Journal: bioRxiv

Article Title: Wnt/β-catenin signaling promotes zebrafish osteoblast dedifferentiation by wnt10a -mediated inhibition of NF-κB

doi: 10.64898/2025.12.29.696582

Figure Lengend Snippet: A) RNASeq (left) and RT-qPCR (right) reveal upregulation of the NF-κB signaling target genes nfkbiaa and nfkbiab in osteoblasts sorted from bglap:GFP fish heterozygous for the wnt10a mutation relative to their wild-type siblings at 1 dpa. nE (qPCR biological replicates) = 3, nA = 10 per replicate. ΔΔCt values are normalized to the mean of the wildtype at 1 dpa. Error bars, Mean ± SEM. Two tailed Student’s t-test. B) Osteoblast dedifferentiation, as measured by bglap downregulation in segment -1 revealed by HCR in situ hybridization, is inhibited in fish treated with the Wnt inhibitor IWR-1, while IP injection of the NF-κB inhibitor Bay-11 slightly but significantly enhances dedifferentiation. Treatment with both inhibitors yields results similar to those of Bay-11 alone. nE = 3 (except for 2 for IWR-1), nA = 18 total per group (12 for IWR-1), nR = 33 (DMSO), 24 (IWR-1), 37 (Bay-11), 33 (both). C) Overexpression of wnt10a using hs:wnt10a fish is sufficient to cause downregulation of bglapl detected by HCR in situ hybridization in non-injured fins, relative to heat-shocked wild-type fish. HCR signals were quantified in a bony segment (“B”) that is located at the same proximal-distal position as “segment -1” in amputated fins. nE = 2, nA = 12 total per group, nR = 22 total per group. Dashed line, joints. Scale bar, 100 µm. D) Immunofluorescence on cryosections of hs:wnt10a transgenic hearts reveals increased embryonic myosin heavy chain (embMHC) expression in Myl7+ cardiomyocytes at the wound border at 7 days post injury (dpi). Plots show the ventricular area covered by anti-embMHC staining relative to the 150 µm wound border zone area occupied by Myl7+ myocardium. nE = 2, nA = 13 wild-type, 11 hs:wnt10a . Scale bar, 100 µm. (B, C, D) Error bars, mean ± 95% CI. Two tailed Student’s t-test.

Article Snippet: Primary antibodies against embMHC Mouse monoclonal (MYH7 DSHB, N2.261, RRID:AB_531790), and Myl7 Rabbit polyclonal (GeneTex, GTX128346, RRID:AB_2885759) were diluted in PEMTx/normal goad serum and applied overnight at 4 °C.

Techniques: Quantitative RT-PCR, Mutagenesis, Two Tailed Test, In Situ Hybridization, Injection, Over Expression, Immunofluorescence, Transgenic Assay, Expressing, Staining

Journal: Genes & Diseases

Article Title: Druggable target ATAD2 enhances the malignant progression and cooperates with E2F1 to up-regulate PDK1 expression in glioma

doi: 10.1016/j.gendis.2025.101810

Figure Lengend Snippet: The expression of the druggable target ATAD2 in glioma. (A) Visualization of DepMap database-based druggability analysis of CTARS-related proteins using a network Venn diagram. (B) Analysis using the GEPIA database analysis revealed significant upregulation of ATAD2 mRNA expression in glioblastoma (GBM). (C) The UALCAN database analysis showed significant up-regulation of ATAD2 protein in GBM. (D) The expression of ATAD2 mRNA across different clinicopathological characteristics was analyzed in the CGGA cohort. Statistical analyses utilized the Kruskal–Wallis H test followed by post hoc Dunn's test for WHO grade comparisons and the Wilcoxon rank-sum test for other parameters. (E) Representative cases showing immunohistochemical staining across various clinicopathological tissue types of gliomas. Scale bar: 100 μm. (F) Statistical analysis of the IHC scores based on the staining intensity and the percentage of positive cells. Statistical analysis was performed with the Kruskal–Wallis H test and by post hoc Dunn's test. Significance levels are indicated as follows: ns, not significant; ∗, P < 0.05; ∗∗∗∗, P < 0.0001.

