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Nanjing Jiancheng Bioengineering Research Institute Co Ltd hepg2 cells assay kits
Hepg2 Cells Assay Kits, supplied by Nanjing Jiancheng Bioengineering Research Institute Co Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hepg2+cells+assay+kits/assay+cells+hepg2+kits/pm42160717-73-5-62
Average 86 stars, based on 1 article reviews

hepg2 cells assay kits - by Bioz Stars, 2026-10

86/100 stars

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AST Assay:

Article Title: Protective effect of cornel iridoid glycoside against isoniazid- and rifampicin- induced liver injury via regulating JAK/STAT pathway.
Article Snippet: Background: Hepatotoxicity associated with Isoniazid (INH) and Rifampicin (RIF) is one of the major i mpediments in antituberculosis therapy.. Purpose: We aimed to investigate the protective effects of the cornel iridoid glycoside (CIG) on liver injury c aused by INH and RIF in vivo and in vitro.. Methods: Liver injury model was induced by INH and RIF (150 mg/kg).

Multiple Displacement Amplification:

Image Search Results

A: Cell viability of HepG2 cells was assessed by CCK-8 assay after transfection with the indicated miRNA mimics. Data are presented as the mean ± SD from three independent experiments. B: Representative western blot protein bands (left) and quantitative expression (right) of SEC61G in HepG2 cells after transfection with miRNAs mimics. Data are presented as the mean ± SD from three independent experiments. C: Dual-luciferase reporter assay of interactions between miR-651-3p and SEC61G; D: Dual-luciferase reporter assay for determining the interactions between miR-488-3p and SEC61G. *P < 0.05.

Journal: PLOS One

Article Title: MicroRNA-488-3p-loaded engineered exosomes inhibit proliferation, migration and invasion of hepatocellular carcinoma by targeting SEC61G

doi: 10.1371/journal.pone.0341056

Figure Lengend Snippet: A: Cell viability of HepG2 cells was assessed by CCK-8 assay after transfection with the indicated miRNA mimics. Data are presented as the mean ± SD from three independent experiments. B: Representative western blot protein bands (left) and quantitative expression (right) of SEC61G in HepG2 cells after transfection with miRNAs mimics. Data are presented as the mean ± SD from three independent experiments. C: Dual-luciferase reporter assay of interactions between miR-651-3p and SEC61G; D: Dual-luciferase reporter assay for determining the interactions between miR-488-3p and SEC61G. *P < 0.05.

Article Snippet: Viability of HepG2 cells in each group were evaluated using CCK-8 assay (#HY-K0301, MedChemExpress, New Jersey, USA) as the manual described.

Techniques: CCK-8 Assay, Transfection, Western Blot, Expressing, Luciferase, Reporter Assay

Representative immunofluorescence images of PKH-67-labelled exosomes after co-incubation with HepG2 cells (scale bars: 20 µm).

Journal: PLOS One

Article Title: MicroRNA-488-3p-loaded engineered exosomes inhibit proliferation, migration and invasion of hepatocellular carcinoma by targeting SEC61G

doi: 10.1371/journal.pone.0341056

Figure Lengend Snippet: Representative immunofluorescence images of PKH-67-labelled exosomes after co-incubation with HepG2 cells (scale bars: 20 µm).

Article Snippet: Viability of HepG2 cells in each group were evaluated using CCK-8 assay (#HY-K0301, MedChemExpress, New Jersey, USA) as the manual described.

Techniques: Immunofluorescence, Incubation

A: Cell viability of HepG2 cells determined by CCK-8 assay after co-incubated with Exo-miR-488-3p or Exo-NC or controls; Data are presented as the mean ± SD from three independent experiments. B and C: Representative images and quantitative analysis of HepG2 cells colony formation after co-incubated with Exo-miR-488-3p or Exo-NC or controls; D: Quantitative analysis of apoptosis. Data are presented as the mean ± SD from three independent experiments. * P < 0.05. E: Representative flow cytometry plots of apoptotic HepG2 cells.

Journal: PLOS One

Article Title: MicroRNA-488-3p-loaded engineered exosomes inhibit proliferation, migration and invasion of hepatocellular carcinoma by targeting SEC61G

doi: 10.1371/journal.pone.0341056

Figure Lengend Snippet: A: Cell viability of HepG2 cells determined by CCK-8 assay after co-incubated with Exo-miR-488-3p or Exo-NC or controls; Data are presented as the mean ± SD from three independent experiments. B and C: Representative images and quantitative analysis of HepG2 cells colony formation after co-incubated with Exo-miR-488-3p or Exo-NC or controls; D: Quantitative analysis of apoptosis. Data are presented as the mean ± SD from three independent experiments. * P < 0.05. E: Representative flow cytometry plots of apoptotic HepG2 cells.

Article Snippet: Viability of HepG2 cells in each group were evaluated using CCK-8 assay (#HY-K0301, MedChemExpress, New Jersey, USA) as the manual described.

