Journal: bioRxiv

Article Title: Histone succinylation directly inhibits Jumonji domain demethylases and stabilizes repressive chromatin states

doi: 10.64898/2026.05.29.728167

Figure Lengend Snippet: (A) Global histone succinylation measured by LC-MS/MS in HepG2/C3A cells following 48 hours of sodium succinate treatment. (B) Global histone succinylation in 3D cultured HepG2/C3A spheroids following 48 hours and 1 week of sodium succinate treatment. (C) Comparison of the relative abundance of succinyl-lysine residues in H3 and H4, in either treated or untreated, cultured cells (2D) or spheroids (3D). (D) Structure of the nucleosome with H3K64 residues highlighted to demonstrate solvent accessibility. (E) Abundance of succinyl-CoA in the nuclear fraction (left) and non-nuclear fraction (right) following sodium succinate treatment. (F) Total count of HepG2/C3A cells treated with 10 mM sodium succinate continuously for 48 to 144 hours, cell count taken every 48 hours. Data are shown as mean ± SEM; * p-value <0 . 05 in two-tailed Student’s t-test .

Article Snippet: The human hepatocellular carcinoma HepG2/C3A cell line was obtained from the American Type Culture Collection (ATCC, CRL-10741).

Techniques: Liquid Chromatography with Mass Spectroscopy, Cell Culture, Comparison, Solvent, Cell Characterization, Two Tailed Test

Journal: bioRxiv

Article Title: Histone succinylation directly inhibits Jumonji domain demethylases and stabilizes repressive chromatin states

doi: 10.64898/2026.05.29.728167

Figure Lengend Snippet: (A) Schematic of peptide pull-down technique used to identify potential protein interactors with histone succinylation. (B) Volcano plot of fold change and significance of enrichment of nuclear proteins from HepG2/C3A cells to a Ksu synthetic peptide vs. Kac synthetic peptide. (C) Volcano plot of fold change and significance of enrichment of bromodomain proteins to Ksu synthetic histone peptide as compared to the Kac peptide, and (D) Ksu synthetic histone peptide as compared to an unmodified peptide. (E) Volcano plot of fold change and significance of enrichment of JmjC domain demethylases to Ksu synthetic histone peptide as compared to an Kac peptide and (F) Ksu synthetic histone peptide as compared to an unmodified peptide. (G) Crystal structure (PDB 6F6D) of an unmodified histone H3 peptide in the catalytic site of KDM6B. (H) Boltz2 model of a Ksu peptide (AARK(succinyl)A) in the catalytic site of KDM6B. (I) Enrichment of JmjC-domain demethylases to the chromatin-bound proteome of HepG2/C3A cells treated with 10 mM sodium succinate for 48 hours. A -log2 p-value greater than 4 indicates significant enrichment .

Article Snippet: The human hepatocellular carcinoma HepG2/C3A cell line was obtained from the American Type Culture Collection (ATCC, CRL-10741).

Techniques:

Journal: bioRxiv

Article Title: Histone succinylation directly inhibits Jumonji domain demethylases and stabilizes repressive chromatin states

doi: 10.64898/2026.05.29.728167

Figure Lengend Snippet: (A) Known targets of JmjC domain demethylase family enzymes. (B) Relative abundance of methylation on targets of JmjC domain demethylases in HepG2/C3A cells treated with sodium succinate. (C) Relative abundance of methylation on targets of JmjC domain demethylases in HepG2/C3A 3D cells treated with sodium succinate. (D) ChIP-MS shows co-enrichment of succinylated histone peptides with H3K27me3 peptides. Data are shown as mean ± SEM; *p-value <0 . 05, **p-value <0 . 01 in two-tailed Student’s t-test .

Article Snippet: The human hepatocellular carcinoma HepG2/C3A cell line was obtained from the American Type Culture Collection (ATCC, CRL-10741).