Article Snippet: The cells were incubated with an ATAD2 primary antibody (BOSTER, cat# M03855, 1:200), washed with PBS containing 0.1% Tween 20, followed by incubation with a secondary antibody (CoraLite594-Goat Anti-Rabbit, Proteintech, cat# SA00013-4, 1:300).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Genes & Diseases

Article Title: Druggable target ATAD2 enhances the malignant progression and cooperates with E2F1 to up-regulate PDK1 expression in glioma

doi: 10.1016/j.gendis.2025.101810

Figure Lengend Snippet: The impact of ATAD2 on the malignant phenotype of glioma cells. (A) The Western blot analysis shows the differential expression of ATAD2 in a normal astrocyte cell line (HA1800) and various glioma cell lines. (B) The Western blot analysis demonstrates the efficacy of three ATAD2 shRNAs in reducing ATAD2 expression in the LN229 and U251MG cell lines. (C) The Western blot analysis revealed the expression levels of ATAD2 and the FLAG-tagged protein following ATAD2 overexpression in U118MG cells. (D – F) The CCK-8 assay shows changes in cell proliferation after knockdown of ATAD2 in LN229 and U251MG cells, and its overexpression in U118MG cells. (G, H) Colony formation assay and statistical analysis. (I, J) Migration and invasion assays and their statistical analysis. The data are presented as means ± SD. For panels (D – F), two-way ANOVA was performed, while unpaired two-tailed Student's t -tests were used for panels (H, I). Statistical significance is denoted as follows: ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001.

Article Snippet: The cells were incubated with an ATAD2 primary antibody (BOSTER, cat# M03855, 1:200), washed with PBS containing 0.1% Tween 20, followed by incubation with a secondary antibody (CoraLite594-Goat Anti-Rabbit, Proteintech, cat# SA00013-4, 1:300).

Techniques: Western Blot, Quantitative Proteomics, Expressing, Over Expression, CCK-8 Assay, Knockdown, Colony Assay, Migration, Two Tailed Test

Journal: Genes & Diseases

Article Title: Druggable target ATAD2 enhances the malignant progression and cooperates with E2F1 to up-regulate PDK1 expression in glioma

doi: 10.1016/j.gendis.2025.101810

Figure Lengend Snippet: The impact of ATAD2 knockdown on Glioma tumorigenesis in vivo . (A) Gross morphological observations were conducted on subcutaneous tumors induced by LN229 cells in the Shco and Sh-3 groups of nude mice. (B) Statistical analysis of the subcutaneous tumor size measurements. (C) Statistical analysis of the subcutaneous xenograft tumor weights. (D) Representative images of IHC staining for ATAD2 and Ki67. Scale bar:100 μm. (E, F) The Image-Pro Plus software (version 6) was used to compute the sum integrated optical density of ATAD2 and Ki-67 IHC staining, followed by statistical analysis. (G) Representative HE staining images of intracranial orthotopic xenografts formed by LN229 cells in the Shco and Sh-3 groups. Scale bar: 1 mm. (H) Statistical analysis of intracranial orthotopic tumor size measurements. (I) Kaplan–Meier survival curve of nude mice with orthotopic intracranial tumors (Log-rank test). The data are presented as means ± SD, two-tailed paired Student's t -test was used to compare the results in (B), (C), (E), and (F); and two-tailed unpaired Student's t -test was used to compare the results in (H). Each group included n = 6 mice. Statistical significance is denoted as follows: ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001.

Article Snippet: The cells were incubated with an ATAD2 primary antibody (BOSTER, cat# M03855, 1:200), washed with PBS containing 0.1% Tween 20, followed by incubation with a secondary antibody (CoraLite594-Goat Anti-Rabbit, Proteintech, cat# SA00013-4, 1:300).

Techniques: Knockdown, In Vivo, Immunohistochemistry, Software, Staining, Two Tailed Test

Journal: Genes & Diseases

Article Title: Druggable target ATAD2 enhances the malignant progression and cooperates with E2F1 to up-regulate PDK1 expression in glioma

doi: 10.1016/j.gendis.2025.101810

Figure Lengend Snippet: ATAD2-E2F1 positive feedback loop regulates the expression of PDK1. (A) Volcano plot of differentially expressed genes from RNA-seq analysis of ATAD2 knockdown and control conditions in LN229 cells. (B) Volcano plot of differentially expressed proteins from proteome analysis of ATAD2 knockdown and control conditions in LN229 cells. (C) The Venn diagram illustrates the overlap between the up-regulated and down-regulated mRNAs and proteins. (D) The fold change ranking plot illustrates 20 commonly down-regulated proteins. (E, F) Western blot analysis confirmed that ATAD2 enhances the expression of PDK1. (G, H) Western blot analysis confirmed that E2F1 up-regulates the expression of ATAD2. (I–K) Western blot analysis validated that E2F1 enhances the expression of ATAD2. (L, M) Western blot analysis showed that ATAD2 cooperates with E2F1 to up-regulate the expression of PDK1. (N) The dual luciferase reporter gene assays revealed that ATAD2 and E2F1 synergistically enhance the promoter activity of PDK1. The data are presented as means ± SD. Statistical comparisons were performed using unpaired two-tailed Student's t -test for figures (F), (H), and (K), and one-way ANOVA followed by Tukey's post hoc test for figures (J), (M), and (N). Statistical significance is denoted as follows: ns, not significant; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001.

