Journal: Antioxidants (Basel, Switzerland)

Article Title: Aster-B Modulates Oxidative Stress Responses and Carotenoid Distribution in ARPE-19 Cells.

doi: 10.3390/antiox14050575

Figure Lengend Snippet: Figure 1. CRISPR/dCas9 activation of endogenous GRAMD1B gene expression in ARPE19 GRAMD1B-null cells. (A) ARPE-19 cells were transfected with scrambled DNA at 0.5, 1, and 1.5 µg DNA per well and GRAMD1B plasmid DNA at 0.5, 1, and 1.5 µg DNA per well, respectively. Western blot analysis was conducted with 50 µg of total protein per lane and showed that GRAMD1B is expressed before clonal selection and becomes enriched in post-clonal ARPE-19 cells (25 and 50 µg per well) treated with GRAMD1B plasmid DNA (1.5 µg per well). In contrast, GRAMD1B expression is absent in control cells treated with scrambled control DNA (1.5 µg per well). A549 cells were used as a positive control for GRAMD1B expression. β-Actin was used as a loading control. (B) Confocal imaging analysis with ARPE-19 cells transfected with plasmid DNA confirmed that GRAMD1B protein is expressed and mostly localized in the cytoplasm. Similar results were obtained from WB and ICC in more than three independent experiments. The scale bar is 10 µm.

Article Snippet: The antibodies used included GRAMD1B (Proteintech), COXIV, Caspase-3, PARP, and Histone H3 (Cell Signaling Technology) at a dilution of 1:1000.

Techniques: CRISPR, Activation Assay, Gene Expression, Transfection, Plasmid Preparation, Western Blot, Selection, Expressing, Control, Positive Control, Imaging

Journal: Antimicrobial Agents and Chemotherapy

Article Title: First Penicillin-Binding Protein Occupancy Patterns of β-Lactams and β-Lactamase Inhibitors in Klebsiella pneumoniae

doi: 10.1128/AAC.00282-18

Figure Lengend Snippet: Principal component analysis of the log-transformed PBP IC50 data for our 13 tested compounds in two K. pneumoniae strains. The plot shows the clustering of compounds according to their positions on the first and second eigenvector. These two vectors explained 86.5% of the total variance. Compounds were grouped into two general clusters. The first cluster contained β-lactams that primarily targeted PBP3 but differed in their secondary targets. The second cluster was comprised of compounds that primarily targeted PBP2 or both PBPs 2 and 4. Of note, among compounds in the second cluster, the carbapenems, amoxicillin, and mecillinam had substantially lower IC50 for their primary targets than the β-lactamase inhibitors. Symbols for β-lactamase inhibitors are smaller due to their much higher IC50 for their primary PBP target relative to the primary PBP target IC50 of the β-lactams.

Article Snippet: Biapenem was purchased from TRC Canada (Toronto, Ontario, Canada); doripenem and avibactam were from MedChem Express (Monmouth Junction, NJ); imipenem and meropenem were from AK Scientific (Union City, CA); sulbactam was from TCI America (Portland, OR); tazobactam, amoxicillin, piperacillin, aztreonam, cefepime, and cefsulodin were from Chem-Impex International, Inc. (Wood Dale, IL); and mecillinam was from Molekula (Newcastle Upon Tyne, United Kingdom).

Techniques: Transformation Assay

Journal: Cell Death Discovery

Article Title: Knockdown of SUCLG2 inhibits glioblastoma proliferation and promotes apoptosis through LMNA acetylation and the mediation of H4K16la lactylation

doi: 10.1038/s41420-025-02856-4

Figure Lengend Snippet: A H4K16la and L-Lac expression patterns in shNC and shSUCLG2 cells of U251 and LN229 were determined using western blotting. B Cell precipitates were collected for cleavage under targets and tagmentation (CUT&Tag) assay to identify H4K16la binding peaks, and the heatmap shows the distribution of H4K16la peaks near the translation start site (TSS). C Comparison of shNC and shSUCLG2 peaks in LN229 cells. D Genomic distribution of H4K16la, which was annotated for its localisation (promoter, exon, intron, or intergenic) and quantified in relative and absolute amounts. E Volcano plots of upregulated versus downregulated genes after CUT&Tag assay for H4K16la binding. F The heat map shows genes downregulated around TSS in LN229 cells containing shNC and shSUCLG2 as detected by the CUT&Tag assay. G KEGG analysis of CUT&Tag-detected downregulated genes at H4K16la. H GO analysis of CUT&Tag-detected downregulated genes. I Volcano plot of differential gene expression in RNA-seq. J KEGG analysis of downregulated genes in shSUCLG2 versus shNC using RNA-seq. K GO analysis of shSUCLG2 versus shNC downregulated genes using RNA-seq. L Combined CUT&Tag and RNA-seq analyses were performed to identify potential downstream target intersections of H4K16la. M Integrated genomics viewer trajectory track of CUT&Tag data showing enrichment of H4K16la at BEST1, GRAMD4H, and MBD6 promoter regions. N LN229 and U251 cells were treated with shNC or shRNA, and DNA fragments were immunoprecipitated with H4K16la antibody and analysed by qPCR. O Representative western blotting images show the quantification of BEST1, MBD6, and GRAMD4 protein levels. P Western blotting of IL-6 and IL-8 in shNC or shRNA lines of LN229 and U251. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: The following antibodies were used: SUCLG2 (A8976, 1:1 200, ABclonal, Wobum, MA), Bax (60267-1-Ig, 1:1000, Proteintech, Rosemont, IL), PCNA (HRP-60097, 1:1 000, Proteintech), Caspase-3 (68773-1-Ig, 1:1000, Proteintech), Bcl-2 (12789-1-AP, 1:1000, Proteintech), β-actin (11313-2-AP, 1:1 500, Proteintech), Cyclin D1 (60186-1-Ig, 1:1500, Proteintech), DLAT (ab172617, 1:1000, Abcam, Cambridge, UK), LMNA (ab172617, 1:1000, Abcam), L-Lac (PTM-1401RM, 1:1000, Jingjie Bio, Nanjing, China), D-Lac (PTM-1429RM, 1:1000, Jingjie Bio), HIF-1α (H1alpha67, 1:1000, Abcam), H4K16la (PTM-122, 1:1000, Jingjie Bio), H4 (PTM-1015RM, Jingjie Bio), Total oxidative phosphorylation Rodent Antibody Cocktail (ab110413, 1:1000, Abcam), BEST1 (1:1000, ab259836, Abcam); GRAMD4 (1:1000, 24299-1-AP9, Proteintech), MBD6 (1:1000, ab204403, Abcam); MFN1 (A21293, 1:1200, ABclonal); MFN2 (A19678, 1:1200, ABclonal); DR1 (A13298,1:1200, ABclonal); IL-6 (A26791, 1:1000, ABclonal); IL-8 (RP00052, 1:1000, ABclonal); anti-rabbit IgG (H + L) (ab205718, 1:5000, Abcam), and anti-mouse IgG (H + L) (ab205719, 1:5000, Proteintech).

Techniques: Expressing, Western Blot, Binding Assay, Comparison, Gene Expression, RNA Sequencing, shRNA, Immunoprecipitation