Techniques: Methylation, Two Tailed Test

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: C3A viability and LC 50 values of the 40 and 80 nm a AuNP and b PC AuNP. Data represent mean ± S.D. ( n = 3). PC human plasma protein corona, ND not determined, BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, LC 50 median lethal concentration

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Clinical Proteomics, Concentration Assay

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: Time-dependent cellular uptake of the 40 nm a BPEI-AuNP, b LA-AuNP, and c PEG-AuNP, and the 80 nm d BPEI-AuNP, e LA-AuNP, and f PEG-AuNP in the absence and presence of PC in C3A cells up to 24 h. Data represent mean ± S.D. ( n = 3). Letters were significantly different according to Tukey’s HSD test. BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, PC human plasma protein corona, MSD minimum significant difference. * p < 0.05; ** p < 0.005; *** p < 0.0001

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Clinical Proteomics

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: Time- and concentration-dependent ROS/RNS production in C3A cells exposed to a the 40 nm BPEI-AuNP and b the 40 nm PEG-AuNP up to 24 h. Data represent mean ± S.D. ( n = 3). Letters were significantly different according to Tukey’s HSD test. BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, CTRL control, MSD a minimum significant difference, PCN pyocyanin (ROS inducer). ** p < 0.005; *** p < 0.0001

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Concentration Assay, Control

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: An inhibitory effect of AuNP on CYP3A4 activity in C3A cells exposed to the 40 and 80 nm BPEI-, LA-, and PEG-AuNP in the absence and presence of PC for 24 h. Values represent mean ± S.D. ( n = 3). BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, PC human plasma protein corona

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Activity Assay, Clinical Proteomics

Journal: Journal of Extracellular Vesicles

Article Title: Small Extracellular Vesicles Engineered Using Click Chemistry to Express Chimeric Antigen Receptors Show Enhanced Efficacy in Acute Liver Failure