Article Snippet: The cells were incubated with an ATAD2 primary antibody (BOSTER, cat# M03855, 1:200), washed with PBS containing 0.1% Tween 20, followed by incubation with a secondary antibody (CoraLite594-Goat Anti-Rabbit, Proteintech, cat# SA00013-4, 1:300).

Techniques: Expressing, RNA Sequencing, Knockdown, Control, Western Blot, Luciferase, Activity Assay, Two Tailed Test

Journal: Genes & Diseases

Article Title: Druggable target ATAD2 enhances the malignant progression and cooperates with E2F1 to up-regulate PDK1 expression in glioma

doi: 10.1016/j.gendis.2025.101810

Figure Lengend Snippet: The clinical significance of the ATAD2-E2F1-PDK1 axis in glioma. (A, B) Analysis using the GlioVis database showed a positive correlation between the expressions of ATAD2, E2F1, and PDK1, as determined by the Spearman correlation test. (C) Representative cases showed the expression of E2F1 and PDK1 in glioma clinical specimens with low and high expression of ATAD2. Scale bar: 100 μm. (D) Spearman's correlation analysis of ATAD2, E2F1, and PDK1 expression levels in glioma clinical specimens. (E) Kaplan–Meier analysis and log-rank test of survival rates across distinct co-expression groups of ATAD2, E2F1, and PDK1 in the CGGA cohort. P values were adjusted for multiple group comparisons using the Bonferroni method. (F) Graphical diagram illustrating that ATAD2 promotes glioma progression and synergizes with E2F1 to increase PDK1 expression. The figure was created using Figdraw ( www.figdraw.com ). Statistical significance is denoted as follows: ∗∗∗, P < 0.001.

Article Snippet: The cells were incubated with an ATAD2 primary antibody (BOSTER, cat# M03855, 1:200), washed with PBS containing 0.1% Tween 20, followed by incubation with a secondary antibody (CoraLite594-Goat Anti-Rabbit, Proteintech, cat# SA00013-4, 1:300).

Techniques: Expressing

Journal: Translational Cancer Research

Article Title: OX40L and IL-2 combination strategy for gastric cancer immunotherapy

doi: 10.21037/tcr-2025-707

Figure Lengend Snippet: Immunohistochemical and flow cytometric analysis of gastric cancer tissue. (A) H&E histochemical staining of tumor-free area (left), tumor margin (middle), and gastric tumor site (right). (B) Representative IHC images for CD3 + , CD4 + , CD8 + , FOXP3, OX40, and OX40L in tumor-free area (left column), tumor margin (middle column), and gastric tumor site (right column) sections. (C) Immunofluorescence staining for the co-expression of CD3 + and OX40 in gastric cancer with anti-CD3 and anti-OX40 antibodies and DAPI for nuclear staining. (D) Flow cytometry analysis of OX40 expression in CD3 cells obtained from PBMC of gastric cancer patients (upper panel) and OX40 expression on CD3 on TILs in gastric cancer patients (low panel). Statistical analysis of all data were performed using one-way ANOVA. Data are shown as mean ± standard deviation. ns, nonsignificant; *, P<0.05; ***, P<0.001. ANOVA, analysis of variance; DAPI, 4',6-diamidino-2-phenylindole; H&E, hematoxylin & eosin; IHC, immunohistochemistry; OX40L, OX40 ligand; PBMC, peripheral blood mononuclear cells; TILs, tumor-infiltrating lymphocytes.

Article Snippet: Immunohistochemistry (IHC) analyses were performed using specific primary rabbit anti-human CD3 monoclonal antibody (Proteintech Group, Wuhan, China), mouse anti-human OX40 and rabbit anti-human OX40L monoclonal antibodies (CST, Danvers, MA, USA), and rabbit anti-human CD4 (Proteintech Group, Wuhan, China), mouse anti-human CD8 (Proteintech Group, Wuhan, China) antibody, rabbit anti-human FOXP3 monoclonal antibodies (CST, Danvers, MA, USA).

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Expressing, Flow Cytometry, Standard Deviation, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: Determination of the interaction affinity between GM1 and APOE. A , schematic illustration of determining the binding affinity between lipid structure and APOE using MST. B , the binding affinity (K D ) between APOE3 or APOE4 and lipid structures containing GM1 or cholesterol (n = 3). C , the binding affinity between APOE3 or APOE4 and lipid structures with different GM1 concentrations. (n = 3). D - H , negative staining images of lipid structures with varying GM1 concentrations. p value: ns (0.05 < p ≤ 1), ∗ (0.01 < p ≤ 0.05, ∗∗ (0.001 < p ≤ 0.01, ∗∗∗ (0.0001 < p ≤ 0.001, ∗∗∗∗ ( p ≤ 0.0001). ( A ) is created with BioRender.com . APOE, apolipoprotein E; MST, microscale thermophoresis.

