Journal: bioRxiv

Article Title: Light-dependent cell fixing with DNA-targeting fluorophores

doi: 10.64898/2026.03.27.714905

Figure Lengend Snippet: (A) Localization of PAL (green) at mtDNA within MitoRed ® -stained mitochondria (red) in live Hep3B cells prior to irradiation (scale bar: 10 µm, crop: 5 µm). (B) Top: snapshots of time-lapse PAL nuclear fluorescence upon WF irradiation (scale bar: 20 µm). Bottom: PAL labeling upon irradiation at distinct phases of the cell cycle ( a : interphase; b : prophase; c : prometaphase; d : metaphase; e : anaphase. Scale bar: 5 µm). Right : kinetics of PAL fluorogenesis during 5 min irradiation. Mean intensities are rescaled from 0 to 1 within a 95% confidence interval. (C) PAL nuclear fluorescence (in green) of a targeted cell islet within a population of live PAL-treated cells upon WF irradiation (PAL + hv) (R1: dotted line; R2: dashed line; NA: non-affected area. Scale bar: 50 µm). Crops in R1 (orange squares) showing stable nuclear morphology and persistence up to 9 days post-irradiation within a live-cell proliferative environment. Crops in R2 (blue squares) showing compromised nuclei. Calculated irradiation powers were 3.5 to 15 W/cm 2 in R1 from limit to center of the beam. Only evanescent or residual scattered light was present in R2 (scale bar: 20 µm). (D) Co-staining of PAL-treated cells (green) with propidium iodide (PI, red) or NucView ® 530 Red Caspase-3 dye (NucView, magenta). Top: PI stains R1 cells (dotted lines), showing a permeabilized status immediately post-irradiation (d0). After 24 h, R2 cells show important delayed PI staining demonstrating a compromised state. Bottom: NucView faintly stains R1 cells 24 h post-irradiation, showing strong signal in compromised R2 cells. The apoptosis inducer staurosporine is used as a positive control. (E) Quantification of nuclear size in different conditions: live PAL-treated cells without irradiation (PAL), FA-fixed cells (FA), PAL-free irradiated cells (hv) or PAL-treated irradiated cells (PAL + hv, R1 and R2) (n>200 per condition). Data shown in violin plots reflecting size distribution, circles indicate the medians and whiskers indicate standard deviations.

Article Snippet: The human cell line Hep3B was obtained from ATCC ® (HB-8064), the human cell lines T24, PC3 and C4-2B were a courtesy of Pr.

Techniques: Staining, Irradiation, Fluorescence, Labeling, Positive Control

Journal: bioRxiv

Article Title: Light-dependent cell fixing with DNA-targeting fluorophores

doi: 10.64898/2026.03.27.714905

Figure Lengend Snippet: (A) Left: Optofixing procedure of a whole Hep3B cell population using a portable LED lamp towards biochemical analysis. Top right: Visualization of abundance of precipitates (UVA irradiation). Right bottom: Corresponding SDS-PAGE analysis of soluble protein contents following treatments with formaldehyde (FA), 4-hydroxynonenal (4HNE), acrolein (ACR) or LED irradiation in presence or absence of PAL. (B) Left panel: WF imaging of PAL nuclear fluorogenesis at day 0 after irradiation by a WF microscope. Right panel: stability of fixed state after 3 days of PAL-treated cells in presence of inhibitors (scale bar: 10µm). (C) Left: fluorogenesis kinetics during 5 min irradiation with conditions described in B . Fluorescence intensities were normalized as [(I-Io)/Io]. Right : quantification of nuclear sizes with conditions described in B (n>60 per condition) 3 days post-irradiation. ( D) Lipid peroxidation (LPO)-quantification in PAL-treated cells using BODIPY™ 581/591-C11 probe. Top left: representative image of probe staining 30 min after irradiation of PAL-treated cells in zones NA, R1 and R2. Top right: plot of ratiometric measurements along the yellow dashed line. Red curve represents Red/Ox ratio, green curve represents the Ox/Red ratio. Bottom: quantification of the oxidation ratio (Ox/Red) 30 min after irradiation of PAL-treated cells (R1, R2) compared to irradiated cells in absence of PAL (hv) or cells treated with PAL without irradiation (PAL) (n>100 per condition). (E) ROS quantification in PAL-treated cells using ROS Brite TM 670 probe. Top: representative image of ROS staining (in magenta) obtained after irradiation of PAL-treated cells (in green) in R1 and R2 (scale bar: 10 µm). Bottom: Quantification of ROS Brite fluorescence intensity in the same conditions than D .