Techniques: CCK-8 Assay, Incubation, Flow Cytometry

HepG2 cells migration (A) and invasion (B) were determined by Transwell assay after co-incubated with Exo-miR-488-3p or Exo-NC or controls. Data are presented as the mean ± SD from three independent experiments. C, HepG2 cells migration determined by wound-healing assay after co-incubation with Exo-miR-488-3p or Exo-NC or controls. *P < 0.05. Data are presented as the mean ± SD from three independent experiments.

Journal: PLOS One

Article Title: MicroRNA-488-3p-loaded engineered exosomes inhibit proliferation, migration and invasion of hepatocellular carcinoma by targeting SEC61G

doi: 10.1371/journal.pone.0341056

Figure Lengend Snippet: HepG2 cells migration (A) and invasion (B) were determined by Transwell assay after co-incubated with Exo-miR-488-3p or Exo-NC or controls. Data are presented as the mean ± SD from three independent experiments. C, HepG2 cells migration determined by wound-healing assay after co-incubation with Exo-miR-488-3p or Exo-NC or controls. *P < 0.05. Data are presented as the mean ± SD from three independent experiments.

Article Snippet: Viability of HepG2 cells in each group were evaluated using CCK-8 assay (#HY-K0301, MedChemExpress, New Jersey, USA) as the manual described.

Techniques: Migration, Transwell Assay, Incubation, Wound Healing Assay

Fig. 4 ACSL4 promotes the proliferation of hepatoblastoma cells in vitro and tumor growth in vivo. (A) The viability of HepG2 and Huh6 cells transfected with siACSL4 or NC was detected by CCK8 assays. (B) Colony formation assays were performed in ACSL4-silenced and control cells. *p < 0.05, **p < 0.01, ***p < 0.001

Journal: BMC cancer

Article Title: ACSL4 promotes the formation of the proliferative subtype in hepatoblastoma.

doi: 10.1186/s12885-025-13592-4

Figure Lengend Snippet: Fig. 4 ACSL4 promotes the proliferation of hepatoblastoma cells in vitro and tumor growth in vivo. (A) The viability of HepG2 and Huh6 cells transfected with siACSL4 or NC was detected by CCK8 assays. (B) Colony formation assays were performed in ACSL4-silenced and control cells. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Total RNA was extracted from ACSL4 knockdown and negative control lentivirus-infected HepG2 cells using the RNAstorm Fresh Cell and Tissue RNA Isolation Kit from Cell Data Sciences (USA).

Techniques: In Vitro, In Vivo, Transfection, Control

Fig. 6 ACSL4 promotes the formation of proliferative HB subtype via MAVS/IRF3/STAT1 signailing pathway. (A) GSEA enrichment analysis results of dif ferential genes between ACSL4 high-expression and ACSL4 low-expression hepatoblastoma tissues in GSE133039 dataset. (B) Enrichment results for IFNα response via GSEA of the hepatoblastoma tissues in GSE133039 dataset. (C) Enrichment results for IFNγ response via GSEA of the hepatoblastoma tissues in GSE133039 dataset. (D) The enrichment results of the interferon-γ response signaling pathway between the ACSL4-high hepatoblastoma subgroup and the ACSL4-low hepatoblastoma subgroup in GSE186975 dataset. (E) The enrichment results of the interferon-γ response signaling pathway between ACSL4 knockdown HepG2 cell line and negative control cells. (F) The MAVS/IRF3/STAT1 interferon response pathway-associated proteins were detected by Western blot in ACSL4 knockdown cells and controls. (G) Inferred intercellular communication network among ACSL4 high tumor cells and other cell clusters, shown in a circle plot. The thickness of the lines represents the strength of cell communication. (H) Circle plots showing cell-cell communications between ACSL4 low tumor cells and other cell clusters

Journal: BMC cancer

Article Title: ACSL4 promotes the formation of the proliferative subtype in hepatoblastoma.

doi: 10.1186/s12885-025-13592-4

Figure Lengend Snippet: Fig. 6 ACSL4 promotes the formation of proliferative HB subtype via MAVS/IRF3/STAT1 signailing pathway. (A) GSEA enrichment analysis results of dif ferential genes between ACSL4 high-expression and ACSL4 low-expression hepatoblastoma tissues in GSE133039 dataset. (B) Enrichment results for IFNα response via GSEA of the hepatoblastoma tissues in GSE133039 dataset. (C) Enrichment results for IFNγ response via GSEA of the hepatoblastoma tissues in GSE133039 dataset. (D) The enrichment results of the interferon-γ response signaling pathway between the ACSL4-high hepatoblastoma subgroup and the ACSL4-low hepatoblastoma subgroup in GSE186975 dataset. (E) The enrichment results of the interferon-γ response signaling pathway between ACSL4 knockdown HepG2 cell line and negative control cells. (F) The MAVS/IRF3/STAT1 interferon response pathway-associated proteins were detected by Western blot in ACSL4 knockdown cells and controls. (G) Inferred intercellular communication network among ACSL4 high tumor cells and other cell clusters, shown in a circle plot. The thickness of the lines represents the strength of cell communication. (H) Circle plots showing cell-cell communications between ACSL4 low tumor cells and other cell clusters

Techniques: Expressing, Knockdown, Negative Control, Western Blot

Effects of different concentrations of AAG-3-I on the viability of HepG2 cells ( A ); effects of different concentrations of insulin on the glucose consumption of HepG2 cells ( B ). Data are presented as mean ± SD. Different lowercase letters indicate significant differences ( p < 0.05) in multifactorial analyses among the groups.

