Journal: Clinical Cancer Research

Article Title: E7386 Enhances Lenvatinib’s Antitumor Activity in Preclinical Models and Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.CCR-25-0725

Figure Lengend Snippet: E7386 induces ISR and ATF4 upregulation. A, Heatmap representation of gene signatures of pathways involving ATF4 and CBP partners in the in vivo model. Statistics: Welch t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. B, Boxplot depicting the quantification of ATF4-positive IHC staining in the murine model ( n = 4–5/arm; top), with representative images (bottom). Scale bar, 250 μm. Statistics: Kruskal–Wallis test followed by the Dunn test adjusted by the Benjamini–Hochberg test; *, P < 0.05. Error bars indicate SD. C, Heatmap representation of gene signatures associated with the ATF4 pathway in HCC cell lines treated with E7386 0.1 μmol/L or DMSO for 24 hours: sensitive to E7386 (top in green) and resistant to E7386 (bottom in red). Statistics: Student t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. D, Representative Western blot analysis and quantification of ATF4 in E7386-treated sensitive (green) and resistant (red) cell lines treated with E7386 at 0.1 μmol/L or DMSO for 24 hours. Vinculin was used for normalization. FC was calculated based on protein levels in DMSO-treated cells. Values represent the average of two independent experiments ( n = 2). Molecular weight ladder markers are displayed beside the protein of interest. Error bars indicate SD. Statistics: One-way Student t test; *, P < 0.05; **, P < 0.01. Statistically significant P values ( P < 0.05) are shown in bold and trends ( P < 0.1) are shown in italics.

Article Snippet: Samples were incubated for 2 hours at room temperature with the anti-CD31 antibody from Abcam (ab28364; RRID: AB_726362; 1:50) or overnight with the anti-ATF4 antibody from Novus Biologicals (SD20-32; RRID: AB_2877169; 1:200) and the anti-CHOP antibody (15204-1-AP; RRID: AB_2292610; 1:200), the anti-CHAC1 antibody (15207-1-AP; RRID: AB_2878118; 1:500), the anti-REDD1 antibody (10638-1-AP; RRID: AB_2245711; 1:500), and the anti-geminin antibody (10802-1-AP; RRID: AB_2110945; 1:200) from Proteintech.

Techniques: In Vivo, Immunohistochemistry, Western Blot, Molecular Weight

Journal: Clinical Cancer Research

Article Title: E7386 Enhances Lenvatinib’s Antitumor Activity in Preclinical Models and Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.CCR-25-0725

Figure Lengend Snippet: E7386 induces ISR via GCN2/eIF2α activation. A, Heatmap representation of gene pathways associated with ISR in the in vivo model. Statistics: Welch t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. B, Heatmap representation of gene pathways associated with ISR in HCC cell lines: sensitive to E7386 (top in green) and resistant to E7386 (bottom in red) treated with E7386 0.1 μmol/L or DMSO for 24 hours. Statistics: Student t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. C, Representative Western blot analysis of ATF4 levels following siRNA-mediated knockdown of EIF2AK1 , EIF2AK2 , EIF2AK3 , EIF2AK4 , and ATF4 with or without E7386 treatment (0.3 μmol/L for 3 hours). β-Actin was used as a loading control. D, Representative Western blot analysis of the ISR pathway–related proteins in Hep3B cells treated with DMSO or increasing concentrations of E7386 (0.03–1 μmol/L). β-Actin was used as a loading control. Molecular weight ladder markers are displayed beside the protein of interest. Statistically significant P values ( P < 0.05) are shown in bold and trends ( P < 0.1) are shown in italics.

