Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs preferentially induced mtApoptosis and immunogenic cell death in cancer cells (A) Flow cytometry analysis of cells positive for Annexin V and propidium iodide (PI) in cancer and non-cancer cell lines after treatment with PBS (control), NPs, naked Puma mRNA, and mPuma@NPs for 12 h ( n = 3). (B) Western blot analysis of mitochondrial apoptois pathway in CT-26 cells treated with control, NPs, mPuma@NPs, and mBim@NPs. Bax, Bcl-2, Bcl-x L , Mcl-1, caspase-3, cleaved caspase-3 (C-Cas3), caspase9, and cleaved caspase-9 (C-Cas9) proteins were detected. β-Actin was used as the loading control ( n = 3). (C) Confocal laser scanning microscopy (CLSM) images of the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) probe in CT-26 cells after treatment with PBS (control), NPs, mPuma@NPs, and mBim@NPs for 12 h. Increased JC-1 monomer signal (green) and decreased JC-1 aggregate signal (red) indicate a decrease in mitochondrial membrane potential ( n = 3). Scale bars, 200 μm. (D) Flow cytometry analysis of cellular oxygen species (ROS) levels using 2,7-dichlorofluorescein diacetate (DCFH-DA) staining in CT-26 cells after 12 h incubation with PBS (control), NPs, naked mRNA, mPuma@NPs, and mBim@NPs ( n = 3). (E) CLSM images of CRT expression in B16-F10 and CT-26 cells after 12 h incubation with PBS (control), NPs, mPuma@NPs, and mBim@NPs ( n = 3). Scale bars, 20 μm. (F) Extracellular ATP and HMGB1 expression levels were analyzed by ELISA in B16-F10 cells after 12-h incubation with PBS (control), NPs, mPuma@NPs, and mBim@NPs ( n = 3). (G) Flow cytometry analysis and quantification of immune stimulation in BMDCs co-cultured with B16-F10 cells pretreated with PBS (control), NPs, mPuma@NPs, and mBim@NPs for 12 h, followed by 48-h co-culture ( n = 3). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also .

Article Snippet: Cleaved-Caspase 9 (Asp353) Antibody , Abmart , Cat#TA5240S; RRID:AB_3740854.

Techniques: Flow Cytometry, Control, Western Blot, Confocal Laser Scanning Microscopy, Membrane, Staining, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay

Journal: iScience

Article Title: CGF induces ROS-mediated metabolic reprogramming and mitochondrial dysfunction to suppress colorectal cancer progression

doi: 10.1016/j.isci.2026.115273

Figure Lengend Snippet: CGF’s effect on cell cycle and apoptosis in CRC (A) Flow cytometry was used to analyze how CGF affects the cell cycle of HCT116 and HT29 cells at certain concentrations, with the percentage of cells in G1, S, and G2 phases shown in each panel. (B) Western blot analysis of the changes in cell cycle-related proteins CDK1, p-CDK1, and cyclin B1 in HCT116 and HT29 cells after CGF treatment. (C) RT-qPCR analysis of the relative expression levels of PUMA and NOXA genes in HCT116 and HT29 cells treated with different concentrations of CGF. (D) Western blot analysis of the changes in apoptosis-related proteins BCL2, PUMA, Noxa, C-caspase 9, and C-caspase 3 in HCT116 and HT29 cells after CGF treatment. (E) Flow cytometry was used to analyze apoptosis in HCT116 and HT29 cells treated with CGF. On the left is a representative plot showing apoptosis, utilizing Annexin V-FITC and PI double staining. Right: Analysis of early and late apoptosis in cells from each group using quantitative methods. (A–C and E) Data presentation is in the form of mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: Cleaved Caspase-9 (Asp315) (D8I9E) Rabbit mAb , CST , Cat # 20750; RRID:AB_2798848.

