Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 1. Knockout of Rnaseh2a gene of HEK293 cells with the CRISPR/Cas9 system.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Knock-Out, CRISPR

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 2. Screening of Rnaseh2a-/- homozygote HEK293 cells.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques:

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 7. Knockout of Rnaseh2a gene of HEK293

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Knock-Out

Journal:

Article Title:

doi:

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-JunB, 1:1000 , Bethyl , Cat# A302-704A, RRID:AB_10749029.

Techniques: Recombinant, SYBR Green Assay, Mutagenesis, RNA Sequencing Assay, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Plasmid Preparation, Software

Journal: BMC Biology

Article Title: Machine learning combined with omics-based approaches reveals T-lymphocyte cellular fate imbalance in abdominal aortic aneurysm

doi: 10.1186/s12915-025-02400-x

Figure Lengend Snippet: Identification of TIRS regulatory mechanisms and key biomarkers. A LASSO-based feature selection, with the optimal lambda determined when the partial likelihood deviance reached the minimum value (left). SVM-RFE-based feature selection, with root mean square error (RMSE) reached the minimum value and R -squared reached the max value (mid). Venn diagram presented the intersection of key biomarkers obtained through both algorithms (right). B Aberrant expression profiles for key biomarkers in Abdominal Aortic Wall Dataset 1 (AAA n = 80 patients, control n = 10 healthy individuals; Student’s t -test). C ROC curve demonstrating the diagnostic efficacy of FOSB, JUNB, CST7, and TBC1D4 in Abdominal Aortic Wall Dataset 1. D Clinical impact plot illustrating the clinical utility of key biomarkers. The “Number high risk” curve closely aligns with the “Number high risk with the event” curve at each threshold probability, indicating exceptional predictive power. E Aberrant expression profiles for key biomarkers in Abdominal Aortic Wall Dataset 2 (AAA n = 9 patients, control n = 10 healthy individuals; Student’s t -test). F ROC curve validating the diagnostic efficacy of FOSB, JUNB, CST7, and TBC1D4 in Abdominal Aortic Wall Dataset 2. G Clinical impact plot demonstrating the clinical utility of key biomarkers. Again, the “Number high risk” curve is closely aligned with the “Number high risk with the event” curve at each threshold probability, highlighting the biomarkers’ strong predictive power. H Aberrant expression profiles for key biomarkers in Perivascular Adipose Tissue Dataset 3 (dilated n = 30, non-dilated n = 30; Student’s t -test). I ROC curve verifying the diagnostic efficacy of FOSB, JUNB, CST7, and TBC1D4 in Perivascular Adipose Tissue Dataset 3. J Impact plots reiterated superior predictive performance probability, indicating outstanding predictive capability

Article Snippet: The primary antibodies against FOSB (1:500, catalog No. ab184938, Abcam) and JUNB (1:50, catalog No. 10486–1-AP, Proteintech) were incubated overnight at 4 °C, followed by incubation with horseradish peroxidase conjugated secondary antibodies.

Techniques: Selection, Expressing, Control, Diagnostic Assay

Journal: BMC Biology

Article Title: Machine learning combined with omics-based approaches reveals T-lymphocyte cellular fate imbalance in abdominal aortic aneurysm

doi: 10.1186/s12915-025-02400-x

Figure Lengend Snippet: Verification of key biomarkers. A Abdominal aortic wall and peripheral blood samples obtained from AAA patients. B Aberrant expression profiles for key biomarkers in the abdominal aortic wall (Inhouse Dataset 1; AAA n = 5 patients, control n = 4 healthy individuals). C ROC curve validating the diagnostic efficacy of FOSB, JUNB, CST7, and TBC1D4 in the abdominal aortic wall (Inhouse Dataset 1). D Clinical impact plot demonstrating the clinical utility of key biomarkers. The “Number high risk” curve closely aligns with the “Number high risk with the event” curve at each threshold probability, indicating exceptional predictive power. E Aberrant expression profiles for key biomarkers in peripheral blood (Inhouse Dataset 2; AAA n = 24 patients, control n = 15 healthy individuals). F ROC curve validating the diagnostic efficacy of FOSB, JUNB, CST7, and TBC1D4 in peripheral blood (Inhouse Dataset 2). G Clinical impact plot illustrating the clinical utility of key biomarkers. Again, the “Number high risk” curve remains closely aligned with the “Number high risk with the event” curve at each threshold probability, highlighting the biomarkers’ strong predictive capability. H Mice were infused with saline or Ang II (1000 ng/kg/min) + BAPN. Gross abdominal aorta images were shown. Scale bar is 1 cm. I Representative images of immunohistochemical stains for elastin fiber (Van Gieson) and representative photomicrographs of hematoxylin and eosin (H&E) staining. Scale bar is 200 μm. J – L Representative immunohistochemical staining of FOSB and JUNB in aortic cross sections. Scale bar is 50 μm. Data are expressed as mean ± SEM (control n = 3 mice, AAA n = 5 or 6 mice). Student’s t -test was utilized to compare continuous variables between the two groups