Journal: Foods

Article Title: Structural Characterization and Hypoglycemic Activity of a Glycoprotein Extracted from Auricularia Auricula

doi: 10.3390/foods13233859

Figure Lengend Snippet: Effects of different concentrations of AAG-3-I on the viability of HepG2 cells ( A ); effects of different concentrations of insulin on the glucose consumption of HepG2 cells ( B ). Data are presented as mean ± SD. Different lowercase letters indicate significant differences ( p < 0.05) in multifactorial analyses among the groups.

Article Snippet: The effect of the AAG-3-I on HepG2 cell viability was measured using a conventional Cell Counting Kit-8 (Dojindo, Tokyo, Japan).

Techniques:

Effects of different concentrations of AAG-3-I on the glycogen content of insulin-resistant HepG2 cells ( A ). Data are presented as mean ± SD. Different lowercase letters indicate significant differences ( p < 0.05) in multifactorial analyses among the groups. Effects of different concentrations of AAG-3-I on the glycogen PAS staining of insulin-resistant HepG2 cells ( B ).

Journal: Foods

Article Title: Structural Characterization and Hypoglycemic Activity of a Glycoprotein Extracted from Auricularia Auricula

doi: 10.3390/foods13233859

Figure Lengend Snippet: Effects of different concentrations of AAG-3-I on the glycogen content of insulin-resistant HepG2 cells ( A ). Data are presented as mean ± SD. Different lowercase letters indicate significant differences ( p < 0.05) in multifactorial analyses among the groups. Effects of different concentrations of AAG-3-I on the glycogen PAS staining of insulin-resistant HepG2 cells ( B ).

Article Snippet: The effect of the AAG-3-I on HepG2 cell viability was measured using a conventional Cell Counting Kit-8 (Dojindo, Tokyo, Japan).

Techniques: Staining

Effects of different concentrations of AAG-3-I on the glucose consumption of insulin-resistant HepG2 cells ( A ); effects of different concentrations of AAG-3-I on the HK and PK activities of insulin-resistant HepG2 cells ( B ). Data are presented as mean ± SD. Different letters indicate significant differences ( p < 0.05) in multifactorial analyses among the groups.

Journal: Foods

Article Title: Structural Characterization and Hypoglycemic Activity of a Glycoprotein Extracted from Auricularia Auricula

doi: 10.3390/foods13233859

Figure Lengend Snippet: Effects of different concentrations of AAG-3-I on the glucose consumption of insulin-resistant HepG2 cells ( A ); effects of different concentrations of AAG-3-I on the HK and PK activities of insulin-resistant HepG2 cells ( B ). Data are presented as mean ± SD. Different letters indicate significant differences ( p < 0.05) in multifactorial analyses among the groups.

Article Snippet: The effect of the AAG-3-I on HepG2 cell viability was measured using a conventional Cell Counting Kit-8 (Dojindo, Tokyo, Japan).

Techniques:

( a ) Phylogenetic tree of A. baumannii peptidoglycan hydrolases homolog to P307. ( b ) Multiple sequence alignment of PHAb7, PHAb8, PHAb9, PHAb10, PHAb11, PlyF307, and AcLys using MEGAX software and ClustalW algorithm. Secondary structures are annotated by Espript 3.0 website.

Journal: eLife

Article Title: Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

doi: 10.7554/eLife.98266

Figure Lengend Snippet: ( a ) Phylogenetic tree of A. baumannii peptidoglycan hydrolases homolog to P307. ( b ) Multiple sequence alignment of PHAb7, PHAb8, PHAb9, PHAb10, PHAb11, PlyF307, and AcLys using MEGAX software and ClustalW algorithm. Secondary structures are annotated by Espript 3.0 website.

Article Snippet: The cytotoxicity of PHAb10 against HepG2 cells was determined by a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s protocol.

