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: bioRxiv

Article Title: Extracellular Vesicles from Senescent Tumor Cells Are Necessary and Sufficient to Drive Paracrine Senescence

doi: 10.64898/2026.03.25.713920

Figure Lengend Snippet: (a) Representative images of cell morphology and SA-β-Gal staining of proliferative (A549-C) and senescent (A549-S) A549 cells (scale, 100 μm; scale in zoomed area, 200 μm). Quantification of the staining. (b) Heatmap representation of RNA-seq analysis of A549-C and A549-S. (c) UMAP showing group clustering. (d) Volcano plot of differentially expressed genes. (e) Dot plot analysis of differentially expressed pathways. Red dots represent upregulated pathways, and blue dots represent downregulated pathways. (f) Schematic representation of proliferative (CM-C) and senescent (CM-S) A549 CM treatment over proliferative A549 cells. (g) Representative images of cell morphology and SA-β-Gal staining, large scale, 200 μm, short scale, 100 μm. (h) Cell size measurement. (i) Cell counting. (j) Quantification of SA-β-Gal positive cells using X-gal substrate. (k) Quantification of SA-β-Gal positive cells using C 12 FDG substrate. (l) RT-qPCR analysis of CDKN1A . GAPDH was used as housekeeping gene. (m) Schematic representation of proliferative (CM-C) and senescent (CM-S) A549 CM over proliferative MCF7. (n) Representative images of cell morphology and SA-β-Gal staining, large scale, 200 μm, short scale, 100 μm. (o) Quantification of SA-β-Gal positive cells using X-gal substrate. (p) RT-qPCR analysis of CDKN1A . GAPDH was used as housekeeping gene. (q) RT-qPCR analysis of SERPINE1 . GAPDH was used as housekeeping gene. Significance was calculated using Student’s t-test showing the exact p-value. Less than 0.05 was considered statistically significant (n= 3).

Article Snippet: Membranes were blocked with 5% milk solution and incubated with primary antibodies for GAPDH (sc-32233, Santa Cruz Biotechnology 1:1000), β-Actin (sc-8432, Santa Cruz Biotechnology, 1:1000), Vinculin (sc-76314, Santa Cruz Biotechnology 1:2000), RAB27A (#69295, Cell Signaling Technology, 1:1000), p53 (sc-6243, Santa Cruz Biotechnology, 1:1000), and p21 (#2947, Cell Signaling Technology, 1:1000), overnight at 4 °C.

Techniques: Staining, RNA Sequencing, Cell Counting, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Extracellular Vesicles from Senescent Tumor Cells Are Necessary and Sufficient to Drive Paracrine Senescence

doi: 10.64898/2026.03.25.713920

Figure Lengend Snippet: (a) Cell size measurement of proliferative (A549-C) and senescent (A549-S) cells. (b) Flow cytometry plots and quantification of SA-β-Gal positive cells using C 12 FDG substrate. (c) RT-qPCR analysis of CDKN1A . GAPDH was used as housekeeping gene. (d) Western blot analysis of senescence markers (p53 and p21). GAPDH and Actin were used as loading controls. (e) Clonogenicity assay and quantification. (f) EdU incorporation assay and quantification (scale, 200 μm). (g) GSEA plots of the Reactome_Cell_Cycle and Fridman_Senescence_up gene sets. (h) Heatmap and list of differentially expressed genes. (i) Dot plot analysis of differentially expressed genes analyzed using the Reactome pathways and KEGG databases. (j) Dot plot analysis of differentially expressed genes using the Biological process terms in Gene Ontology. (k) Flow cytometry plots of cells treated with control (CM-C) and senescent (CM-S) conditioned media. Significance was calculated using Student’s t-test showing the exact p-value. Less than 0.05 was considered statistically significant (n=3, except d, n=1).

Article Snippet: Membranes were blocked with 5% milk solution and incubated with primary antibodies for GAPDH (sc-32233, Santa Cruz Biotechnology 1:1000), β-Actin (sc-8432, Santa Cruz Biotechnology, 1:1000), Vinculin (sc-76314, Santa Cruz Biotechnology 1:2000), RAB27A (#69295, Cell Signaling Technology, 1:1000), p53 (sc-6243, Santa Cruz Biotechnology, 1:1000), and p21 (#2947, Cell Signaling Technology, 1:1000), overnight at 4 °C.

Techniques: Flow Cytometry, Quantitative RT-PCR, Western Blot, Control

Journal: bioRxiv

Article Title: Extracellular Vesicles from Senescent Tumor Cells Are Necessary and Sufficient to Drive Paracrine Senescence

doi: 10.64898/2026.03.25.713920

Figure Lengend Snippet: (a) Cytokine array analyses of CMs from proliferative (CM-C), senescent (CM-S), and GW4869-treated senescent cells (CM-S+GW6849) and their quantification. Red squares mark reference dots. (b) NTA analysis of particles in the CM. (c) Schematic representation of treatments using CMs on proliferative A549. (d) Representative images of morphology and SA-β-Gal staining (scale, 100 μm; scale in zoomed area, 200 μm). (e) Cell size measurement. (f) Cell counting. (g) Quantification of SA-β-Gal positive cells using X-gal substrate. (h) Quantification of SA-β-Gal positive cells using C 12 FDG substrate. (i) RT-qPCR analysis of CDKN1A . GAPDH was used as housekeeping gene. Significance was calculated using one-way ANOVA showing the exact p-value. Less than 0.05 was considered statistically significant (b-i, n=3; a, n=1).

Article Snippet: Membranes were blocked with 5% milk solution and incubated with primary antibodies for GAPDH (sc-32233, Santa Cruz Biotechnology 1:1000), β-Actin (sc-8432, Santa Cruz Biotechnology, 1:1000), Vinculin (sc-76314, Santa Cruz Biotechnology 1:2000), RAB27A (#69295, Cell Signaling Technology, 1:1000), p53 (sc-6243, Santa Cruz Biotechnology, 1:1000), and p21 (#2947, Cell Signaling Technology, 1:1000), overnight at 4 °C.

Techniques: Staining, Cell Counting, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Extracellular Vesicles from Senescent Tumor Cells Are Necessary and Sufficient to Drive Paracrine Senescence

doi: 10.64898/2026.03.25.713920

Figure Lengend Snippet: (a) RT-qPCR analysis of RAB27A . GAPDH was used as housekeeping gene. (b) Western blot analysis of RAB27A. Vinculin was used as loading control. (c) NTA analysis of particles in CM. (d) Representative images of morphology and SA-β-Gal staining (scale, 100 μm; scale in zoomed area, 200 μm). (e) Cell counting. (f) Cell size measurement. (g) Quantification of SA-β-Gal positive cells using X-gal substrate. (h) Quantification of SA-β-Gal positive cells using C 12 FDG substrate. (i) RT-qPCR analysis of CDKN1A . GAPDH was used as housekeeping gene. Significance was calculated using one-way ANOVA showing the exact p-value. Less than 0.05 was considered statistically significant (a, c-i, n=3; b, n=1).

Article Snippet: Membranes were blocked with 5% milk solution and incubated with primary antibodies for GAPDH (sc-32233, Santa Cruz Biotechnology 1:1000), β-Actin (sc-8432, Santa Cruz Biotechnology, 1:1000), Vinculin (sc-76314, Santa Cruz Biotechnology 1:2000), RAB27A (#69295, Cell Signaling Technology, 1:1000), p53 (sc-6243, Santa Cruz Biotechnology, 1:1000), and p21 (#2947, Cell Signaling Technology, 1:1000), overnight at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot, Control, Staining, Cell Counting