doi: 10.1002/jev2.70044

Figure Lengend Snippet: Validation of the production and targeting of DBCO‐scFv in the HepG2/C3A cell model. (A) Sandwich ELISAs were performed to detect serial dilutions of scFv that bound to ASGR1. scFv was then detected using an HRP‐conjugated anti‐6X His tag antibody, and the absorbance at 450 nm was quantified using an absorbance microplate reader. Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. The figure to the right presents a schematic representation of the sandwich ELISAs for assessing the binding affinity of scFv to ASGR1. (B) Confocal microscopy images (630×) showed that after 30 min, scFv, which was stained with an Alexa Fluor 488‐conjugated anti‐His tag antibody, specifically targeted ASGR1 expressed in the membrane of HepG2/C3A cells. ASGR1 was gradually internalized by the cells as the incubation time increased (0.5, 1, 3, and 6 h). Images of the nuclei (DAPI, blue), cell membrane (PKH26, red), and scFv (green) were merged. Scale bar = 50 µm. (C) Quantification of the mean fluorescence intensity of the scFv targeting ASGR1 in HepG2/C3A cells incubated for different durations (0.5, 1, 3, and 6 h). The mean fluorescence intensity per field was calculated across a total of six fields. The fluorescence intensities are presented as arbitrary units with SEMs. (D) Different molar ratios of galactose to scFv were mixed and added to ELISA wells coated with the ASGR1 antigen. The scFv signal was gradually inhibited concomitant with increasing proportions of galactose. Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. (E) Confocal microscopy images (×400) showing that scFv (1 µM) stained with an Alexa Fluor 488‐conjugated anti‐His tag antibody specifically targeted ASGR1 expressed on the membrane of HepG2/C3A cells and that the targeting was inhibited by preincubation with galactose (500 mM) for 1 h. Images of the nuclei (DAPI, blue), cell membrane (PKH26, red), and scFv (green) were merged. Scale bar = 50 µm. (F) The quantification results revealed that the mean fluorescence intensity of the scFv (1 µM) targeting ASGR1 in HepG2/C3A cells was inhibited after preincubation with galactose (500 mM) for 1 h. The mean fluorescence intensity per field was calculated across a total of six fields. The fluorescence intensities are presented as arbitrary units with SEMs. (G) Schematic representation of DBCO‐scFv production through the conjugation of scFv (S19C) and DBCO‐PEG4‐maleimide by a site‐specific cysteine‐cyclooctyne reaction. (H) Sandwich ELISAs were performed to detect serial dilutions of DBCO‐scFv bound to ASGR1. DBCO‐scFv was detected with biotin‐PEG3‐azide (10 µM) via a click reaction and subsequently interacted with streptavidin HRP and TMB. The absorbance was measured at 450 nm by an absorbance microplate reader. Three replicates per sample were performed. The data are presented as the mean ± SEM. The figure to the right presents a schematic representation of the sandwich ELISAs for assessing the binding affinity of DBCO‐scFv for ASGR1. (I) SDS‐PAGE of DBCO‒scFv conjugates was performed under nonreducing conditions. Coomassie blue staining (left) and fluorescence image (right) of the SDS‐PAGE gel of the DBCO‒scFv conjugates. M: marker ladder. Lane 1: scFv protein control group. Lane 2: Calfluor 488 azide control group. Lane 3: scFv + Calfluor 488 azide control group. Lane 4: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 1:1:1. Lane 5: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 1:2:2. Lane 6: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 1:4:4. Lane 7: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 2:4:4. Lane 8: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 4:4:4. (J) Confocal microscopy images (×400) showing that after 30 min, DBCO‐scFv, detected by click reaction with Calfluor 647 Azide (10 µM), specifically targeted ASGR1 expressed on the membrane of HepG2/C3A cells. DBCO‐scFv was gradually internalized by the cells after a prolonged incubation time (6 h). Images of nuclei (DAPI, blue), cell membranes (FM1‐43, green), and DBCO‐scFv (red) were merged. Scale bar = 50 µm. (K) The mean fluorescence intensity was significantly higher ( p < 0.01) in the DBCO‐scFv‐treated HepG2/C3A cells than in the control cells without scFv treatment. The mean fluorescence intensity per field was calculated across a total of six fields. The fluorescence intensities are presented as arbitrary units with SEMs. (L) Confocal microscopy images (×630) showing that after 3 h of incubation, DBCO‐scFv, detected by click reaction with Calfluor 647 Azide (10 µM), specifically targeted ASGR1 and was internalized into the cytoplasm by HepG2/C3A cells. Images of nuclei (DAPI, blue), cell membranes (FM1‐43, green), and DBCO‐scFv (red) were merged. Scale bar = 50 µm. All data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01.

Article Snippet: The human hepatoblastoma cell line C3A (HepG2/C3A) was obtained from the Bioresource Collection and Research Center (BCRC), Taiwan.

Techniques: Biomarker Discovery, Microplate Reader Absorbance Measurement, Binding Assay, Confocal Microscopy, Staining, Membrane, Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay, Conjugation Assay, SDS Page, Marker, Control

Journal: Journal of Extracellular Vesicles

Article Title: Small Extracellular Vesicles Engineered Using Click Chemistry to Express Chimeric Antigen Receptors Show Enhanced Efficacy in Acute Liver Failure

