (D) Percent of GFP + IL6 + CD19+ cells; paired T ‐test p = 0.03 comparing WT and ∆K2 infection in the untreated cultures. (E) Correlation between total infection (GFP + CD19 + ) and GFP within plasmablast using pearson's method. (F) Correlation between total infection (GFP + CD19 + ) and GFP within classical memory using pearson's method. (G) lack of correlation between total infection (GFP + CD19 + ) and GFP within IL6 + B cells. (H) Percent of GFP + MZ‐like cells; Two‐way repeated measures ANOVA p = 0.006, F = 10.6 for Nab treatment, post hoc paired T ‐test p = 0.03 comparing no treatment to IL6 neutralization in KSHV‐∆K2 infection. (I) Correlation between total infection (GFP + CD19 + ) and GFP within MZ‐like using pearson's method.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Isolation, Derivative Assay, Flow Cytometry, Neutralization, Virus

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 impacts hIL6 expression in B cell subsets. Naïve B cells were isolated from 15 unique tonsils and Mock infected or infected with BAC16‐derived KSHV‐WT or KSHV‐ΔK2. Cultures were treated with IL6 neutralizing antibodies or left untreated. At 3 dpi supernatants were analyzed for secreted hIL6 and cells were analyzed for hIL6 expression within B cell subsets by ICCS. (A) Frequency of IL6 + B cells. Two‐way repeated measures ANOVA p = 0.002, F = 7.6 for virus strain, post hoc paired T ‐test p = 0.006 comparing KSHV‐WT to ∆K2 in the no treatment condition. (B) Secreted hIL6 showed the same statistical effect as IL6+ within B cells ( p = 0.01). (C) Frequency of IL6+ within B cell subsets with the following significant effects. Plasma cell: Two‐way repeated measures ANOVA p = 0.02, F = 4.4 for virus strain, post hoc paired T ‐test p = 0.05 comparing KSHV‐WT to ∆K2 in the hIL6 neutralized condition. Double negative memory: paired T ‐test p = 0.0 comparing KSHV‐WT to ∆K2 in the no treatment condition. Naive: Two‐way repeated measures ANOVA p = 0.03, F = 3.9 for virus strain, post hoc paired T ‐test p = 0.02 comparing KSHV‐WT to ∆K2 in the no treatment condition. Transitional: Two‐way repeated measures ANOVA p = 0.003, F = 7.3 for virus strain, post hoc paired T ‐test p = 0.04 comparing KSHV‐WT to ∆K2 in the no treatment condition. Germinal Center: Two‐way repeated measures ANOVA p = 0.002, F = 7.7 for virus strain, post hoc paired T ‐tests p = 0.02 comparing KSHV‐WT to ∆K2 in the hIL6 neutralized condition, p = 0.01 comparing KSHV‐WT to ∆K2 in the no treatment condition.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Expressing, Isolation, Infection, Derivative Assay, Virus, Clinical Proteomics

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 and hIL6 differentially alter secretion of TNF‐α, BAFF and IL‐10 during KSHV infection. Supernatants from 11 unique tonsils infected and treated as in experiments described in Figure were harvested at 3 dpi and clarified of cellular debris by centrifugation. Concentrations of 13 cytokines related to B cell activation and differentiation were determined using the Biolegend Legendplex Human B cell panel for each supernatant. Cytokines significantly altered by experimental parameters were (A) BAFF with 2‐way repeated measures ANOVA showing significant interaction of virus infection and hIL‐6 neutralization ( p = 0.03, F = 4.4) and significantly different pairwise comparison via T‐test comparing Mock+IL6 NAb and KSHV‐∆K2 + IL6 Nab ( p = 0.039). (B) TNF‐α with 2‐way repeated measures ANOVA showing significant effect of virus condition ( p = 0.05, F = 3.6) and significant pairwise comparisons via T ‐test comparing Mock and KSHV‐WT ( p = 0.008) and KSHV‐WT with KSHV‐∆K2 ( p = 0.01) with no hIL6 neutralization; (C) IL‐10 with significant pairwise comparisons via T‐test comparing Mock and KSHV‐WT ( p = 0.02) with no hIL6 neutralization.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Centrifugation, Activation Assay, Virus, Neutralization, Comparison

