Journal: Nature Communications

Article Title: A combinatorial synthetic strategy for developing genome-editing protein-delivery agents targeting mouse retina

doi: 10.1038/s41467-026-69077-w

Figure Lengend Snippet: a Schematic diagram of the color-switching construct of the rd12 reporter-cell line. An intervening sequence from the Rpe65 - rd12 genomic sequence containing the mutation (c.130 C  >  T; p.R44X) was inserted into a gene expressing both mCherry and eGFP. Expression of eGFP in the reporter cells is prevented by a STOP codon, which can be restored via proper action of ABE. b Representative fluorescence-microscopic images of rd12 -reporter cells 48 h after adding the RNP-CBB complex. Scale bar, 400 μm. c , d Quantitative assessment of ABE-RNP delivery into rd12 -reporter cells, mediated by lipidoid complexes of ABE8e with CBB compounds ( c ) by flow cytometry; or CBBZ compounds ( d ) by fluorescence-microscopic imaging only. [ABE8e]/[CBB(Z)] = 1:8. A, ABE8e-RNP only. NE, no enzyme. Quantification of ABE8e RNP delivery mediated by Lipofectamine 3000 (LF) is included as a positive control. The ratio (eGFP/mCherry) reflects the relative editing efficiency. The CBB compounds were considered positive hits if the ratio of green-to-red (eGFP/mCherry) was more than twice that of A (the dashed line). Data are presented as mean ± SD, n = 4 replicates ( c ) and 3 replicates ( d ). e Schematic diagram of the ABE-activity assay. EndoV = endonuclease V. Blue, SpCas9 PAM; red, target base; orange, bystander base; arrowhead, nick site; FAM, fluorescein. f Representative urea-PAGE-gel image of products of ABE deamination cleaved by EndoV. The gel was imaged with a fluorescein filter. N, no enzyme; P, positive control: 60 bp DNA with inosine in the middle; A, ABE-RNP in TAS; D, ABE-RNP in TAS with 10% DMSO. Numbers indicate molar excess of the CBB compound versus ABE. g Fluorescence quantification of substrate and products of deamination and EndoV cleavage. Two independent samples per data point were assayed in parallel. Source data are provided as a Source Data file.

Article Snippet: Purified DNA was nicked with 5 units of Endonuclease V (EndoV, NEB) for 2-3 h at 37 °C, after which the reaction was quenched by addition of TriTrack DNA-loading dye (1 × final) (Thermo Fisher, R1161) and incubation at 95 °C for 2 min.

Techniques: Construct, Sequencing, Mutagenesis, Expressing, Fluorescence, Flow Cytometry, Imaging, Positive Control, Activity Assay

Journal: Cancer Cell International

Article Title: APX3330 reverses the immunosuppressive tumor microenvironment during colorectal carcinogenesis

doi: 10.1186/s12935-026-04176-8

Figure Lengend Snippet: Expression of APE1/Ref-1 in Tumor Tissues of Patients with CRC ( A ) Flowchart illustrating the case screening process. ( B ) Representative images demonstrating APE1/Ref-1 expression assessed through IHC staining, with black horizontal lines indicating 200 μm. The red arrows denote specific locations that exemplify the range of APE1/Ref-1 expression. The accompanying histogram depicts the distribution of tumor stage and differentiation levels corresponding to each intensity of APE1/Ref-1 staining. ( C ) Visualization of patient baseline characteristics alongside APE1/Ref-1 IHC staining intensity, organized according to the American Joint Committee on Cancer (AJCC) stage. Abbreviations: AC: Ascending colon; TC: Transverse colon; DC: Descending colon; SC: Sigmoid colon. ( D ) A percentage stacked histogram illustrating the distribution of APE1/Ref-1 IHC staining intensity across various stages and degrees of differentiation. The category classified as “Poor” encompasses poorly differentiated adenocarcinoma, medullary carcinoma, and signet-ring cell carcinoma. Abbreviations: MC: Medullary carcinoma; SC: Signet-ring cell carcinoma; MA: Mucinous adenocarcinoma. E - F . Violin plots depicting the expression levels of APE1/Ref-1 in both tumor and normal tissues in TCGA database. G - H . Differential expression of APE1/Ref-1 was verified by IHC staining, with black horizontal lines indicating 200 μm. Normol, n = 3. Tumor, n = 176

Article Snippet: The specific antibodies employed in this study were APE1/Ref-1 (Proteintech, IL, USA, #10203-1-AP, dilution 1:1000) and β-Tubulin (CST, MA, USA, #2146S, dilution 1:1000).