Article Snippet: The primary antibodies against FOSB (1:500, catalog No. ab184938, Abcam) and JUNB (1:50, catalog No. 10486–1-AP, Proteintech) were incubated overnight at 4 °C, followed by incubation with horseradish peroxidase conjugated secondary antibodies.

Techniques: Expressing, Control, Diagnostic Assay, Saline, Immunohistochemical staining, Staining

Journal: eLife

Article Title: Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi–Goutières syndrome

doi: 10.7554/eLife.08007

Figure Lengend Snippet: ( A ) Genomic DNA was treated with RNase H2 to reveal the presence of ribonucleotides as single-strand nicks. An untreated control was used to reveal background single-stranded breaks in genomic DNA preparations. The intensity of labeling after gel electrophoresis was measured. ( B , C ) Relative ribonucleotide loads are reported as fold-increase of radiolabel incorporation of RNase H2-treated over untreated sample. At least three independent replicates were performed and the error bars indicate SEM. DOI: http://dx.doi.org/10.7554/eLife.08007.005

Article Snippet: Whole-cell protein was extracted and Western blot was performed using antibodies against RNase H2A (1:1000, ProSci) or GFP (1:500, UC Davis/NIH NeuroMab Facility, Davis, CA), and tubulin (1:1000, Sigma–Aldrich, St. Louis, MO).

Techniques: Control, Labeling, Nucleic Acid Electrophoresis

Journal: eLife

Article Title: Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi–Goutières syndrome

doi: 10.7554/eLife.08007

Figure Lengend Snippet: ( A ) Western blot using an anti-RNase H2A antibody shows successful gene knockout in HeLa-GFP cells. Scramble cells were generated with a CRISPR vector carrying a scrambled guide RNA sequence. Tubulin was used as a loading control. ( B ) Immunocytochemistry confirms elevated DNA damage response in RNASEH2A KO cells; DAPI (blue), gamma H2AX antibody (green). ( C ) Bright field and green fluorescent protein (GFP) microscopy images of RNASEH2A KO and scramble control cells. Knockout of the RNASEH2A gene triggers the reactivation of the silent GFP reporter in HeLa-GFP cells. ( D ) (Top) Western blot showing GFP expression in HeLa-GFP cells (RNASEH2A KO and scramble); (bottom) bar graph quantification of Western blot. ( E ) Bisulfite methylation sequencing for two different LINE-1 loci in RNASEH2A KO and scramble control cells. Black and white circles represent methylated and unmethylated CpG sites, respectively. Missing bubbles indicate that a CpG site is absent from the sequence of that particular molecule. The coordinates for each fragment analyzed are indicated at top. ( F ) Quantification of percent methylation at each CpG sites surveyed in panel E . p-value was calculated using a paired Wilcoxon test with the alternative hypothesis that RNASEH2A KO is less methylated than scramble. DOI: http://dx.doi.org/10.7554/eLife.08007.012

Article Snippet: Whole-cell protein was extracted and Western blot was performed using antibodies against RNase H2A (1:1000, ProSci) or GFP (1:500, UC Davis/NIH NeuroMab Facility, Davis, CA), and tubulin (1:1000, Sigma–Aldrich, St. Louis, MO).

Techniques: Western Blot, Gene Knockout, Generated, CRISPR, Plasmid Preparation, Sequencing, Control, Immunocytochemistry, Microscopy, Knock-Out, Expressing, Methylation