Journal: PLoS ONE

Article Title: Sustained Spindle-Assembly Checkpoint Response Requires De Novo Transcription and Translation of Cyclin B1

doi: 10.1371/journal.pone.0013037

Figure Lengend Snippet: A) Analysis of Cyclin B1, Securin and CDK1 levels by western blotting. NIH3T3 cells were incubated with nocodazole (noc) for 14 h and treated with actinomycin D (ActD; 8 µM) or cycloheximide (CHX; 35.5 µM) with or without the inhibitor of the proteasome MG132 (25 µM), for 4 h; β-actin was used as loading control. B) Quantification of Cyclin B1 and Securin proteins in noc-, noc-ActD-, noc-ActD-MG, noc-CHX and noc-CHX-MG-cells. Normalization of Cyclin B1 and Securin was calculated on the ratio of these proteins per β-actin protein present in each condition. Data presented as mean ± S.D. from three independent experiments. C and D) Immunofluorescence microscopy: asynchronous population (control) and noc-cells treated with ActD for 4 h; DAPI stains the DNA (blue) and Cyclin B1 (C) and Securin (D) are stained in green. Arrowheads point mitotic cells and arrows point cell that undergone mitotic slippage. The adapted cells shown are representative of both ActD and CHX induced mitotic slippage. Scale bar = 25 µm.

Article Snippet: The treatments with actinomycin D (8 μM; Sigma), cycloheximide (35.5 μM; Sigma) and MG132 (25 μM; Tocris) were performed at 37°C for 4 h for NIH3T3 and for 6 h for HEK293 cells.

Techniques: Western Blot, Incubation, Control, Immunofluorescence, Microscopy, Staining

Journal: PLoS ONE

Article Title: Sustained Spindle-Assembly Checkpoint Response Requires De Novo Transcription and Translation of Cyclin B1

doi: 10.1371/journal.pone.0013037

Figure Lengend Snippet: A) Real time quantitative PCR analysis of several spindle checkpoint transcripts (Mad1, Mad2, BubR1, Bub3 and Cdc20) present in nocodazole (noc) cells treated with or without the inhibitor of transcription actinomycin D (ActD; 8 µM), for 4 h. Results from each gene amplification were normalized using L19 expression and presented as fold increase from nocodazole cells. Data presented as mean ± S.D. from three independent experiments; * p<0.05 noc-ActD versus noc. B and D) Western blotting analysis of Bub3 (B) and Mad2 (D) in nocodazole (noc) cells treated with ActD or cycloheximide (CHX; 35.5 µM) in the presence or absence of the proteasome inhibitor MG132 for 4 h; β-actin was used as loading control. C and E) Quantification of Bub3 (C) and Mad2 (E) protein in noc-, noc-ActD- and noc-CHX-cells. Normalization of Bub3 or Mad2 was calculated on the ratio of these proteins per β-actin protein present in each condition. Data presented as mean ± S.D. from three independent experiments.

Article Snippet: The treatments with actinomycin D (8 μM; Sigma), cycloheximide (35.5 μM; Sigma) and MG132 (25 μM; Tocris) were performed at 37°C for 4 h for NIH3T3 and for 6 h for HEK293 cells.

Techniques: Real-time Polymerase Chain Reaction, Amplification, Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Sustained Spindle-Assembly Checkpoint Response Requires De Novo Transcription and Translation of Cyclin B1

doi: 10.1371/journal.pone.0013037

Figure Lengend Snippet: Analysis of the level of transcripts of Cyclin B1 and Securin by real-time quantitative PCR. mRNA was isolated from a G1/S enriched population of cells treated with thymidine (Thy), and from nocodazole- arrested cells (noc-cells) treated with or without actinomycin D (ActD; 8 µM) and MG132 for 4 h. Results from Cyclin B1 and Securin amplification were normalized using L19 expression and presented as fold increase of Cyclin B1 or Securin expression from noc-cells. Data presented as mean ± S.D. from three independent experiments; *** p<0.005; ** p<0.01; * p<0.05 Thy or Noc+ActD or Noc+ActD+MG versus Noc.

Article Snippet: The treatments with actinomycin D (8 μM; Sigma), cycloheximide (35.5 μM; Sigma) and MG132 (25 μM; Tocris) were performed at 37°C for 4 h for NIH3T3 and for 6 h for HEK293 cells.

Techniques: Real-time Polymerase Chain Reaction, Isolation, Amplification, Expressing

Journal: Life

Article Title: Casomorphine-10 (CM-10) Peptide Orchestrates Circadian and Neurodevelopmental Gene Clusters via δ-Opioid Receptor Signaling: Insights from Transcriptome Analysis with δ-Opioid Receptor-Expressing HEK293 Cells

doi: 10.3390/life15101636

Figure Lengend Snippet: δ-opioid receptor (DOR)-expressing HEK293 cells and HEK293 cells stained with anti-DOR antibody and followed with FITC-conjugated anti-mouse IgG ( A ). Volcano plot of identified differentially expressed genes (DEGs) in DOR-HEK293 cells after 1 h treatment with CM-10 ( B ). Red boxes show changed gene expression over 1.4-time higher and less than 0.71 with p -value predicted by EdgeR of less than 0.05.