Techniques: Sequencing, Software

( a ) Purified proteins were analyzed on a 12% SDS-PAGE gel. M: standard protein marker. ( b ) Halo of the five PHAbs on a lawn of A. baumannii 3437. The concentrations of each peptidoglycan hydrolase are 2.5 μg/ml, 5 μg/ml, and 10 μg/ml. ( c ) pH stability of PHAb10 and PHAb11. Log-phase A. baumannii 3437 cells were incubated with 50 μg/ml peptidoglycan hydrolases at 37°C for 1 hr at the indicated pH conditions. pH buffer: 20 mM boric acid and 20 mM phosphoric acid were mixed in equal volumes and adjusted to different pH values with HCl. ( d ) Log-phase A. baumannii 3437 cells were incubated with 50 μg/ml peptidoglycan hydrolase in 20 mM Tris-HCl (pH 7.4) in the presence of different concentrations of NaCl (left) and urea (right) for 1 hr at 37°C. ( e ) Relative activity of five PHAbs against A. baumannii 3437 after 1 hr storage at different temperatures. In all cases, viable bacteria were counted by serial dilution and plating assay. Experiments were carried out in triplicate (n=3-9); error bars indicate standard deviation. Figure 2—figure supplement 1—source data 1. PDF file containing original SDS-PAGE gel for , indicating the relevant bands. Figure 2—figure supplement 1—source data 2. Original file for SDS-PAGE analysis displayed in .

Journal: eLife

Article Title: Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

doi: 10.7554/eLife.98266

Figure Lengend Snippet: ( a ) Purified proteins were analyzed on a 12% SDS-PAGE gel. M: standard protein marker. ( b ) Halo of the five PHAbs on a lawn of A. baumannii 3437. The concentrations of each peptidoglycan hydrolase are 2.5 μg/ml, 5 μg/ml, and 10 μg/ml. ( c ) pH stability of PHAb10 and PHAb11. Log-phase A. baumannii 3437 cells were incubated with 50 μg/ml peptidoglycan hydrolases at 37°C for 1 hr at the indicated pH conditions. pH buffer: 20 mM boric acid and 20 mM phosphoric acid were mixed in equal volumes and adjusted to different pH values with HCl. ( d ) Log-phase A. baumannii 3437 cells were incubated with 50 μg/ml peptidoglycan hydrolase in 20 mM Tris-HCl (pH 7.4) in the presence of different concentrations of NaCl (left) and urea (right) for 1 hr at 37°C. ( e ) Relative activity of five PHAbs against A. baumannii 3437 after 1 hr storage at different temperatures. In all cases, viable bacteria were counted by serial dilution and plating assay. Experiments were carried out in triplicate (n=3-9); error bars indicate standard deviation. Figure 2—figure supplement 1—source data 1. PDF file containing original SDS-PAGE gel for , indicating the relevant bands. Figure 2—figure supplement 1—source data 2. Original file for SDS-PAGE analysis displayed in .

Techniques: Purification, SDS Page, Marker, Incubation, Activity Assay, Bacteria, Serial Dilution, Standard Deviation

( a ) Heat map showing the halo size formed by different concentrations of peptidoglycan hydrolases on different bacterial lawns. ( b ) Bactericidal activity of five peptidoglycan hydrolases (50 μg/ml) in 20 mM Tris-HCl (pH 7.4) at 37°C for 1 hr against different exponential bacteria. ( c ) Time-dependent bactericidal activity of 50 μg/ml PHAb10 or PHAb11 against exponential A. baumannii in 20 mM Tris-HCl (pH 7.4). ( d ) Bactericidal activity of 50 μg/ml PHAb10 or PHAb11 in 20 mM Tris-HCl (pH 7.4) at 37°C for 1 hr against different bacteria in exponential and stationary phases. ( e–f ) Dose-dependent bactericidal activity of PHAb10 and PHAb11 against multiple exponential bacteria in 20 mM Tris-HCl (pH 7.4) at 37°C for 1 hr. ( g–h ) Susceptibility of various Gram-negative ( g ) and Gram-positive ( h ) bacterial strains to PHAb10 and PHAb11. Exponential cultures of each bacterium are treated with 50 μg/ml PHAb10 or PHAb11 for 1 hr at 37°C and residual viable bacterial cells are counted by plating serial dilutions onto agar plates. For Enterococcus faecalis , 10 μg/ml of each peptidoglycan hydrolase is used. ( i ) Thermal stability of PHAb10 and PHAb11. Each enzyme is stored at different temperatures for 1 hr, cooled to room temperature, and then incubated with exponential A. baumannii 3437 in 20 mM Tris-HCl (pH 7.4) at a final concentration of 50 μg/ml at 37°C for 1 hr. Viable bacteria are counted after each treatment by plating serial dilutions on Lysogeny Broth (LB) agar. All assays were performed with at least three biological replicates (n=3-9). Dash lines represent the limit of detection and data below the limit of detection is not shown.

