Journal: Current Issues in Molecular Biology

Article Title: In Vitro Digestive-Enzyme Inhibition and Antiglycation Activity of Cannabis sativa L. Essential Oil: Experimental Evaluation and Molecular Docking Analysis

doi: 10.3390/cimb48090938

Figure Lengend Snippet: Inhibitory effect of CSEO and Gallic acid (positive control) on glycation activity of hemoglobin. Results are shown as mean ± SEM ( n = 3). Graphs marked by different letters (a, b, c) are significantly different (the significance started at p < 0.05).

Article Snippet: Advanced glycation end-product (AGE) formation was quantified by fluorescence spectroscopy (excitation 355 nm, emission 460 nm; Horiba FluoroMax-4, Kyoto, Japan).

Techniques: Positive Control, Activity Assay

Journal: Current Issues in Molecular Biology

Article Title: In Vitro Digestive-Enzyme Inhibition and Antiglycation Activity of Cannabis sativa L. Essential Oil: Experimental Evaluation and Molecular Docking Analysis

doi: 10.3390/cimb48090938

Figure Lengend Snippet: IC 50 values of the multiple stages of albumin glycation. Data are presented as mean ± SEM ( n = 3).

Article Snippet: Advanced glycation end-product (AGE) formation was quantified by fluorescence spectroscopy (excitation 355 nm, emission 460 nm; Horiba FluoroMax-4, Kyoto, Japan).

Techniques: Inhibition

Journal: Life Science Alliance

Article Title: CDK4/6 inhibition induces a senescence-associated secretory phenotype via delayed NF-κB activation

doi: 10.26508/lsa.202603790

Figure Lengend Snippet: (A) Diagram summarizing CDK4/6i (Palbo) versus doxorubicin (Doxo) mechanisms of cell cycle arrest. (B) Schematic overview of comparative time course between CDK4/6i and doxorubicin and multi-omics approaches used to profile the transcriptome (RNA-seq), chromatin accessibility (ATAC-seq), and epigenome (CUT&RUN/Tag) in LS8817 cells. At least two biological replicates per timepoint and drug treatment were profiled. (C) Left: percent nuclei counted that had cellular SA-β-gal+ staining at different timepoints. The number of nuclei counted was >300 per condition at 10× objective magnification. Each point represents a field of view, with different shapes indicating biological replicates. Error bars denote SD. Right: representative SA-β-gal staining images at day 3 and day 14 shown at 10× objective magnification with scale bar = 50 μm (D) . Crystal violet staining after clonogenic outgrowth for 10–12 d. 800 cells were seeded per well in a 6-well plate in drug-free media after completion of drug treatment at each timepoint. (E) Number of ATRX foci (left) and macroH2A foci (indicative of SAHFs; right) per nucleus was counted for ∼100 nuclei per timepoint. (F) Representative ATRX and SAHF images are shown at day 14 of each drug treatment at 60× objective magnification. The scale bar is 15 μm. (G) Number of γH2AX foci and 53BP1 foci per nucleus was counted for ∼100 nuclei per timepoint and stratified into 0–4, 5–10, or >10 foci/nuclei. (H) . Representative images of γH2AX and 53BP1 staining are shown at day 14 of each drug treatment at 60× objective magnification. The scale bar is 15 μm. (I) Western blots of serine 15–phosphorylated p53 (phospho-p53) and total p53 in Cycling, Palbo-, and Doxo-treated LS8817 cell lysates harvested 18 d after treatment started. GAPDH was used as a loading control. Samples are from a separate experiment, independent of multi-omics data. Source data are available for this figure.

Article Snippet: Total RNA was sent to Plasmidsaurus for single-end RNA sequencing.

Techniques: Biomarker Discovery, RNA Sequencing, Staining, Western Blot, Control

Journal: Life Science Alliance

Article Title: CDK4/6 inhibition induces a senescence-associated secretory phenotype via delayed NF-κB activation