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 and hIL6 cooperatively support B cell viability during KSHV infection but have differential effects on subset frequencies. Naïve B cells were extracted from a distinct set of 15 tonsils and subjected to infection with either Mock, KSHV‐WT, or KSHV‐ΔK2. The cultures were either supplemented with IL6‐neutralizing antibodies or remained untreated. At 3dpi, the cultures were examined by flow cytometry to assess B cell viabilities and the frequencies of B cell subsets. (A) Percent of viable CD19 + . Two‐way repeated measures ANOVA p = 0.01, F = 5.3 for virus strain, p = 0.04, F = 5.5 for hIL6 neutralization and p = 0.03, F = 3.9 for the interaction of the two variables. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.01 comparing no treatment to hIL6 neutralization in Mock cultures, p = 0.01 comparing Mock with KSHV‐WT in untreated cultures and p = 0.004 comparin Mock with ∆K2 in untreated cultures. (B) Frequency of naïve B cells Two‐way repeated measures ANOVA p = 0.05, F = 4.7 for hIL6 neutralization and p = 0.04, F = 3.6 for the interaction of virus and hIL6 neutralization. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.018 comparing Mock to ∆K2 in the no treatment condition and p = 0.01 comparing untreated to hIL6 neutralization in Mock infected cultures. (C) Frequency of germinal center B cells. Two‐way repeated measures ANOVA p = 0.03, F = 4.1 for virus strain. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.019 comparing Mock to ∆K2 in the no treatment condition. (D) Frequency of plasmablasts. Two‐way repeated measures ANOVA p = 0.00006, F = 14.1 for virus strain and p = 0.001, F = 9.0 for the interaction of virus and hIL6 neutralization. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.03 comparing Mock and WT, p = 0.03 comparing Mock and ∆K2 in the no treatment conditions and p = 0.002 comparing Mock and WT, p = 0.04 comparing Mock and ∆K2 and p = 0.004 comparing WT and ∆K2 in the hIL6 neutralized conditions.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Flow Cytometry, Virus, Neutralization

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: hIL6 and vIL6 have separable effects on the differentiation of plasmablast and germinal center B cells from naïve B cells during infection. I B cells loaded with tracking dye before infection and coculture with unlabeled lymphocyte fractions to track differentiation of naïve B cells over time during Mock, KSHV‐WT or KSHV‐∆K2 infection with or without neutralization of hIL6. At 3 dpi cells were harvested and analyzed for B cell subset markers, KSHV infection (GFP+ populations) and differentiation (dye+ populations). (A) proportional distribution of dye+ B cells amongst B cell subsets (B) frequency of undifferentiated naïve B cells. Two‐way repeated measures ANOVA p = 0.02, F = 3.6 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.031 comparing Mock to ∆K2 in the untreated condition. (C) Frequency of GC B cells differentiated from naïve. Two‐way repeated measures ANOVA p = 0.02, F = 3.6 for virus strain, p = 0.03, F = 3.8 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.039 comparing Mock to ∆K2 and p = 0.035 comparing WT to ∆K2 in the untreated conditions. (D) Frequency of plasmablast differentiated from naïve. Two‐way repeated measures ANOVA p = 0.00004, F = 10.2 for virus strain, p = 0.05, F = 3.9 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.012 comparing Mock to ∆K2 and p = 0.014 comparing Mock to WT in the untreated conditions, p = 0.007 comparing Mock to WT and p = 0.011 comparing WT to ∆K2 in the hIL6 neutralized conditions. (E) Correlation between differentiation of GCB and total frequency of GCB using pearson's method. (F) Correlation between differentiation of plasmablast and total frequency of plasmablast via pearson's method. (G) Correlation between differentiation of GCB and IL6 + GCB within each virus condition using pearson's method. (H) Correlation between GCB differentiation and total KSHV infection using pearson's method. (I) Correlation between differentiation of GCB and KSHV infection of plasmablast using pearson's method. (J) Correlation between differentiation of GCB and KSHV infection of classical memory using pearson's method.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Neutralization, Virus

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: KSHV infection drives functional maturation of B cells during ex vivo infection, but vIL6 suppresses differentiation of IgG+ plasma cells during KSHV infection. Naive B cells loaded with tracking dye before infection and coculture with unlabeled lymphocyte fractions to track differentiation of naïve B cells over time during Mock, KSHV‐WT or KSHV‐∆K2 infection with or without neutralization of hIL6. At 3 dpi cells were harvested and analyzed for B cell subset markers including Ig isotype, KSHV infection (GFP+ populations) and differentiation (dye+ populations). (A) Frequency of differentiated IgG+ B cells normalized to each sample's mock value. Two‐way repeated measures ANOVA conducted before data normalization: p = 0.00008, F = 15.4 for virus strain, p = 0.04, F = 5.1 for hIL6 neutralization and p = 0.002, F = 7.4 for interaction of virus strain and hIL6 neutralization. (B) frequency of differentiated IgG+ plasma cells. Paired T ‐test p = 0.03 comparing WT and ∆K2 in the untreated condition. (C) Concentrations of IgG isotypes at 3 dpi were determined in clarified supernatants from eight samples via Legendplex immunoassay. Two‐way repeated measures ANOVA showed significant effect of virus strain on supernatant concentrations of IgG1 ( p = 0.036, F = 6.4) and significant effect of virus strain ( p = 0.04, F = 5.99) and significant interaction of virus strain and IL6 neutralization ( p = 0.04, F = 6) on supernatant concentrations of IgG3.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Functional Assay, Ex Vivo, Clinical Proteomics, Neutralization, Virus