Article Snippet: Samples were incubated for 2 hours at room temperature with the anti-CD31 antibody from Abcam (ab28364; RRID: AB_726362; 1:50) or overnight with the anti-ATF4 antibody from Novus Biologicals (SD20-32; RRID: AB_2877169; 1:200) and the anti-CHOP antibody (15204-1-AP; RRID: AB_2292610; 1:200), the anti-CHAC1 antibody (15207-1-AP; RRID: AB_2878118; 1:500), the anti-REDD1 antibody (10638-1-AP; RRID: AB_2245711; 1:500), and the anti-geminin antibody (10802-1-AP; RRID: AB_2110945; 1:200) from Proteintech.

Techniques: Activation Assay, In Vivo, Western Blot, Knockdown, Control, Molecular Weight

Journal: Clinical Cancer Research

Article Title: E7386 Enhances Lenvatinib’s Antitumor Activity in Preclinical Models and Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.CCR-25-0725

Figure Lengend Snippet: ATF4 targets altered by E7386 treatment in sensitive cell lines. A, Heatmap representation of genes associated with ISR, endoplasmic reticulum stress, amino acid synthesis, tRNA synthetases, cell cycle, and angiogenesis in HCC cell lines: sensitive to E7386 (in green) and resistant to E7386 (in red) treated with E7386 and DMSO. FC was corrected based on DMSO values. Only FC ≥ 1.5 (for overexpression) or FC ≤ 0.8 (for downregulation) was included. Statistics: Student t test for two group comparisons. B, Representative Western blot analysis of cyclin D1, cyclin D2, and geminin in Hep3B and SNU398 cell lines treated with E7386 and DMSO. Vinculin was used as a loading control. Quantification of the Western blot is provided in Supplementary Fig. S3. Molecular weight ladder markers are displayed beside the protein of interest. C, Quantification of VEGF secretion from Hep3B and SNU398 cells treated with DMSO, E7386, lenvatinib, or a combination of E7386 with lenvatinib. Statistics: Kruskal–Wallis test followed by the Dunn test adjusted by the Benjamini–Hochberg test; *, P < 0.05; **, P < 0.01. Error bars indicate the SD of the average from at least two independent experiments and technical duplicates. D, Heatmap representation of gene signatures associated with angiogenesis in the in vivo model. Statistics: Welch t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. E, Box plot depicting the quantification of CD31-positive IHC staining in the murine model ( n = 4–5/arm; left), with representative images (right). Scale bar, 250 μm. Statistics: Kruskal–Wallis test followed by the Dunn test adjusted by the Benjamini–Hochberg; *, P < 0.05. Error bars indicate SD. Statistically significant P values ( P < 0.05) are shown in bold and trends ( P < 0.1) are shown in italics.

Article Snippet: Samples were incubated for 2 hours at room temperature with the anti-CD31 antibody from Abcam (ab28364; RRID: AB_726362; 1:50) or overnight with the anti-ATF4 antibody from Novus Biologicals (SD20-32; RRID: AB_2877169; 1:200) and the anti-CHOP antibody (15204-1-AP; RRID: AB_2292610; 1:200), the anti-CHAC1 antibody (15207-1-AP; RRID: AB_2878118; 1:500), the anti-REDD1 antibody (10638-1-AP; RRID: AB_2245711; 1:500), and the anti-geminin antibody (10802-1-AP; RRID: AB_2110945; 1:200) from Proteintech.

Techniques: Over Expression, Western Blot, Control, Molecular Weight, In Vivo, Immunohistochemistry

Journal: Clinical Cancer Research

Article Title: E7386 Enhances Lenvatinib’s Antitumor Activity in Preclinical Models and Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.CCR-25-0725

Figure Lengend Snippet: E7386 and lenvatinib induce ATF4 upregulation and antiangiogenic effects in patients with uHCC treated in the context of a phase Ib/II clinical trial. A, Clinical trial schematic overview and time points for obtaining samples for RNA sequencing. Overall, seven matched cases pre- and on-treatment were included in the transcriptomic analysis. B, Heatmap representation of gene signatures associated with the ATF4 pathway and angiogenesis in matched patient samples pre- and on-treatment with the combination of E7386 and lenvatinib. Patients V and VII, highlighted in a red box, carry gain-of-function missense mutations in CTNNB1 , whereas the rest are WT. Patient VII also harbors an AXIN1 mutation. For on-treatment samples, the percentage of MTS is indicated on the red–green scale. Statistics: Student t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. QD, once a day; BID, twice a day. Statistically significant P values ( P < 0.05) are shown in bold and trends ( P < 0.1) are shown in italics.