Article Snippet: The fixed DOR-HEK293 and HEK293 cells were then incubated with a rabbit anti-DOR antibody (GeneTex, Irvine, CA, USA) after dilution with 1% casein in PBS (1/1000) and stained with a secondary antibody (Cy3-conjugated anti-rabbit IgG, Rockland Inc., PA, USA).

Techniques: Expressing, Staining, Gene Expression

Journal: Life

Article Title: Casomorphine-10 (CM-10) Peptide Orchestrates Circadian and Neurodevelopmental Gene Clusters via δ-Opioid Receptor Signaling: Insights from Transcriptome Analysis with δ-Opioid Receptor-Expressing HEK293 Cells

doi: 10.3390/life15101636

Figure Lengend Snippet: Predicted DOR agonistic signaling networks in DOR-HEK293 cells after 1 h treatment with CM-10. Fourteen genes with suggested involvement in cAMP-dependent protein kinases, transcriptional regulators in response to cAMP, circadian rhythm, stress and depression are shown in and were applied for network analysis by STRING. Red: circadian rhythm, Green: regulation of transcription of Notch receptor target, Yellow: PKA activation in glucagon signalling.

Article Snippet: The fixed DOR-HEK293 and HEK293 cells were then incubated with a rabbit anti-DOR antibody (GeneTex, Irvine, CA, USA) after dilution with 1% casein in PBS (1/1000) and stained with a secondary antibody (Cy3-conjugated anti-rabbit IgG, Rockland Inc., PA, USA).

Techniques: Activation Assay

Journal: Scientific Reports

Article Title: The local anesthetic, lidocaine, suppresses the growth of colon cancer SW480 cells by decreasing the voltage-gated sodium channel subunit Na v 1.5

doi: 10.1038/s41598-025-29505-1

Figure Lengend Snippet: Na v 1.5 levels decreased in lidocaine-treated SW480 cells. ( A ) Na v 1.5 levels decreased in lidocaine-treated SW480 cells. SW480 cells were treated with or without 5 mM lidocaine for 24 h, and the cell lysates were subjected to western blot analysis using polyclonal anti-Na v 1.5 antibodies. The arrow indicates Na v 1.5. The relative expression levels of Na v 1.5 were normalized to GAPDH levels in each sample. The results of each sample assayed in triplicate are presented as the mean ± SD. * p < 0.05. ( B ) The level of Na v 1.5 did not increase after treatment with a proteasome inhibitor, MG 132, in lidocaine-treated SW480 cells. SW480 cells were treated with 5 mM lidocaine, together with or without 5 µM MG132 for 24 h. The cell lysates were subjected to western blot analysis using polyclonal anti-Na v 1.5 and p21 antibodies. p21 was used as a positive control of MG132 treatment . The arrow indicates Na v 1.5. The relative expression levels of Na v 1.5 and p21 were normalized to a-tubulin levels in each sample. The results of each sample assayed in triplicate are presented as the mean ± SD. * p < 0.05, ** p < 0.01. ( C ) The level of Na v 1.5 did not increase after treatment with an autophagy inhibitor, Bafilomycin A1, in lidocaine-treated SW480 cells. SW480 cells were treated with 5 mM lidocaine with or without 200 nM Bafilomycin A1 (BAF) for 24 h . The arrow indicates Na v 1.5. The relative expression levels of Na v 1.5 and p62 were normalized to a-tubulin levels in each sample. p62 was used as a positive control of Bafilomycin A1 treatment. The results of each sample assayed in triplicate are presented as the mean ± SD. * p < 0.05, ** p < 0.01. ( D ) Na v 1.5 levels in the membrane fraction decreased in lidocaine-treated SW480 cells. SW480 cells were treated with or without 5 mM lidocaine for 24 h, and cell lysates of the cytosol and membrane fractions were prepared using Mem-PER™ Plus Membrane Protein Extraction Kit. Each cell lysate was subjected to western blot analysis. ATP1A1 was used as the membrane marker protein and GDF-15 was used as the lidocaine-inducible protein, which increased in the membrane fraction after lidocaine treatment. The relative expression levels of Na v 1.5 and GDF-15 were normalized to ATP1A1 levels in each sample. The results of each sample assayed in triplicate are presented as the mean ± SD. ** p < 0.01.

Article Snippet: MG132 and Bafilomycin A1 were purchased from Fujifilm-Wako (Osaka, Japan) and LKT Laboratories (MN, USA), respectively.

Techniques: Western Blot, Expressing, Positive Control, Membrane, Protein Extraction, Marker