Journal: eLife

Article Title: Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

doi: 10.7554/eLife.98266

Figure Lengend Snippet: ( a ) Heat map showing the halo size formed by different concentrations of peptidoglycan hydrolases on different bacterial lawns. ( b ) Bactericidal activity of five peptidoglycan hydrolases (50 μg/ml) in 20 mM Tris-HCl (pH 7.4) at 37°C for 1 hr against different exponential bacteria. ( c ) Time-dependent bactericidal activity of 50 μg/ml PHAb10 or PHAb11 against exponential A. baumannii in 20 mM Tris-HCl (pH 7.4). ( d ) Bactericidal activity of 50 μg/ml PHAb10 or PHAb11 in 20 mM Tris-HCl (pH 7.4) at 37°C for 1 hr against different bacteria in exponential and stationary phases. ( e–f ) Dose-dependent bactericidal activity of PHAb10 and PHAb11 against multiple exponential bacteria in 20 mM Tris-HCl (pH 7.4) at 37°C for 1 hr. ( g–h ) Susceptibility of various Gram-negative ( g ) and Gram-positive ( h ) bacterial strains to PHAb10 and PHAb11. Exponential cultures of each bacterium are treated with 50 μg/ml PHAb10 or PHAb11 for 1 hr at 37°C and residual viable bacterial cells are counted by plating serial dilutions onto agar plates. For Enterococcus faecalis , 10 μg/ml of each peptidoglycan hydrolase is used. ( i ) Thermal stability of PHAb10 and PHAb11. Each enzyme is stored at different temperatures for 1 hr, cooled to room temperature, and then incubated with exponential A. baumannii 3437 in 20 mM Tris-HCl (pH 7.4) at a final concentration of 50 μg/ml at 37°C for 1 hr. Viable bacteria are counted after each treatment by plating serial dilutions on Lysogeny Broth (LB) agar. All assays were performed with at least three biological replicates (n=3-9). Dash lines represent the limit of detection and data below the limit of detection is not shown.

Techniques: Activity Assay, Bacteria, Incubation, Concentration Assay

( a ) Schematic representation of PHAb10 and PHAb11 domains. ( b ) Bactericidal activity of peptides derived from PHAb10 and PHAb11. Bacterial cells are treated with 50 μg/ml P10-CP, P11-NP, or P11-CP in 20 mM Tris-HCl at 37°C for 1 hr. ( c–d ) Bactericidal activity of different truncations of PHAb10 and PHAb11. Exponential A. baumannii 3437 cells are incubated with 50 μg/ml of each truncated fragment in 20 mM Tris-HCl for 1 hr at 37°C. Dash lines represent the limit of detection and data below the limit of detection is not shown. ( e ) Peptidoglycan hydrolytic activity of different truncations of PHAb10 and PHAb11. 0.1 μg of each truncated fragment is dropped onto autoclaved lawns of A. baumannii 3437 and S. aureus N315 and incubated at 37°C for 4 hr. The groups treated with an equal volume of 20 mM Tris-HCl served as controls. ( f ) Schematic diagram of the bactericidal mechanisms of PHAb10 against Gram-negative bacteria (top) and Gram-positive bacteria (bottom). All assays ( b-e ) were performed with at least three biological replicates (n=3-9).

Journal: eLife

Article Title: Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

doi: 10.7554/eLife.98266

Figure Lengend Snippet: ( a ) Schematic representation of PHAb10 and PHAb11 domains. ( b ) Bactericidal activity of peptides derived from PHAb10 and PHAb11. Bacterial cells are treated with 50 μg/ml P10-CP, P11-NP, or P11-CP in 20 mM Tris-HCl at 37°C for 1 hr. ( c–d ) Bactericidal activity of different truncations of PHAb10 and PHAb11. Exponential A. baumannii 3437 cells are incubated with 50 μg/ml of each truncated fragment in 20 mM Tris-HCl for 1 hr at 37°C. Dash lines represent the limit of detection and data below the limit of detection is not shown. ( e ) Peptidoglycan hydrolytic activity of different truncations of PHAb10 and PHAb11. 0.1 μg of each truncated fragment is dropped onto autoclaved lawns of A. baumannii 3437 and S. aureus N315 and incubated at 37°C for 4 hr. The groups treated with an equal volume of 20 mM Tris-HCl served as controls. ( f ) Schematic diagram of the bactericidal mechanisms of PHAb10 against Gram-negative bacteria (top) and Gram-positive bacteria (bottom). All assays ( b-e ) were performed with at least three biological replicates (n=3-9).