doi: 10.26508/lsa.202603790

Figure Lengend Snippet: (A) Distance measures between samples ordered by treatment and timepoint for RNA-seq (left) and ATAC-seq (right). (B) Principal component analysis on RNA-seq (top) and ATAC-seq (bottom) samples. (C, D) Venn diagram showing the number of peaks with decreased accessibility that contain E2F family of motifs relative to all down-regulated ATAC-seq peaks (D). Motif enrichment results of E2F family of motifs in down-regulated ATAC-seq peaks relative to up-regulated peaks (based on differential accessibility relative to Cycling) within each drug. The timepoints indicated represent the peak sets chosen as input for MEME-AME. Dot intensity represents −log10(Padj) values, whereas dot size reflects the difference between true-positive and false-positive peaks (%TP - %FP). (E) Motif enrichment results of top 10 most enriched motifs in ATAC-seq peaks up-regulated in palbociclib (Palbo-Up: left), both treatments (Shared-Up: middle), or doxorubicin (Doxo-Up: right), merged across all timepoints relative to Cycling and day 3, using unique up-regulated peaks from the alternate condition as a background. (F) Venn diagrams showing the number of up-regulated H3K27ac peaks that overlap with up-regulated ATAC-seq peaks, defined as drug-activated enhancers (see Methods). The color of text indicates the number of peaks from each peak set (H3K27ac: left; ATAC-seq: right). (G) Principal component analysis of H3K27ac CUT&Tag samples. (H) Log2 fold change (log2FC) values across all significantly changing p53 target genes in day 14 Palbo and day 14 Doxo, relative to Cycling. Paired t test shows difference between Palbo and Doxo is significant, with P -value = 4.468 × 10-8. (I) Normalized RNA-seq counts of the p53 target gene, CDKN1A (p21), over time. (J) Tn5 insertion sites around aggregated p53 motifs overlapping Doxo-, Shared-, or Palbo-up-regulated ATAC-seq peaks relative to Cycling (peaks from ). The number of overlapping peaks with p53 motifs per condition is shown.

Article Snippet: Total RNA was sent to Plasmidsaurus for single-end RNA sequencing.

Techniques: RNA Sequencing

Journal: Life Science Alliance

Article Title: CDK4/6 inhibition induces a senescence-associated secretory phenotype via delayed NF-κB activation

doi: 10.26508/lsa.202603790

Figure Lengend Snippet: (A) −Log10( Padj ) values of top 5 IPA predicted upstream regulator complexes and transcriptional regulators of Shared-up-regulated genes relative to other RNA-seq clusters in LS8817 cells. (B) Left: Log2 fold change (log2FC) values of NF-κB target genes that are part of the Shared-up-regulated genes (RNA Cluster 7) in day 3 and day 14 Palbo and Doxo relative to Cycling in LS8817 cells. Median log2FC values for each condition are shown as a bold white line. The dotted gray line at log2FC = 0 indicates no change from Cycling. A paired t test was used to assess significance between log2FC distributions. Right: gene IDs of NF-κB target genes that are also defined as SASP genes based on whether they were classified as such in manually curated databases provided in and and the SASP Atlas . (C) Quantification of log10 ratio of mean intensity of p65 in the nucleus to perinuclear region in LS8817 cells. Statistical significance was calculated using Dunnett’s test. Each point is a cell that was quantified. (D) K-means clustered heatmap of z-scores from rlog-normalized counts for significantly changing genes across treatment and timepoints (days of treatment) based on RNA-seq performed in MCF-7 cells. SASP and cell cycle genes are annotated in dark green and blue, respectively. NF-κB target genes found in each up-regulated gene cluster are shown left of the heatmap. Genes in dark green are also annotated as SASP. (E) GO enrichment results of NF-κB related terms from each RNA-seq cluster in MCF7 cells. (F) GSEA running enrichment score for enriched Hallmark term “TNFA SIGNALING VIA NFKB” for palbociclib, doxorubicin, and palbociclib+fulvestrant treatment in MCF7 cells. Padj values from GSEA are shown. (G) Quantification of log10 ratio of mean intensity of p65 in the nucleus to perinuclear region in MCF7 cells across different biological replicates. Statistical significance was calculated using Dunnett’s test. Source data are available for this figure.

Article Snippet: Total RNA was sent to Plasmidsaurus for single-end RNA sequencing.

Techniques: RNA Sequencing

Journal: Life Science Alliance

Article Title: CDK4/6 inhibition induces a senescence-associated secretory phenotype via delayed NF-κB activation