Techniques: Flow Cytometry, Western Blot, Quantitative RT-PCR, Expressing, Double Staining

Journal: iScience

Article Title: CGF induces ROS-mediated metabolic reprogramming and mitochondrial dysfunction to suppress colorectal cancer progression

doi: 10.1016/j.isci.2026.115273

Figure Lengend Snippet: The effect of CGF-induced ROS on the MAPK/ERK 1/2/c-MYC signaling pathway (A) Western blotting was used to analyze the expression of MEK, P-MEK, ERK 1/2, P-ERK 1/2, and c-MYC proteins in HCT116 and HT29 cells after treatment with specified CGF concentrations. (B) Immunofluorescence staining of HCT116 and HT29 cells after CGF treatment, showing changes in p-ERK (green). PMA was used to promote nuclear entry of p-ERK (200 nM, 4 h), and DAPI was used to stain the nucleus (blue), with merged images shown (scale bars, 10 μM). (C) Western blot was used to analyze the levels of MEK, P-MEK, ERK 1/2, P-ERK 1/2, and c-MYC proteins in HCT116 and HT29 cells following treatment with CGF (40 μM, 24 h) and NAC (500 μM, 24 h). (D) Untargeted metabolomics analysis of glycolytic metabolites after CGF treatment in HCT116 cells. Metabolites marked in blue represent those inhibited by CGF. (E and F) ELISA analysis of lactate secretion in the cell supernatant of HCT116 (E) and HT29 (F) cells after CGF treatment. (G and H) Analysis of mRNA expression levels of genes related to glycolysis in HCT116 and HT29 cells exposed to certain concentrations of CGF using RT-qPCR. (I) Western blot was used to analyze the expression levels of apoptosis-associated proteins (BCL2, PUMA, NOXA, C-caspase 9, and C-caspase 3) and EMT markers (N-cadherin and vimentin) in HCT116 and HT29 cells after they were treated with CGF. (J) Western blot analysis of the reversal of EMT markers N-cadherin, vimentin, SLUG, and SNAIL proteins in HCT116 and HT29 cells overexpressing c-MYC. (E–J) Data presentation is in the form of mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: Cleaved Caspase-9 (Asp315) (D8I9E) Rabbit mAb , CST , Cat # 20750; RRID:AB_2798848.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Pancreas

Article Title: Knockdown of Lymphoid Enhancer-binding Factor 1 Inhibits Pancreatic Adenocarcinoma Growth and Neoangiogenesis by Curbing Notch1 and Nuclear Factor Kappa B Signaling Pathways

doi: 10.1097/MPA.0000000000002562

Figure Lengend Snippet: Knockdown of LEF1 induces apoptosis in PAAD cells. A–C, Flow cytometry detection of apoptosis and apoptosis rate statistics: apoptosis rate was higher in AsPC-1/shLEF1 and BxPC-3/shLEF1. D–F, Calcein AM/PI staining assay results and statistics: the live cells decreased while the dead cells increased in AsPC-1/shLEF1 and BxPC-3/shLEF1. G, ATP production assay: the ATP production decreased in AsPC-1/shLEF1 and BxPC-3/shLEF1. H, LDH release assay showed an increase in LDH release in AsPC-1/shLEF1 and BxPC-3/shLEF1. I–M, WB results and quantitative analysis: the level of Bcl-2 protein decreased in AsPC-1/shLEF1 and BxPC-3/shLEF1, while BAX, Cleaved caspase-3 and Cleaved caspase-9 increased (n = 3, * P <0.05, ** P <0.01).

Article Snippet: The first antibodies were as follows: LEF1 (ab137872, 1:1000, Abcam), proliferating cell nuclear antigen (PCNA, ab29, 1:1000, Abcam), MMP-2 (4022S, 1:1000, CST), MMP-9 (3852S, 1:1000, CST), BAX (2772S, 1:1000, CST), Bcl-2 (15071S, 1:1000, CST), Cleaved caspase-3 (9661S, 1:1000, CST), Cleaved caspase-9 (9509S, 1:1000, CST), vascular endothelial growth factor-A (VEGFA, ab214424, 1:1000, Abcam), total Notch1 (ab52627, 1:1000, Abcam), NICD (4147, 1:1000, CST), Hes1 (ab71559, 1:1000, Abcam), Hey1 (ab235173, 1:1000, Abcam), Jagged1 (ab300561, 1:1000, Abcam), P65 (ab32536, 1:1000, Abcam), p-P65 (3033S, 1:1000, CST), GAPDH (5174S, 1:5000, CST), HRP-anti-Rabbit (A0362, 1:1000, Beyotime), HRP-anti-Mouse (A0350, 1:1000, Beyotime).

Techniques: Knockdown, Flow Cytometry, Staining, Lactate Dehydrogenase Assay