Article Snippet: Samples were incubated for 2 hours at room temperature with the anti-CD31 antibody from Abcam (ab28364; RRID: AB_726362; 1:50) or overnight with the anti-ATF4 antibody from Novus Biologicals (SD20-32; RRID: AB_2877169; 1:200) and the anti-CHOP antibody (15204-1-AP; RRID: AB_2292610; 1:200), the anti-CHAC1 antibody (15207-1-AP; RRID: AB_2878118; 1:500), the anti-REDD1 antibody (10638-1-AP; RRID: AB_2245711; 1:500), and the anti-geminin antibody (10802-1-AP; RRID: AB_2110945; 1:200) from Proteintech.

Techniques: RNA Sequencing, Mutagenesis

Journal: Clinical Cancer Research

Article Title: E7386 Enhances Lenvatinib’s Antitumor Activity in Preclinical Models and Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.CCR-25-0725

Figure Lengend Snippet: Proposed novel mechanism of action for E7386. E7386 treatment triggers ATF4 overexpression and ATF4-dependent ISR, along with downregulation of cell-cycle proteins, VEGFA upregulation, and secretion. Concomitant treatment of E7386 with lenvatinib reverses ATF4/VEGFA-related angiogenesis and potentiates antitumor responses. (Created with BioRender.com. Llovet, J. [2025] https://BioRender.com/io86bef .)

Article Snippet: Samples were incubated for 2 hours at room temperature with the anti-CD31 antibody from Abcam (ab28364; RRID: AB_726362; 1:50) or overnight with the anti-ATF4 antibody from Novus Biologicals (SD20-32; RRID: AB_2877169; 1:200) and the anti-CHOP antibody (15204-1-AP; RRID: AB_2292610; 1:200), the anti-CHAC1 antibody (15207-1-AP; RRID: AB_2878118; 1:500), the anti-REDD1 antibody (10638-1-AP; RRID: AB_2245711; 1:500), and the anti-geminin antibody (10802-1-AP; RRID: AB_2110945; 1:200) from Proteintech.

Techniques: Over Expression

Journal: Biomaterials Research

Article Title: Versatile Nanotherapeutics for Enhancing Sonodynamic Therapy/Chemotherapy of Thyroid Cancer through Remodeling Tumor Microenvironment and Synergistic Reactive Oxygen Species Augment

doi: 10.34133/bmr.0338

Figure Lengend Snippet: FL@M enhances oxidative stress and induces apoptosis. (A) ROS production in 8505C cells after different treatments was detected by DCFH-DA. (B) Quantification of intracellular fluorescence intensity of DCFH-DA shown in (A). (C) Lipid oxidation levels in 8505C cells after different treatments were detected by BODIPY. (D) Quantification of intracellular fluorescence intensity of lipid oxidation level shown in (C). (E) The changes in the MMP of 8505C cells after different treatments were detected by JC-1. (F) Quantification of intracellular fluorescence intensity of MMP shown in (E). (G) Flow cytometry was used to assess ROS production after different treatments detected by DCFH-DA. (H) Flow cytometry was used to assess lipid oxidation levels after different treatments detected by BODIPY. (I) Flow cytometry was used to assess MMP after different treatments detected by JC-1. (J) Apoptosis of 8505C cells was assessed by flow cytometry with Annexin V-PE/7AAD staining. (K) Western blotting analysis of GPX4, MMP2, MMP9, MMP1, and E-Cad protein in 8505C cells with various treatments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The primary antibodies used in this study were as follows: GPX4 (ab125066, Abcam), MMP2 (10373-2-AP, Proteintech), MMP9 (10375-2-AP, Proteintech), MMP1 (10371-2-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), HIF-1α (20960-1-AP, Proteintech), Galectin-3 (82024-1-RR, Proteintech), Vinculin (A2752, ABclonal), and ACTB (AC026, ABclonal).