Techniques: Activity Assay, Derivative Assay, Incubation, Bacteria

( a ) Overall structure of PHAb8 monomer. ( b ) Dimeric structure of PHAb10. Chain A is colored in green, Chain B is colored in magenta, and the gray stick denotes the rotation axis. The electrostatic surface of PHAb10 with two potential substrate-binding cavities are shown (negative = red; positive = blue). ( c ) Dimeric structure of PHAb11. Chain A is colored in wheat, Chain B is colored in green-cyan, and the red stick denotes the rotation axis. The electrostatic surface of PHAb11 with two potential substrate binding cavities is also shown (negative = red; positive = blue). ( d ) Superimposition of monomeric PHAb8, PHAb10, and PHAb11. PHAb8 is shown in light blue, PHAb10 in green, and PHAb11 in wheat. Residues involved in the catalytic triad are shown in sticks. ( e–f ) Residual bactericidal activity of PHAb10, PHAb11, and their truncation variants. Each truncation variant is treated at 100°C for 1 hr, stored at 25°C for 1 hr, and then tested for bactericidal activity by a log-killing assay. All assays were performed with at least three biological replicates (n=3-9). Dash lines represent the limit of detection and data below the limit of detection is not shown. ( g ) Thermal unfolding curves of PHAb8, PHAb10, and PHAb11 from 20°C to 100°C as determined by nano-differential scanning fluorimetry (nanoDSF). Values on the y-axis represent the first derivative of the fluorescence ratio at 350 nm and 330 nm. Peaks represent the transition temperature of each protein. ( h–j ) Circular dichroism (CD) spectra of PHAb8 ( h ), PHAb10 ( i ), and PHAb11 ( j ) at 90°C and temperatures close to their transition temperatures. ( k–o ) CD spectra of PHAb10, PHAb11, and their truncation variants before and after heat treatment. Each domain fragment is treated at 100°C for 1 hr, stored at 25°C for additional hour prior to CD detection.

Journal: eLife

Article Title: Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

doi: 10.7554/eLife.98266

Figure Lengend Snippet: ( a ) Overall structure of PHAb8 monomer. ( b ) Dimeric structure of PHAb10. Chain A is colored in green, Chain B is colored in magenta, and the gray stick denotes the rotation axis. The electrostatic surface of PHAb10 with two potential substrate-binding cavities are shown (negative = red; positive = blue). ( c ) Dimeric structure of PHAb11. Chain A is colored in wheat, Chain B is colored in green-cyan, and the red stick denotes the rotation axis. The electrostatic surface of PHAb11 with two potential substrate binding cavities is also shown (negative = red; positive = blue). ( d ) Superimposition of monomeric PHAb8, PHAb10, and PHAb11. PHAb8 is shown in light blue, PHAb10 in green, and PHAb11 in wheat. Residues involved in the catalytic triad are shown in sticks. ( e–f ) Residual bactericidal activity of PHAb10, PHAb11, and their truncation variants. Each truncation variant is treated at 100°C for 1 hr, stored at 25°C for 1 hr, and then tested for bactericidal activity by a log-killing assay. All assays were performed with at least three biological replicates (n=3-9). Dash lines represent the limit of detection and data below the limit of detection is not shown. ( g ) Thermal unfolding curves of PHAb8, PHAb10, and PHAb11 from 20°C to 100°C as determined by nano-differential scanning fluorimetry (nanoDSF). Values on the y-axis represent the first derivative of the fluorescence ratio at 350 nm and 330 nm. Peaks represent the transition temperature of each protein. ( h–j ) Circular dichroism (CD) spectra of PHAb8 ( h ), PHAb10 ( i ), and PHAb11 ( j ) at 90°C and temperatures close to their transition temperatures. ( k–o ) CD spectra of PHAb10, PHAb11, and their truncation variants before and after heat treatment. Each domain fragment is treated at 100°C for 1 hr, stored at 25°C for additional hour prior to CD detection.

Techniques: Binding Assay, Activity Assay, Variant Assay, Nano Differential Scanning Fluorimetry, Fluorescence, Circular Dichroism

( a–c ) Structures of monomeric PHAb8 ( a ), PHAb10 ( b ), and PHAb11 ( c ). ( d ) Structural superposition of PHAb8, PHAb10, PHAb11, and five classical T4L-like lysozymes that share high similarity from Dali search. PHAb8 is shown in light blue, PHAb10 in green, PHAb11 in wheat, AcLys in red, LysF1 in blue, SpmX-Mur- Ae in yellow, P22 in purple, and R21 in orange. ( e–h ) Structural alignment of PHAb8, PHAb10, PHAb11 with various lysozymes including hen egg white lysozyme (HEWL) (e, cyan), goose egg white lysozyme (GEWL) (f, deep salmon), phage lambda lytic transglycosylase (g, yellow), and T4 lysozyme (h, orange). Residues in the conserved catalytic triad are shown as sticks. ( j ) Nano-differential scanning fluorimetry (nanoDSF) scattering plots of PHAb8, PHAb10, and PHAb11 at different temperatures.