Article Snippet: We then blocked the cells with 2% bovine serum albumin for 30 min. Next, we added primary APOE recombinant rabbit monoclonal antibody (diluted 1:1000 in 2% bovine serum albumin, Invitrogen) and incubated it for 2 h at room temperature.

Techniques: Binding Assay, Negative Staining, Microscale Thermophoresis

Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: Analysis of GM1 localization and levels following the cellular uptake of lipid structures. A , schematic illustration of the method used to determine GM1 following the cellular uptake. B , GM1 levels in differentiated PC-12 cells following cellular uptake, determined using the SpectraMax i3 or by quantifying confocal microscopy images. (n ≥ 3) ( C ) representative images showing GM1 and APOE localization in differentiated PC-12 cells following cellular uptake, as determined by confocal microscopy. (n ≥ 6) ( D ) GM1 levels in HEK-293 cells following cellular uptake, determined using the SpectraMax i3 or by quantifying confocal microscopy images. (n ≥ 6) ( E ) (1) cellular uptake of DiD-labeled cholesterol and GM1 lipid structures in U-87 MG cells, and (2) changes in cholesterol levels in U-87 MG cells after cellular uptake of APOE3 and APOE4-enriched cholesterol lipoproteins, as well as a mixture of APOE3 and APOE4-enriched cholesterol and GM1 lipoproteins. (n ≥ 6). p value: ns (0.05 < p ≤ 1), ∗ (0.01 < p ≤ 0.05, ∗∗ (0.001 < p ≤ 0.01, ∗∗∗ (0.0001 < p ≤ 0.001, ∗∗∗∗ ( p ≤ 0.0001). ( A ) is created with BioRender.com . Created in BioRender. Dokholyan, N. (2025) https://BioRender.com/g17z389 . APOE, apolipoprotein E; HEK, human embryonic kidney; PC, l -α-phosphatidylcholine.

Article Snippet: We then blocked the cells with 2% bovine serum albumin for 30 min. Next, we added primary APOE recombinant rabbit monoclonal antibody (diluted 1:1000 in 2% bovine serum albumin, Invitrogen) and incubated it for 2 h at room temperature.

Techniques: Confocal Microscopy, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: Western blot results of APOE receptors: LDLR, VLDLR, LRP1, and ApoER2 on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells. A , the expression of LDLR on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n = 4). B , the expression of VLDLR on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n = 4). C , the expression of LRP1 on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n = 3). D , the expression of ApoER2 on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n = 3). E , schematic illustration of determining the changes of APOE secondary structures. F , the CD results of APOE 3 under the effect of different GM1 content on the lipid structures (n ≥ 3). G , the CD results of APOE 4 under the effect of different GM1 content on the lipid structures (n = 3). p value: ns (0.05 < p ≤ 1), ∗ (0.01 < p ≤ 0.05, ∗∗ (0.001 < p ≤ 0.01, ∗∗∗ (0.0001 < p ≤ 0.001, ∗∗∗∗ ( p ≤ 0.0001). ( E ) is created with BioRender.com . APOE, apolipoprotein E; ApoER2, APOE receptor 2; HEK, human embryonic kidney; LDLR, low-density lipoprotein receptor; LRP1, LRPR-related protein 1; PC, l -α-phosphatidylcholine; VLDLR, very low-density lipoprotein receptor.

Article Snippet: We then blocked the cells with 2% bovine serum albumin for 30 min. Next, we added primary APOE recombinant rabbit monoclonal antibody (diluted 1:1000 in 2% bovine serum albumin, Invitrogen) and incubated it for 2 h at room temperature.

Techniques: Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: The binding affinity of APOE-enriched lipoprotein and APOE receptor LDLR using MST. A and B , the binding affinity between APOE3-enriched lipoprotein with varying GM1 concentration to LDLR. (n = 3) ( C and D ) the binding affinity between APOE4-enriched lipoprotein varying GM1 concentration to LDLR (n = 3). p value: ns (0.05 < p ≤ 1), ∗ (0.01 < p ≤ 0.05, ∗∗ (0.001 < p ≤ 0.01, ∗∗∗ (0.0001 < p ≤ 0.001, ∗∗∗∗ ( p ≤ 0.0001). APOE, apolipoprotein E; LDLR, low-density lipoprotein receptor; MST, microscale thermophoresis.

Article Snippet: We then blocked the cells with 2% bovine serum albumin for 30 min. Next, we added primary APOE recombinant rabbit monoclonal antibody (diluted 1:1000 in 2% bovine serum albumin, Invitrogen) and incubated it for 2 h at room temperature.

Techniques: Binding Assay, Concentration Assay, Microscale Thermophoresis