Techniques: Fluorescence, Flow Cytometry, Staining, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Coq4 deficiency induces placental vascular development defects through FSP1/CoQ10 axis-mediated endothelial ferroptosis

doi: 10.3389/fcell.2026.1774201

Figure Lengend Snippet: Coq4 −/− mice exhibit impaired placental vascular development. (A) Light microscopic image of E9.5 placenta (2×),n = 6. (B) Immunofluorescence staining of placenta from WT and Coq4 −/− mouse with DAPI (blue), CD34 (red), and Coq4 (green). Scale bar = 750 μm, n = 6.

Article Snippet: Primary antibody including Coq4 antibody (1:200, Proteintech, Cat: 16654-1-AP), ACSL4 antibody (1:200, Zenbio, Cat: R24265 ) and CD34 antibody (1:200, Proteintech, Cat: 14486-1-AP).

Techniques: Immunofluorescence, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Coq4 deficiency induces placental vascular development defects through FSP1/CoQ10 axis-mediated endothelial ferroptosis

doi: 10.3389/fcell.2026.1774201

Figure Lengend Snippet: Coq4 deficiency suppresses vascular endothelial cell migration and angiogenesis. (A) Established stably transfected HUVEC cell lines with shNc and shCoq4, and the expression levels of Coq4 were detected by Western blotting, compared to shNc group, n = 3. (B) Cell scratch assay for detecting cell migration capacity and quantitative analysis, compared to shNc group, n = 3; (C) Matrigel tube formation assay for measuring angiogenesis capacity and quantitative analysis, compared to shNc group, n = 3.

Article Snippet: Primary antibody including Coq4 antibody (1:200, Proteintech, Cat: 16654-1-AP), ACSL4 antibody (1:200, Zenbio, Cat: R24265 ) and CD34 antibody (1:200, Proteintech, Cat: 14486-1-AP).

Techniques: Migration, Stable Transfection, Transfection, Expressing, Western Blot, Wound Healing Assay, Tube Formation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Coq4 deficiency induces placental vascular development defects through FSP1/CoQ10 axis-mediated endothelial ferroptosis

doi: 10.3389/fcell.2026.1774201

Figure Lengend Snippet: Transcriptome analysis of HUVECs with stabilized knockdown of Coq4. (A) Pearson correlation heatmap, where x-axis and y-axis represent the square of the correlation coefficient between samples. (B) Principal Component Analysis (PCA) plot, where x-axis represents the first principal component (54.1%) and y-axis represents the second principal component (15.4%). (C) Volcano plot of differentially expressed genes. The x-axis represents log2FoldChange, and the y-axis represents -log10 (padj). The two vertical dashed lines indicate the 2-fold expression difference threshold; the horizontal dashed line indicates the padj = 0.05 threshold. Red dots indicate genes upregulated in this combination, blue dots indicate genes downregulated in this group, and gray dots indicate genes with non-significant expression differences. (D) Top 20 significantly enriched pathways from KEGG analysis of differentially expressed genes, categorized by level 1 pathways. The x-axis shows the number of differentially expressed genes enriched in each pathway, while the y-axis displays different KEGG pathway names. (E) Significantly upregulated pathways from KEGG analysis of differentially expressed genes. (F) Significantly differentially expressed genes enriched in ferroptosis pathways. The x-axis represents samples, and the y-axis represents significantly differentially expressed genes.