Journal: eLife

Article Title: Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

doi: 10.7554/eLife.98266

Figure Lengend Snippet: ( a–c ) Structures of monomeric PHAb8 ( a ), PHAb10 ( b ), and PHAb11 ( c ). ( d ) Structural superposition of PHAb8, PHAb10, PHAb11, and five classical T4L-like lysozymes that share high similarity from Dali search. PHAb8 is shown in light blue, PHAb10 in green, PHAb11 in wheat, AcLys in red, LysF1 in blue, SpmX-Mur- Ae in yellow, P22 in purple, and R21 in orange. ( e–h ) Structural alignment of PHAb8, PHAb10, PHAb11 with various lysozymes including hen egg white lysozyme (HEWL) (e, cyan), goose egg white lysozyme (GEWL) (f, deep salmon), phage lambda lytic transglycosylase (g, yellow), and T4 lysozyme (h, orange). Residues in the conserved catalytic triad are shown as sticks. ( j ) Nano-differential scanning fluorimetry (nanoDSF) scattering plots of PHAb8, PHAb10, and PHAb11 at different temperatures.

Techniques: Nano Differential Scanning Fluorimetry

PHAb8, PHAb10, and PHAb11 encoding sequences were retrieved from A. baumannii strain 9201, A. baumannii strain LWSS-03-02-11B, and A. baumannii strain ABUH797 were analyzed by PhageBoost tool. The results show that all three genes are indeed located in the prophage region.

Journal: eLife

Article Title: Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

doi: 10.7554/eLife.98266

Figure Lengend Snippet: PHAb8, PHAb10, and PHAb11 encoding sequences were retrieved from A. baumannii strain 9201, A. baumannii strain LWSS-03-02-11B, and A. baumannii strain ABUH797 were analyzed by PhageBoost tool. The results show that all three genes are indeed located in the prophage region.

Techniques:

( a ) Intermolecular interactions of PHAb10 dimer. The residues involved are shown as sticks. ( b ) SDS-PAGE analysis of PHAb10 with or without chemical cross-linker. ( c ) Native-PAGE analysis of PHAb10 and its variants. Marker: the upper band represents BSA (66.4 kDa), and the lower band represents lysozyme (14 kDa). ( d ) Bactericidal activity of PHAb10 and its variants before and after heat treatment. Each protein is incubated for 1 hr at 4°C or 100°C, stored at 25°C for an additional 1 hr, and then examined for bactericidal activity in 20 mM Tris-HCl (pH 7.4) at a concentration of 50 μg/ml against exponential A. baumannii 3437 cells for 1 hr. All assays were performed with at least three biological replicates (n=6-9). Dash lines represent the limit of detection and data below the limit of detection is not shown. Data are analyzed by two-tailed Student’s t-tests. ns: statistically not significant; *: p<0.05. ( e–f ) Circular dichroism spectra of PHAb10 and its mutants before and after treatment at 100°C for 1 hr. ( g ) Zipper model showing the thermodynamics of PHAb10 dimer. Figure 5—source data 1. PDF file containing original SDS/Native-PAGE gels for , indicating the relevant bands. The left gel corresponds to ; the middle and the right gels correspond to . Figure 5—source data 2. Original file for SDS/Native-PAGE analysis displayed in .

Journal: eLife

Article Title: Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

doi: 10.7554/eLife.98266

Figure Lengend Snippet: ( a ) Intermolecular interactions of PHAb10 dimer. The residues involved are shown as sticks. ( b ) SDS-PAGE analysis of PHAb10 with or without chemical cross-linker. ( c ) Native-PAGE analysis of PHAb10 and its variants. Marker: the upper band represents BSA (66.4 kDa), and the lower band represents lysozyme (14 kDa). ( d ) Bactericidal activity of PHAb10 and its variants before and after heat treatment. Each protein is incubated for 1 hr at 4°C or 100°C, stored at 25°C for an additional 1 hr, and then examined for bactericidal activity in 20 mM Tris-HCl (pH 7.4) at a concentration of 50 μg/ml against exponential A. baumannii 3437 cells for 1 hr. All assays were performed with at least three biological replicates (n=6-9). Dash lines represent the limit of detection and data below the limit of detection is not shown. Data are analyzed by two-tailed Student’s t-tests. ns: statistically not significant; *: p<0.05. ( e–f ) Circular dichroism spectra of PHAb10 and its mutants before and after treatment at 100°C for 1 hr. ( g ) Zipper model showing the thermodynamics of PHAb10 dimer. Figure 5—source data 1. PDF file containing original SDS/Native-PAGE gels for , indicating the relevant bands. The left gel corresponds to ; the middle and the right gels correspond to . Figure 5—source data 2. Original file for SDS/Native-PAGE analysis displayed in .

Techniques: SDS Page, Clear Native PAGE, Marker, Activity Assay, Incubation, Concentration Assay, Two Tailed Test, Circular Dichroism

Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin for 24 hr. After cells were exposed to a series of concentrations of PHAb10 (0, 6.25, 12.5, 25, 50, and 100 μg/ml) for an additional 24 hr, the relative viability of cells after each treatment was determined by Cell Counting Kit-8 (CCK-8) assay. All assays were performed with at least three biological replicates (n=6-9).