doi: 10.26508/lsa.202603790

Figure Lengend Snippet: (A) Tn5 insertion site coverage at the center of the NF-κB subunit, TF65 (p65) motifs overlapping nearby Doxo-up-regulated ATAC-seq peaks, defined as within 50 kb of shared-up-regulated genes. The number of nearby peaks is shown on the left. The scale bar represents the bigWig signal of normalized coverage. (B) Same as (A), but for nearby Palbo-up-regulated ATAC-seq peaks. (C) Top: Tn5 insertion site coverage at the center of the AP-1 family member, FOSB motifs overlapping all up-regulated ATAC-seq peaks, in either drug treatment, within 50 kb of shared-up-regulated genes. Bottom: Motif enrichment results of AP-1 family of motifs (JUN/FOS) in up-regulated ATAC-seq peaks within 50 kb of shared-up-regulated genes (based on differential accessibility relative to Cycling or day 3 treatment controls) relative to nearby static peaks for each drug. The timepoints indicated represent the peak sets chosen as input for MEME-AME. Dot intensity represents −log10(Padj) values, whereas dot size reflects the difference between true-positive and false-positive peaks (%TP - %FP). (D) Top: Tn5 insertion site profiles around FOSB motifs overlapping all ATAC-seq peaks. ATAC-seq peaks increasing in accessibility at any timepoint in response to Doxo or Palbo, compared with Cycling or day 3 controls within each drug. The number of overlapping peaks with FOS motifs is shown. Bottom: Motif enrichment results of AP-1 family of motifs in up-regulated ATAC-seq peaks at each timepoint relative to Cycling or day 3 controls within each drug. (E) Normalized RNA-seq counts of macroH2A2 and macroH2A1 over time. (F) macroH2A1 (mH2A1) signal for all mH2A1 peaks called. The total number of mH2A1 peaks is shown on the left. The scale bar represents the bigWig signal of normalized coverage. (G) Genome browser view of CXCL1 and CXCL8 loci. Normalized bigWig tracks for ATAC-seq, H3K27ac, and macroH2A1 (mH2A) are shown. Up-regulated regions occurring in both treatments are highlighted in blue (Shared changes), whereas Doxo-up-regulated regions are highlighted in green (Changes in Doxo).

Article Snippet: Total RNA was sent to Plasmidsaurus for single-end RNA sequencing.

Techniques: RNA Sequencing

Journal: Life Science Alliance

Article Title: CDK4/6 inhibition induces a senescence-associated secretory phenotype via delayed NF-κB activation

doi: 10.26508/lsa.202603790

Figure Lengend Snippet: (A) Schematic of pharmacological inhibition of NF-κB with BAY11-7082 (BAY) in LS8817 cells (see Methods). 10 μM BAY or equivalent volume of DMSO (negative control) was added to cells for 2–3 d before harvest. (B) Principal component analysis on RNA-seq of day 14 palbociclib- or doxorubicin-treated LS8817 cells with or without NF-κB inhibitor, BAY11-7082 (BAY). (C) RT–qPCR results of palbociclib- or doxorubicin-treated LS8817 cells with or without BAY. Relative fold change values to Cycling are shown. A dotted gray line indicates no change. Diamonds inside boxplot represent the mean fold change value. Circles, triangles, and rectangles represent the fold change of individual biological replicate. Dark shades indicate Palbo and Doxo samples treated with BAY, whereas lighter shades indicate samples that had DMSO added at the same time as BAY samples. Statistical significance was calculated using a two-tailed t test. (D) Crystal violet staining after clonogenic outgrowth after palbociclib or doxorubicin treatment ±BAY. 800 cells were seeded per well in a 6-well plate and grown in drug-free media for 12 d after harvest. Source data are available for this figure.

Article Snippet: Total RNA was sent to Plasmidsaurus for single-end RNA sequencing.

Techniques: Inhibition, Negative Control, RNA Sequencing, Quantitative RT-PCR, Two Tailed Test, Staining

Journal: Life Science Alliance

Article Title: CDK4/6 inhibition induces a senescence-associated secretory phenotype via delayed NF-κB activation

doi: 10.26508/lsa.202603790

Figure Lengend Snippet: (A) Volcano plot from RNA-seq analysis showing shared NF-κB–driven SASP genes and other top 15 most significantly changing genes in day 14 palbociclib or doxorubicin treated with DMSO control. (B) Same as (A) but for day 14 palbociclib or doxorubicin treated with BAY. (C) Venn diagram showing the number of genes down-regulated by BAY relative to day 14 palbociclib- or doxorubicin-treated DMSO controls.

Article Snippet: Total RNA was sent to Plasmidsaurus for single-end RNA sequencing.

Techniques: RNA Sequencing, Control

Journal: Life Science Alliance

Article Title: CDK4/6 inhibition induces a senescence-associated secretory phenotype via delayed NF-κB activation

doi: 10.26508/lsa.202603790

Figure Lengend Snippet: (A) Schematic of the experimental strategy testing whether inhibition of the DNA damage response kinases ATM and ATR affects NF-κB activation in CDK4/6i treatment in LS8817 cells. (B) Second biological replicate of Western blots for phospho-p53 and total p53, corresponding to . (C) . As in (B), but for phospho-Chk2 and total Chk2. (D) Normalized RNA-seq counts of TGFB1 and TGFB2 over time. (E) Normalized RNA-seq counts of TGFBR1 and TGFBR2 over time. (F) Normalized RNA-seq counts of IL1R1 and TLR4 over time.

Article Snippet: Total RNA was sent to Plasmidsaurus for single-end RNA sequencing.

Techniques: Inhibition, Activation Assay, Western Blot, RNA Sequencing