Techniques: Expressing, Immunohistochemistry, Staining, Quantitative Proteomics

Journal: Cancer Cell International

Article Title: APX3330 reverses the immunosuppressive tumor microenvironment during colorectal carcinogenesis

doi: 10.1186/s12935-026-04176-8

Figure Lengend Snippet: APE1/Ref-1 Redox Function Affects Immunosuppressive TME ( A ) Correlation between APE1/Ref-1 expression and the infiltration of MDSCs and CD8 + T cell analyzed within the TCGA database. ( B ) Representative IHC image of high (upper panel) or low (lower panel) APE1/Ref-1 expression. C - F . mIHC staining reveals the infiltration of immune cells in paraffin-embedded tumor tissues from CRC patients, categorized by high APE1/Ref-1 expression (left panel) and low APE1/Ref-1 expression (right panel), along with corresponding statistical analyses ( D - F ). Human PMN-MDSC are defined as CD11B + CD14 − 29,30 . The white horizontal line in the images denotes 200 μm. G . Visualization of luminex liquid suspension chip detection, where the interior of the circle indicates the inhibitory effect of the drug on cytokine expression, while the exterior indicates a promoting effect. The length of the fan segments represents the magnitude of fold change (FC), quantified as log2(FC) following drug treatment; the color of the segments corresponds to the P value, with gray indicating a lack of statistical significance. H - I . Assessment of alterations in the expression levels of tumor necrosis factor-α (TNF-α, H ) and CXCL1 ( I ) subsequent to APX3330 treatment in HT-29

Article Snippet: The specific antibodies employed in this study were APE1/Ref-1 (Proteintech, IL, USA, #10203-1-AP, dilution 1:1000) and β-Tubulin (CST, MA, USA, #2146S, dilution 1:1000).

Techniques: Expressing, Staining, Luminex, Suspension

Journal: Molecular Cancer Therapeutics

Article Title: BH3-only proteins Mcl-1 and Bim as well as endonuclease G are targeted in spongistatin 1–induced apoptosis in breast cancer cells

doi: 10.1158/1535-7163.mct-08-1179

Figure Lengend Snippet: Figure 4. Spongistatin 1 induces the translocation of AIF and EndoG to the nucleus. A, cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 24 h. Nonnucleic and nuclear protein fractions were prepared and AIF and EndoG were detected by specific antibodies using Western blot analysis. Cytochrome c oxidase served as control for the quality of the extraction procedure. B, cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 24 h. Translocation of AIF and EndoG from mitochondria to the nucleus was analyzed by confocal microscopy. Blue, nuclei; green, AIF and EndoG. All experiments were done three times with consistent results. C, AIF and EndoG assume a functional role in spongistatin 1–induced apo- ptosis. MCF-7 cells were transfected or cotransfected with oligonucleotides encoding for either AIF siRNA and EndoG siRNA or a nonsense sequence. Cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 48 h. Apoptotic cells were quantified by flow cytometry. Results are percent specific apoptosis. Downregulation of AIF and EndoG protein levels was verified by Western blot. Equal protein loading was controlled by staining membranes with β-actin. Columns, mean of three independent experiments done in triplicate; bars, SE. ***, P < 0.001 (ANOVA/Bonferroni).

Article Snippet: Cells were blocked with 0.2% bovine serum albumin and incubated with specific antibodies against AIF (rabbit polyclonal IgG; Upstate) and EndoG (rabbit polyclonal antibody; Prosci).

Techniques: Translocation Assay, Western Blot, Control, Extraction, Confocal Microscopy, Functional Assay, Transfection, Sequencing, Flow Cytometry, Staining

Journal: Molecular Cancer Therapeutics

Article Title: BH3-only proteins Mcl-1 and Bim as well as endonuclease G are targeted in spongistatin 1–induced apoptosis in breast cancer cells

doi: 10.1158/1535-7163.mct-08-1179

Figure Lengend Snippet: Figure 6. Bim functions as a proapoptotic regulator upstream of mitochondria. A, MCF-7 cells were transfected with oligonucleotides encoding for either Bim siRNA or nonsense sequence. Cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 24 h. Nonnucleic and nuclear protein fractions were prepared and AIF and EndoG were detected by specific antibodies using Western blot analysis. Cytochrome c oxidase served as control for the quality of the extraction procedure. As a control, Bim protein level in cell lysates from nonsense and Bim siRNA cells was analyzed by Western blot. B, Bim and EndoG are the major regulators of spongistatin 1–induced cell death. MCF-7 cells were cotransfected with oligonucleotides encoding for either Bim siRNA or EndoG siRNA or nonsense sequence. Cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 48 h. Apoptotic cells were quantified by flow cytometry. Results are percent specific apoptosis. Downregulation of Bim and EndoG protein levels was verified by Western blot. Equal protein loading was controlled by staining membranes with β-actin. All experiments were done three times with consistent results. Columns, mean of three independent experiments done in triplicate; bars, SE. ***, P < 0.001 (ANOVA/Bonferroni). C, proposed mechanism of spongistatin 1–induced apoptosis. Thick arrows, main signaling pathway of spongistatin 1. The tubulin depolymerizing agent spongistatin 1 frees Bim from its sequestration both by the microtubule network and by the antiapoptotic protein Mcl-1. Bim triggers the translocation of AIF and EndoG from mitochondria to the nucleus leading to caspase-independent apoptosis. Thin arrows, inferior role of the caspase-dependent cell death induced by spongistatin 1.

Article Snippet: Cells were blocked with 0.2% bovine serum albumin and incubated with specific antibodies against AIF (rabbit polyclonal IgG; Upstate) and EndoG (rabbit polyclonal antibody; Prosci).

Techniques: Transfection, Sequencing, Western Blot, Control, Extraction, Flow Cytometry, Staining, Translocation Assay