Journal: eLife

Article Title: Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

doi: 10.7554/eLife.98266

Figure Lengend Snippet: Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin for 24 hr. After cells were exposed to a series of concentrations of PHAb10 (0, 6.25, 12.5, 25, 50, and 100 μg/ml) for an additional 24 hr, the relative viability of cells after each treatment was determined by Cell Counting Kit-8 (CCK-8) assay. All assays were performed with at least three biological replicates (n=6-9).

Techniques: Cell Culture, Modification, Cell Counting, CCK-8 Assay

( a ) Experimental schema for burn wound infection model. Burn wounds produced by exposing naked skin to 65°C water for 12 s was infected with 10 µl 5×10 8 cfu/ml of A. baumannii 3437. At 24 hr post-colonization, mice are treated with 5 or 10 µg/mouse PHAb10 (10 µl; n=5), 4 µg/mouse minocycline (10 µl; n=5), or an equal volume of Tris buffer (10 µl; n=5). Four hours post-treatment, viable bacteria on the burn wound skin is collected and counted. ( b ) Viable A. baumannii collected from burn wound skin after each treatment. The number of viable cells in each group is normalized and compared with the number of viable cells treated with Tris buffer by one-way analysis of variance (ANOVA). ns: statistically not significant; *: p<0.05; **: p<0.01. ( c ) Experimental schema for abscess model. Mice are infected hypodermically with 25 μl 5×10 8 cfu/ml of A. baumannii 3437 on the right side of the back dorsum. Twenty-four hours after infection, mice are injected hypodermically with 10 µg/mouse PHAb10 (10 µl; n=6), 4 µg/mouse minocycline (10 µl; n=7), or an equal volume of Tris buffer (10 µl; n=8). Each group is injected subcutaneously once a day for 5 consecutive days. Viable bacteria were counted at the infection site 24 hr after the last dose. ( d ) Viable A. baumannii collected from the skin after each treatment. The number of viable cells from each group was normalized and compared with those of the Tris buffer-treated group by ANOVA. ns: statistically not significant; **: p<0.01.

Journal: eLife

Article Title: Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

doi: 10.7554/eLife.98266

Figure Lengend Snippet: ( a ) Experimental schema for burn wound infection model. Burn wounds produced by exposing naked skin to 65°C water for 12 s was infected with 10 µl 5×10 8 cfu/ml of A. baumannii 3437. At 24 hr post-colonization, mice are treated with 5 or 10 µg/mouse PHAb10 (10 µl; n=5), 4 µg/mouse minocycline (10 µl; n=5), or an equal volume of Tris buffer (10 µl; n=5). Four hours post-treatment, viable bacteria on the burn wound skin is collected and counted. ( b ) Viable A. baumannii collected from burn wound skin after each treatment. The number of viable cells in each group is normalized and compared with the number of viable cells treated with Tris buffer by one-way analysis of variance (ANOVA). ns: statistically not significant; *: p<0.05; **: p<0.01. ( c ) Experimental schema for abscess model. Mice are infected hypodermically with 25 μl 5×10 8 cfu/ml of A. baumannii 3437 on the right side of the back dorsum. Twenty-four hours after infection, mice are injected hypodermically with 10 µg/mouse PHAb10 (10 µl; n=6), 4 µg/mouse minocycline (10 µl; n=7), or an equal volume of Tris buffer (10 µl; n=8). Each group is injected subcutaneously once a day for 5 consecutive days. Viable bacteria were counted at the infection site 24 hr after the last dose. ( d ) Viable A. baumannii collected from the skin after each treatment. The number of viable cells from each group was normalized and compared with those of the Tris buffer-treated group by ANOVA. ns: statistically not significant; **: p<0.01.

Techniques: Infection, Produced, Bacteria, Injection

Mice were subcutaneously injected with 25 μl of 5×10 8 cfu/ml of A. baumannii 3437 on the right side of the back dorsum. Twenty-four hours after infection, mice were injected subcutaneously with 10 µg/mouse PHAb10 (10 µl; n=6), 4 µg/mouse minocycline (10 µl; n=7), or an equal volume of Tris buffer (10 µl; n=8). Each group was subcutaneously injected once a day for 5 consecutive days. The body weight of mice in each group was monitored daily.

Journal: eLife

Article Title: Dimer-monomer transition defines a hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome by lysin-derived antimicrobial peptide-primed screening

doi: 10.7554/eLife.98266

Figure Lengend Snippet: Mice were subcutaneously injected with 25 μl of 5×10 8 cfu/ml of A. baumannii 3437 on the right side of the back dorsum. Twenty-four hours after infection, mice were injected subcutaneously with 10 µg/mouse PHAb10 (10 µl; n=6), 4 µg/mouse minocycline (10 µl; n=7), or an equal volume of Tris buffer (10 µl; n=8). Each group was subcutaneously injected once a day for 5 consecutive days. The body weight of mice in each group was monitored daily.

Techniques: Injection, Infection

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hepg2 cells assay kits (Nanjing Jiancheng Bioengineering Research Institute Co Ltd)

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