Article Snippet: The human cell line Hep3B was obtained from ATCC ® (HB-8064), the human cell lines T24, PC3 and C4-2B were a courtesy of Pr.

Techniques: Irradiation, SDS Page, Imaging, Microscopy, Fluorescence, Staining

Journal: bioRxiv

Article Title: Light-dependent cell fixing with DNA-targeting fluorophores

doi: 10.64898/2026.03.27.714905

Figure Lengend Snippet:

Article Snippet: The human cell line Hep3B was obtained from ATCC ® (HB-8064), the human cell lines T24, PC3 and C4-2B were a courtesy of Pr.

Techniques: Labeling

Journal: Cell Death Discovery

Article Title: ATGL sensitizes hepatocellular carcinoma cells to genotoxic drugs by modulating p53 acetylation/phosphorylation status

doi: 10.1038/s41420-026-03048-4

Figure Lengend Snippet: A , B HUH7 and C , D Hep3B cells were transfected with an empty vector (Vehicle) or ATGL-overexpressing construct (ATGL-OE) for 24 h and then treated with 50 µM etoposide or 2 µM doxorubicin for 6 h. Western blot analysis of γH2AX levels was performed. E Western blot analysis of Ac-p53 Lys-382, p-p53 Ser-15 and p53 levels was performed in HepG2 cells after treatment with 50 µM etoposide for 2, 4, 6 and 24 h. F Densitometric ratios of Ac-p53 and p-p53 after treatment with 50 µM etoposide for 2, 4, 6 and 24 h. G – I HepG2 cells were transfected with empty vector (Vehicle) or ATGL-overexpressing (ATGL-OE) construct and, after 24 h, treated with 50 µM etoposide for 6 h with or without 10 µM C646 for 24 h. Western blot analysis of Ac-p53 Lys-382, p-p53 Ser-15 and p53 levels was performed. H Densitometric analysis ratios of Ac-p53 and p-p53 after treatment with 50 µM etoposide for 6 h. J – L HepG2 cells were treated with 50 µM etoposide for 6 h with or without 1 µM GW7647 for 24 h. Western blot analysis of Ac-p53 Lys-382, p-p53 Ser-15, p53 and γH2AX levels was performed. K Densitometric analysis of ratio between Ac-p53 and p-p53 expression after treatment with 50 µM etoposide for 6 h. The images are representative of three independent experiments that yielded similar results. β-Actin and ATGL were used as loading and transfection controls, respectively. The data are presented as the means ± SDs from three independent experiments. Statistical significance was determined by Student t test and one-way ANOVA with Tukey’s post hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001 vs CTRL or as indicated by brackets.

Article Snippet: HepG2, HUH7 and Hep3B cell lines were purchased from American Type Culture Collection (ATCC).

Techniques: Transfection, Plasmid Preparation, Construct, Western Blot, Expressing

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: E6AP downregulates p53 levels in an HCV Core-dependent manner. ( a – d ) HepG2 and Hep3B cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the indicated plasmids, for 48 h, followed by Western blotting. The images of the target proteins were cropped from the original images and band intensities were quantified using ImageJ (NIH). Values indicate p53 and E6AP levels relative to the loading control (γ-tubulin).

Article Snippet: The HepG2 (#88065) and Hep3B (#88064) cell lines were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Control

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: HCV Core stimulates E6AP-mediated proteasomal degradation of p53 but inhibits MDM2-mediated degradation. HepG2 and Hep3B cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the indicated plasmids, for 48 h. For ( a ), HepG2-Core cells stably expressing HCV Core were also included. ( a , b ) Protein levels were measured by Western blot analysis. ( c ) Cells were treated with 50 μM cycloheximide (CHX) for the indicated times before harvesting, followed by Western blotting. Bands of p53 and γ-tubulin were quantified to determine the half-life (t1/2) of p53. The difference in the p53-to-γ-tubulin ratio among samples is shown in a graph. ( d , e ) Cells were treated with 10 μM MG132 for 4 h before harvesting to block further proteasomal degradation. For lanes 5 and 6 in ( e ), cells were treated with Nutlin 3a for 24 h. Total p53 was immunoprecipitated with an anti-p53 antibody and subjected to Western blotting using anti-E6AP, anti-MDM2, anti-HCV Core, and anti-HA antibodies to detect E6AP, MDM2, HCV Core, and HA-Ub-complexed p53, respectively. The input indicates the levels of the designated proteins in the cell lysates. ( f , g ) For mammalian two-hybrid assays, Hep3B cells were transfected with the Gal4 reporter (G5E1b-luc), pSG424-E6AP (or pSG424-MDM2), and pCMV p53-VP16, along with the indicated plasmids, for 48 h, followed by a luciferase assay. For ( f ), cells were treated with 10 μM MG132 for 4 h before harvesting. Luciferase activity from G5E1b-luc was normalized to the β-gal activity measured in the corresponding cell extract. The values show relative luciferase activity compared to the control’s basal level. Results are presented as mean ± SD from four independent experiments ( n = 4). ND, not detected.

