Fig 2: OLFM4 loss leads to abnormal SC dedifferentiation and proliferation by activating MEK/ERK.Bubble chart of KEGG database enrichment analysis for iBAT RNA sequencing in Ctrl and Olfm4CKO mice (n = 3 mice per group). Statistical analysis was performed using a one-sided hypergeometric distribution test with Benjamini–Hochberg false discovery rate (FDR) correction for multiple comparisons (A). Immunoblot (Top) and statistical analysis (Bottom) of p-ERK in iBAT (n = 6 mice per group) (B). (Left) Representative FACS plots of SOX10+/P-ERK+ cells isolated from BAT. (Right) Percentage of SOX10+/P-ERK+ cells in BAT, analyzed in FlowJo_v10.8.1 (n = 3 mice per group) (C). Fluorescence localization (Left) and statistical analysis (Right) of p-ERK and SOX10 in iBAT (n = 6 mice per group) (D). Immunoblot (Top) and statistical analysis (Bottom) of EGR1 and SOX10 in iBAT (n = 6 mice per group) (E). Fluorescence localization (Left) and statistical analysis (Right) of EGR1 and SOX10 in iBAT (scale bar, 20 µm). (Left) Representative FACS plots of SOX10+/EGR1+ cells isolated from BAT. (Right) Percentage of SOX10+/EGR1+ cells in BAT, analyzed in FlowJo_v10.8.1 (n = 3 mice per group) (F). Fluorescence localization (Left) and statistical analysis (Right) of EGR1 and SOX10 in iBAT (scale bar, 10 µm) (n = 6 mice per group) (G). Co-culture pattern diagram of primary adipocytes and primary SCs (H). Immunoblot (Left) and statistical analysis (Right) of EGR1 and Cyclin D1 in primary SCs (n = 4 independent biological replicates) (I). Fluorescence localization (Left) and statistical analysis (Right) of EGR1 and SOX10 in primary SCs (scale bar, 40 µm) (n = 6 independent biological replicates) (J). (Left) Analysis of proliferation in primary SCs and labelled with EdU (scale bar, 40 µm and 20 µm). (Right) The quantity of Edu positive cells in per field, analyzed in ImageJ software (n = 6 independent biological replicates) (K). All Data are mean ± s.e.m. of biologically independent samples. Statistical analysis was performed using two-tailed unpaired Student’s t-test (B–G, I–K). All experiments were independently repeated at least three times with similar results. Source data are provided as a Source Data file.

Fig 3: The MEK/ERK signaling pathway mediates temperature-induced SC remodeling.Mice were randomly assigned to thermoneutral (TN) housing, standard environment (SE) or restoration of standard environment (RSE). The SE group as a control (A). Immunoblot (Left) and statistical analysis (Right) of P0, Periaxin, MBP, and CD44 in iBAT (n = 3 mice per group) (B). Immunoblot (Top) and statistical analysis (Bottom) of TH and CGRP in iBAT (n = 3 mice per group) (C). Immunoblot (Top) and statistical analysis (Bottom) of EGR1 and SOX10 in iBAT (n = 6 mice per group) (D). Fluorescence localization (Left) and relative immunofluorescence intensity of SOX10-positive or SOX10-negative cells (against DAPI) statistical analysis (Right) of EGR1 in iBAT (scale bar, 20 µm) (n = 6 mice per group) (E). Fluorescence localization (Left) and relative immunofluorescence intensity statistical analysis (Right) of P0 (F) and CD44 (G) (against DAPI) in iBAT (scale bar, 10 μm) (n = 6 mice per group). Immunoblot (Top) and statistical analysis (Bottom) of p-ERK in iBAT (H). Fluorescence localization (Left) and relative immunofluorescence intensity of SOX10-positive or SOX10-negative cells (against DAPI) statistical analysis (Right) of p-ERK in iBAT (scale bar, 10 µm) (n = 6 mice per group) (I). Immunoblot (Left) and statistical analysis (Right) of P0, Periaxin, MBP, and CD44 in iBAT (n = 3 mice per group) ( J). Immunoblot (Left) and statistical analysis (Right) of EGR1 and SOX10 in iBAT (n = 6 mice per group) (K). Immunoblot (Left) and statistical analysis (Right) of TH and CGRP in iBAT (n = 3) (L). All Data are mean ± s.e.m. of biologically independent samples. Statistical analysis was performed using two-tailed unpaired Student’s t-test (B, C) and one-way ANOVA (two-sided) with Tukey post hoc test for pairwise comparisons (D−L). All experiments were independently repeated at least three times with similar results. Source data are provided as a Source Data file.

