Journal: Journal of Translational Medicine

Article Title: LDHC4 drives lung adenocarcinoma progression by inducing lactylation of RB1 at lysine 900 to disrupt the RB1–E2F1 complex

doi: 10.1186/s12967-026-08070-9

Figure Lengend Snippet: Influence of RB1–K900lac on the cell cycle pathway. ( A to C ) Cell cycle analysis by flow cytometry in A549 and PC-9 cell lines stably expressing RB1–WT or RB1–K900R via lentiviral vectors. ( D ) Representative immunofluorescence images showing the distribution of CDK1 in A549 and PC-9 cells. CDK1 protein was labeled with red fluorescent Cy3, and nuclei were counterstained with blue fluorescent DAPI. Images were acquired using a high-resolution confocal multiphoton microscopy system (NIKON AX RMP, Japan). ( E , F ) Western blot analysis of cell cycle-related CDK molecule expression. ( G , H ) Expression of cell cycle-related cyclin molecules. ( I , J ) Expression of P21 and Chk1 molecules. ( K ) Schematic diagram illustrating how LDHC4 promotes the cell cycle by inducing RB1 lactylation. ** p < 0.01, *** p < 0.001

Article Snippet: The following antibodies were used in this study: rabbit anti-human LDHC (subunit C) monoclonal antibody (mAb) (Proteintech Group, Inc., 1:1000), rabbit anti-human RB1 mAb (Proteintech Group, Inc., 1:2000), rabbit anti-human L-Lactyl Lysine mAb (PTM Bio, Inc., 1:1000), rabbit anti-human E2F1 mAb (APExBIO Technology, LLC, 1:500), rabbit anti-human Lamin B mAb (Beyotime Biotech, Inc., 1:1000), rabbit anti-human CDK1 mAb (Beyotime Biotech, Inc., 1:800), rabbit anti-human CDK2 mAb (Beyotime Biotech, Inc., 1:800), rabbit anti-human CDK4 mAb (Beyotime Biotech, Inc., 1:1000), rabbit anti-human CDK6 mAb (Beyotime Biotech, Inc., 1:1000), rabbit anti-human cyclin A2 mAb (Beyotime Biotech, Inc., 1:1000), rabbit anti-human cyclin B1 mAb (Beyotime Biotech, Inc., 1:1000), rabbit anti-human cyclin D1 mAb (Beyotime Biotech, Inc., 1:500), rabbit anti-human P21 mAb (Abmart Bio, Inc., 1:500), rabbit anti-human Chk1 mAb (Immunoway Bio, Inc., 1:2000), and rabbit anti-β-Actin monoclonal antibody (Beyotime Biotech, Inc., 1:1000). β-Actin and Lamin B served as loading controls, and protein band intensities were quantified using Image J software.

Techniques: Cell Cycle Assay, Flow Cytometry, Stable Transfection, Expressing, Immunofluorescence, Labeling, Microscopy, Western Blot

Journal: Antioxidants

Article Title: Exploring the Anticancer Potential of the Multistrain Probiotic Formulation OxxySlab in Bladder Cancer Cell Lines

doi: 10.3390/antiox14111282

Figure Lengend Snippet: Senescence induction in BC cells following OxxySlab lysate treatment. Cell lines were treated with OxxySlab lysate (75–150 μg/mL) for 48 h. Senescent cells were identified by β-galactosidase staining (β-gal). Representative phase-contrast images of T24 ( A ), 5637 ( E ) and SV-HUC-1 ( I ) β-gal-positive cells (blue staining) are shown. Quantification of senescence was performed by measuring the Mean Gray Value of blue staining intensity using ImageJ software, with data expressed as Inverted Mean Gray Value (255 − MGV), which directly reflects β-gal activity. Data are expressed as mean ± SEM of two independent experiments in triplicate. Western blot analysis of the senescence markers p21, p53 and p16 was performed in untreated (CNTR) and OxxySlab -treated T24 ( B – D ), 5637 ( F – H ), and SV-HUC1 ( J – L ) cells. Protein levels were quantified by densitometry, normalized to GAPDH, and expressed as fold change relative to CNTR of three independent experiments (mean ± SEM). Statistical significance was assessed by one-way ANOVA followed by Tukey post hoc test (* p < 0.05, ** p < 0.01).