Article Snippet: The HepG2 (#88065) and Hep3B (#88064) cell lines were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea).

Techniques: Transfection, Plasmid Preparation, Expressing, Stable Transfection, Western Blot, Blocking Assay, Immunoprecipitation, Luciferase, Activity Assay

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: E6AP induces ubiquitin-dependent proteasomal degradation of phosphorylated p53 in the presence of HCV Core. HepG2 and Hep3B cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the designated plasmids, for 48 h. For ( c , d ), cells were treated with 10 μM MG132 for 4 h before harvesting. Band intensities were quantified using ImageJ (NIH). ( a , d ) Protein levels were measured by Western blot analysis. ( b ) Phosphorylated p53 at Ser-15 (pSer-15 p53) in cell lysates was immunoprecipitated with an anti-pSer-15 p53 antibody, followed by Western blotting to detect E6AP, MDM2, HCV Core, and HA-Ub-complexed pSer-15 p53. The input shows the levels of the indicated proteins in cell lysates. ( c ) E6AP or MDM2 were immunoprecipitated with the appropriate antibodies and subjected to Western blotting to detect HCV Core, p53, pSer-15 p53, and pSer-20 p53. The input shows the levels of the indicated proteins in cell lysates. ND, not detected.

Article Snippet: The HepG2 (#88065) and Hep3B (#88064) cell lines were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: HCV Core-induced p53 phosphorylation is required for E6AP-mediated proteasomal degradation. ( a ) HepG2 cells transiently transfected with the designated plasmids for 47 h were treated with the indicated concentrations of the ATM inhibitor KU-55933 for 1 h before harvesting, followed by Western blotting. ( b , c ) Cell lysates prepared in ( a ) were immunoprecipitated with anti-p53 or anti-E6AP antibodies, followed by Western blotting. ( d – f ) For mammalian two-hybrid assays, Hep3B cells were transfected with pSG424-E6AP (or pSG424-MDM2), pCMV p53-VP16, and G5E1b-luc, along with the indicated plasmids, for 47 h. Cells were either mock-treated or treated with 10 μM KU-55933 for 1 h before harvesting, followed by a luciferase assay ( n = 4). ND, not detected.

Article Snippet: The HepG2 (#88065) and Hep3B (#88064) cell lines were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea).

Techniques: Phospho-proteomics, Transfection, Western Blot, Immunoprecipitation, Luciferase

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: p53 phosphorylation is sufficient for E6AP to induce p53 ubiquitination. ( a ) HepG2 cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the designated amounts of the E6AP expression plasmid, for 48 h. Cells were treated with the designated concentrations of etoposide for 24 h and MG132 for 4 h before harvesting, followed by Western blotting. ( b – d ) Cell lysates prepared in ( a ) were immunoprecipitated with anti-p53, anti-pSer-15 p53, or anti-E6AP antibodies, followed by Western blotting. ( e – g ) Hep3B cells were transfected with pSG424-E6AP (or pSG424-MDM2), pCMV p53-VP16, and G5E1b-luc, along with the indicated plasmids, for 48 h. Cells were either mock-treated or treated with 10 μM etoposide for 24 h before harvesting, followed by a luciferase assay ( n = 4).

Article Snippet: The HepG2 (#88065) and Hep3B (#88064) cell lines were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Luciferase

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: Phosphorylation of p53 at Ser-15 is crucial for E6AP-mediated protein degradation. Hep3B cells were transfected with either wild-type (WT) p53 or p53 mutants with substitutions at Ser-15 and/or Ser-20, along with the indicated plasmids, for 48 h. ( a , b , d ) Protein levels were determined by Western blotting. ( c , e ) Cell lysates were immunoprecipitated with an anti-p53 antibody, and the immunoprecipitates were analyzed by Western blotting.

Article Snippet: The HepG2 (#88065) and Hep3B (#88064) cell lines were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea).

Techniques: Phospho-proteomics, Transfection, Western Blot, Immunoprecipitation