Fig 4: Elevated expression of nuclear EGR1 in tumors that were poor-responders to bevacizumab therapy. (a,b) Sections were reacted with rabbit anti-EGR1 antibody, as described in the Methods. (a) Box and dot plot depicting nuclear EGR1 expression in tumors that were poor-responders to bevacizumab (five mouse tumors from two PDXs-G64 and G108), and in tumors that were good-responders to bevacizumab (five mouse tumors from two PDXs-G39 and G59). Mouse ids are color-labeled. H-scores were determined for each sample as described in the Methods. Statistical analysis: linear mixed model (p = 0.02). (b) A representative image is shown of immunohistochemical staining for EGR1 in a tumor that was a poor-responder to bevacizumab and in a tumor that was a good-responder to bevacizumab. Arrows denote EGR1+ nuclei. Scale bar denotes 40 μm. (c,d) Sections were reacted with rat mAb anti-EGR1 antibody, as described in the Methods. (c) Box and dot plot depicts nuclear EGR1 expression in tumors that were poor-responders to bevacizumab (five mouse tumors from two PDXs-G64 and G108), and nuclear EGR1 expression in tumors that were good-responders to bevacizumab (five mouse tumors from two PDXs-G39 and G59). Mouse ids are color-labeled. Statistical analysis: linear mixed model (p = 0.03). (d) A representative image is shown of immunofluorescence for nuclear EGR1 in a tumor that was a poor-responder to bevacizumab and in a tumor that was a good-responder to bevacizumab. Arrows denote EGR1+ nuclei. Scale bar denotes 20 μm. (e) Primary cells from PDX tumors were propagated as neurospheres in neural basal media with EGF and bFGF8 and harvested for flow cytometry. Cells were stained with a live/dead marker (LIVE/DEAD™ Fixable Violet Kit, Thermo # L34955) and then fixed and permeabilized (True Nuclear Buffer Set, BioLegend # 424401), and stained with Alexa Fluor® 647 anti-EGR1 Antibody (BioLegend # 943906), followed by analysis on a BD Fortessa (BD Biosciences) flow cytometer. A violin plot is shown. Data were analyzed using the FlowJo (v10.10.0) software, as described in the Methods. Mean EGR1 fluorescence intensity in PDX tumors that were poor-responders (897.9) as compared to the mean fluorescent intensity in PDX tumors that were good-responders (673.0) to bevacizumab therapy. Statistical analysis: Student’s two-sided T-Test (p < 0.001).

Fig 5: Fisetin eliminates senescent B cells and enhances the efficacy of ICB therapy(A) The schematic of anti-aging drug screening.(B) PCA plot of senescence-related markers (left) and statistical analysis (right). Data were normalized to the solvent control using the formula (Value_exp – Value_control)/Value_control, where “Value” refers to either MFI obtained from flow cytometry or cytokine concentrations measured by ELISA. Data are presented as box-and-whisker plots showing the median (center line), interquartile range (box), and 1.5× interquartile range (whiskers). Each dot represents an individual biological sample. Statistical significance was assessed using a Wilcoxon test (∗∗p < 0.001, ∗p < 0.05).(C) Heatmap of senescence-related markers, shown as Z score normalized values.(D) Representative flow cytometry plots and statistical analysis of senescence markers after Fisetin treatment with different concentrations.(E) Schematic representation of the experimental design for C57BL/6 mice treated with anti-PD-1 antibodies, Fisetin, or the combination.(F) Representative esophageal tumors from 4-NQO-treated C57BL/6 mice across four treatment groups (IgG control, Fisetin, anti-PD-1, and combination of anti-PD-1 with Fisetin), with corresponding statistical analysis of tumor burden.(G) Representative H&E staining of esophageal tumors from four different groups.(H) Representative flow cytometric plots depict the immunosuppressive phenotype of EGR1+ B cells and TREM2+ TAMs.(I) Quantification of tumor burden across treatment groups. Data are presented as box-and-whisker plots. Each dot represents an individual biological sample (n = 6 per group). Statistical significance was assessed using a Wilcoxon test.(J) Quantification of EGR1⁺ B cells and TREM2⁺ TAMs. Data are presented as box-and-whisker plots. Each dot represents an individual biological sample (n = 4 per group). Statistical significance was assessed using a Wilcoxon test.(K) B cells, either pretreated with Fisetin or untreated, were adoptively transferred into Ighm−/− mice, followed by subcutaneous ESCC tumor implantation. Mice were subsequently treated with either IgG control or anti-PD-1 antibodies.(L) Tumor weights measured at the experimental endpoint.(M) Quantification of tumor weights. Data are presented as box-and-whisker plots as defined above. Statistical significance was assessed using a two-tailed t test (n = 5).(N) Tumor growth curves depict changes in tumor volume over time for each treatment group (two-tailed t test, n = 5 per group).(O) Representative flow cytometric plots depict the immunosuppressive phenotype of TREM2+ TAMs.(P) Quantification of TREM2⁺ TAMs. Data are presented as box-and-whisker plots as defined above. Each dot represents an individual biological sample (n = 4 per group).Statistical significance was assessed using a two-tailed t test.