Article Snippet: Following incubation with 5% non-fat dry milk in Tris-buffered saline for 1 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies: goat anti-human vimentin polyclonal antibody (Chemicon International, Temecula, CA, USA; dilution 1:100), mouse anti-human E-cadherin monoclonal antibody (Cell Signaling Technology; dilution 1:1000), rabbit anti-human β-catenin monoclonal antibody (Cell Signaling Technology; dilution 1:1000), rabbit anti-human p21 polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA dilution 1:1000); rabbit anti-human phospho-Nrf2 monoclonal antibody (S40) (1:2000, Abcam, Cambridge, UK); rabbit anti-human p16 polyclonal antibody (Santa Cruz Biotechnology, dilution 1:200); mouse anti-human p53 monoclonal antibody (Santa Cruz Biotechnology, dilution 1:1000); mouse anti-human GAPDH monoclonal antibody (Immunological Sciences, Rome, Italy; dilution 1:1000).

Techniques: Staining, Software, Activity Assay, Western Blot

Journal: Cell Reports Medicine

Article Title: Cardiolipin-mimic lipid nanoparticles without antibody modification delivered senolytic in vivo CAR-T therapy for inflamm-aging

doi: 10.1016/j.xcrm.2025.102209

Figure Lengend Snippet: In vivo therapeutic effect of mCAR-uPAR LNPs in collagen-induced arthritis (A) scRNA-seq data of synovial membrane from RA patients from public resources. (B) mIHC staining of uPAR (green), PAI-1 (red), and P21 (cyan) in synovial samples from human RA patients; scale bar, 50 μm. (C) Schematic diagram of the treatment schedule for the CIA mouse model. (D–F) Clinical score and paw thickness of the CIA mice in different treated groups ( n = 5, mean ± SD). (G and H) (G) Representative of H&E and safarin O/Fast green staining in the ankles of CIA mice with all groups. The quantification of inflammation level and cartilage erosion was performed in each mouse (from n = 5 biological independent mice per group, mean ± SD) and shown in (H). (I and J) (I) Paraffin section mIHC staining of uPAR (red), CD3 (cyan), F4/80 (green), and CD206 (magenta) in the ankles of CIA mice. Scale bar, 100 μm. Quantifications of the mIHC stainings were presented in (J), from n = 5 individual samples, mean ± SD. "n" indicated biologically independent samples. Statistical significance was calculated through one-way ANOVA with Dunnett test. All the schematic illustrations were created with BioRender.com .

Article Snippet: anti-human p21 , CST , Cat# 2947T; RRID: AB_823586.

Techniques: In Vivo, Membrane, Staining, Paraffin Section

Journal: International Journal of Molecular Sciences

Article Title: CD26 Is Differentially Expressed throughout the Life Cycle of Infantile Hemangiomas and Characterizes the Proliferative Phase

doi: 10.3390/ijms25189760

Figure Lengend Snippet: Impact of DPP-IV inhibition on infantile hemangioma-derived endothelial cells (Hem-ECs) in vitro. Hem-ECs treated with increasing vildagliptin (VLDG) concentrations for 24 h ( A ) and 72 h ( B ), respectively. Percent viability with respect to untreated cells (CTRL) is reported as mean ± standard error (SE); * p < 0.05 vs. control. Holm-Sidak’s test. ( C ) Immunoblotting analysis documenting a dose-dependent increase in p21 protein expression in Hem-ECs following VLDG exposure. HSP90 serves as the loading control. Densitometric values normalized vs. control (HSP90) are reported at the bottom of each p21 blot. ( D ) Flow cytometric analysis of Hem-EC proliferation. Gating strategy illustrating the percentage of ki67-positive untreated (CTRL) and VLDG-treated Hem-ECs. The density of scatter dot-plots in positive gate decreases in a dose-dependent manner. Mean fluorescent intensity (MFI) of ki67 labeling is also reported. ( E ) Apoptosis assay, as measured by the cytometric analysis of Annexin V, on Hem-ECs after 72 h exposure to 100 μM and 250 μM VLDG. ( F ) Cell cycle analysis of Hem-ECs after 72 h of VLDG treatment. The percentage of cells in the different phases of the cell cycle was calculated using the Watson Pragmatic Model of FlowJo (v10.8) software.

Article Snippet: Membranes were incubated with 1:1000 rabbit anti-human p21 CIP1/WAF1 (hereafter named p21) (Cell Signaling Technology, Danvers, MA, USA; #2947S) and 1:500 mouse anti-HSP90 (Santa Cruz Biotechnology, Dallas, TX, USA; sc-69703).

Techniques: Inhibition, Derivative Assay, In Vitro, Control, Western Blot, Expressing, Labeling, Apoptosis Assay, Cell Cycle Assay, Software