Article Snippet: Primary antibody including Coq4 antibody (1:200, Proteintech, Cat: 16654-1-AP), ACSL4 antibody (1:200, Zenbio, Cat: R24265 ) and CD34 antibody (1:200, Proteintech, Cat: 14486-1-AP).

Techniques: Knockdown, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Coq4 deficiency induces placental vascular development defects through FSP1/CoQ10 axis-mediated endothelial ferroptosis

doi: 10.3389/fcell.2026.1774201

Figure Lengend Snippet: Coq4 deficiency induces ferroptosis in endothelial cells. (A) Immunofluorescence staining of placentas from WT and Coq4 −/− mice with DAPI (blue), CD34 (red), and ACSL4 (green). Scale bar = 750μm, n = 6. (B) CCK-8 assay for cell viability, compared to shNc, n = 6. (C) Commercial kit for GSH content detection, compared to shNc, n = 6. (D) MDA (malondialdehyde) levels measured by commercial kit, compared to shNc group, n = 6. (E) Western blotting for 4-HNE expression and quantitative analysis, compared to shNC group, n = 3. (F) FerroOrange assay for intracellular Fe 2+ , n = 3, Scale bar = 150 μm. (G) Mitochondrial morphology in shNc and shCoq4 cells under transmission electron microscopy, n = 3, Scale bar = 500 μm. (H) Western blotting for ACSL4, SLC7A11, and FTH1 protein expression levels and quantitative analysis, compared to shNc, n = 3.

Article Snippet: Primary antibody including Coq4 antibody (1:200, Proteintech, Cat: 16654-1-AP), ACSL4 antibody (1:200, Zenbio, Cat: R24265 ) and CD34 antibody (1:200, Proteintech, Cat: 14486-1-AP).

Techniques: Immunofluorescence, Staining, CCK-8 Assay, Western Blot, Expressing, Transmission Assay, Electron Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Coq4 deficiency induces placental vascular development defects through FSP1/CoQ10 axis-mediated endothelial ferroptosis

doi: 10.3389/fcell.2026.1774201

Figure Lengend Snippet: Coq4 Knockdown Induces Ferroptosis in Endothelial Cells via the FSP1/CoQ10 Axis but Independently of GPX4. (A) Expression levels of GPX4 and FSP1 were detected by Western blotting and quantitative analysis, compared to shNc, n = 3. (B) Quantitative analysis of intracellular CoQ10 levels using a detection kit, compared to shNc, n = 3. (C) Quantitative analysis of FSP1 protein expression following CoQ10 supplementation via Western blotting, n = 3.

Article Snippet: Primary antibody including Coq4 antibody (1:200, Proteintech, Cat: 16654-1-AP), ACSL4 antibody (1:200, Zenbio, Cat: R24265 ) and CD34 antibody (1:200, Proteintech, Cat: 14486-1-AP).

Techniques: Knockdown, Expressing, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Coq4 deficiency induces placental vascular development defects through FSP1/CoQ10 axis-mediated endothelial ferroptosis

doi: 10.3389/fcell.2026.1774201

Figure Lengend Snippet: FSP1 and CoQ10 Synergistically Improve Ferroptosis Induced by Coq4 Deficiency. (A) Western blotting analysis of FSP1 overexpression and quantitative analysis, n = 3. (B) Western blotting analysis of ACSL4, SLC7A11, and FTH1 protein expression levels and quantitative analysis, n = 5. (C) Quantitative analysis of intracellular MDA (malondialdehyde) levels using a detection kit, n = 6. (D) Cell viability assay using a CCK-8 kit, n = 6.

Article Snippet: Primary antibody including Coq4 antibody (1:200, Proteintech, Cat: 16654-1-AP), ACSL4 antibody (1:200, Zenbio, Cat: R24265 ) and CD34 antibody (1:200, Proteintech, Cat: 14486-1-AP).

Techniques: Western Blot, Over Expression, Expressing, Viability Assay, CCK-8 Assay