doi: 10.1002/jev2.70044

Figure Lengend Snippet: CAR‐sEVs significantly enhanced targeting efficacy in the HepG2/C3A cell model. (A) Schematic representation of the conjugation of N₃‐sEVs with DBCO‐Cy5 (red) and DBCO‐AF488‐scFv (green) using click chemistry to generate CAR‐sEVs. (B) Flow cytometry histograms showing the fluorescence intensity of HepG2/C3A cells treated with increasing doses (10⁷, 10⁸, or 10⁹ particles) of CAR‐sEVs or N 3 ‐sEVs for 18 h. The fluorescence was detected in the Alexa Fluor 488 and PE‐Cy5 channels. Sham treatment (no sEVs) was included as a control. (C) Quantification of targeting efficiency (%) of N 3 ‐sEVs and CAR‐sEVs at different doses (10⁷, 10⁸, or 10⁹ particles). (D) Confocal microscopy images (630×) showing the cellular uptake of N₃‐sEVs and CAR‐sEVs in HepG2/C3A cells at various time points (0.5, 1, 2, 4, and 6 h). The images illustrate the nuclei (DAPI, blue), sEV particles with azido groups (DBCO‐Cy5, red), and DBCO‐scFv (labelled with Alexa Fluor‐488, green). The merged DIC (differential interference contrast) images to fluorescent images. Scale bar = 10 µm. (E) Flow cytometry was used to measure the mean fluorescence intensity in HepG2/C3A cells treated with N₃‐sEVs (grey) or CAR‐sEVs (red) over 6 h. All data are presented as the mean ± SEM. *** p < 0.001.

Article Snippet: The human hepatoblastoma cell line C3A (HepG2/C3A) was obtained from the Bioresource Collection and Research Center (BCRC), Taiwan.

Techniques: Conjugation Assay, Flow Cytometry, Fluorescence, Control, Confocal Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Small Extracellular Vesicles Engineered Using Click Chemistry to Express Chimeric Antigen Receptors Show Enhanced Efficacy in Acute Liver Failure

doi: 10.1002/jev2.70044

Figure Lengend Snippet: Compared to unmodified sEVs, CAR‐sEVs enhanced the therapeutic effects against the APAP challenge in C3A cells. (A) (i) APAP dose‐dependently reduced the viability of HepG2/C3A cells, with an IC50 of 10 mM APAP. (ii) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with pcMSC‐CM in the presence of APAP (10 mM), as determined by a luminescent cell viability assay. (iii) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with sEVs from pcMSCs in the presence of APAP (10 mM), as determined by a luminescent cell viability assay. (iv) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with pcMSC‐CM in the presence of increasing doses of APAP (0, 10, 20, 40 mM). Three replicates per sample were performed, and the experiment was repeated three times. (v) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with sEVs from pcMSCs in the presence of increasing doses of APAP (0, 10, 20, 40 mM). Three replicates per sample were performed, and the experiment was repeated three times. (vi) Compared with corresponding doses of sEVs, CAR‐sEVs significantly increased the viability of HepG2/C3A cells in a dose‐dependent manner after 48 h of incubation in the presence of APAP (10 mM). Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) The heatmap displays the predicted interactions between pcMSC‐sEVs miRNAs and genes of interest categorized into four major functional groups: anti‐inflammation (TGF‐β1, TGF‐β2, TGF‐β3, IL‐6, IL‐1β, TNF‐α, and NKB1), liver regeneration promotion (PTEN, MKK4, MFAP4), anti‐apoptosis (caspase‐3, caspase‐7, caspase‐8, caspase‐9, Bax and Bim), and anti‐fibrosis (COL1A1 and COL3A1). TargetScanHuman 8.0 was used to calculate the targetability score, with only the top miRNAs having a TargetScore context++ score of ≤ −0.1 selected. The miRNAs were selected using TargetScanHuman 8.0, with a TargetScore context++ score ≤ −0.1. The miRNA expression is shown as log2 RPM (reads per million), with higher values indicating greater miRNA expression. (C) (i) The line chart showing the highly expressed miRNAs significantly involved in specific biological processes of negative regulation of hepatocyte proliferation (GO:2000346) and positive regulation of the apoptotic process (GO:0043065). (ii) The Venn diagram illustrates the overlap of miRNAs involved in two biological processes. The overlapping area represents 15 miRNAs predicted to target genes involved in both processes. (iii) The top miRNAs from this overlap are listed in the table below.

Article Snippet: The human hepatoblastoma cell line C3A (HepG2/C3A) was obtained from the Bioresource Collection and Research Center (BCRC), Taiwan.

Techniques: Incubation, Cell Viability Assay, Functional Assay, Expressing