Journal: Cell Death & Disease

Article Title: GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner

doi: 10.1038/s41419-020-03311-1

Figure Lengend Snippet: A The scheme of isobaric iodoTMT switch labeling-based mass spectrometry assay and results of MS/MS spectrum of palmitoylated peptide of GNA13. IodoTMT 6 -127 labeling on Cys14 and Cys18 (the two C in lowercase) of GNA13 peptide sequence was shown in peaks graph (upper panel). B Total, membrane (Mem) and cytosolic (Cyto) fractions of HeLa cells expressing HA-tagged WT GNA13, C14S, C18S, or C14/18S mutant of GNA13 were immunoblotted with an anti-HA antibody. α-Tubulin was used as a loading control for total cellular proteins, while Na-K-ATPase and GAPDH were used as markers of the membrane and cytosol, respectively. C HeLa cells overexpressing WT GNA13 or C14/18S mutant were incubated with cycloheximide (CHX) and analyzed by western blot at the indicated time points. D Protein levels of WT GNA13 and C14/18S mutant in HeLa cells treated with or without indicated caspase inhibitors for 24 h were detected by immunoblotting with an anti-HA antibody. α-Tubulin was used as a loading control.

Article Snippet: The cycloheximide (CHX), autophagy inhibitors, Hydroxychloroquine sulfate (HCQ), ULK-101, and the pan-caspase inhibitor Z-VAD(OMe)-FMK were purchased from TargetMol (Wellesley Hills, MA, USA).

Techniques: Labeling, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing, Membrane, Expressing, Mutagenesis, Control, Incubation, Western Blot

Journal: The EMBO Journal

Article Title: Mitotic checkpoint gene expression is tuned by codon usage bias

doi: 10.15252/embj.2021107896

Figure Lengend Snippet: A Cellular protein noise (coefficient of variation, CV = std / mean) in live‐cell microscopy images of S. pombe ; n = 7 images (Nmt1‐GFP), 11 (Mad1‐GFP), 19 (Mad2‐GFP), 10 (Mad3‐GFP); single images had 16–79 GFP‐positive and 6–94 GFP‐negative (control) cells. Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Mad1, Mad2, and Mad3 all showed significantly lower noise than Nmt1 (Wilcoxon rank sum test; all P < 0.001). B Simulations of stochastic gene expression noise from selected mRNA/protein half‐life combinations assuming a constantly active promoter (see Methods). Synthesis rates were set to obtain a mean mRNA number of 4 per cell, and a mean protein number of 6,000 per cell. The x‐axis of each graph shows time, the y‐axis shows mRNA number per cell (blue) or protein number per cell (black). C Theoretical prediction for the coefficient of variation (CV = std/mean) of the protein number per cell, assuming different mRNA and protein half‐lives, using the same underlying model as in B. Synthesis rates were adjusted to maintain a mean mRNA number per cell of 3.5, and a mean protein number per cell of 6,000 (approx. 100 nM). D mRNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Lines are regression curves from generalized linear mixed model fits, excluding the measurements at t = 0 in order to accommodate for noninstantaneous labeling by 4tU. Act1 + and ecm33 + were used as long and short half‐life controls, respectively; qPCR was performed for the endogenous mRNAs. Half‐lives (95% confidence interval): mad1 + 5.6 min (4.3–8.4), mad2 + 7.7 min (6.2–10.4), mad3 + 5.2 min (4.3–6.9), act1 + 61.8 min (37.2–172.3), ecm33 + 5.0 min (4.5–5.7). E Protein abundances after translation shut‐off with cycloheximide (CHX); n = 3 experiments, error bars = std. Lines indicate fit to a one‐phase exponential decay. Cdc2 and Cdc13 were used as long and short half‐life controls, respectively. Immunoblots for the endogenous proteins (no tag). A representative experiment shown in Appendix Fig . Source data are available online for this figure.

Article Snippet: Cycloheximide (from Streptomyces griseus) , Chem Impex , Cat # 00083.

Techniques: Microscopy, Negative Control, Expressing, Labeling, Western Blot

Journal: The EMBO Journal

Article Title: Mitotic checkpoint gene expression is tuned by codon usage bias

doi: 10.15252/embj.2021107896

Figure Lengend Snippet:

Article Snippet: Cycloheximide (from Streptomyces griseus) , Chem Impex , Cat # 00083.

Techniques: Recombinant, In Vitro, Sequencing, Labeling, Protease Inhibitor, Isolation, Magnetic Beads, Bicinchoninic Acid Protein Assay, Software, Membrane

Journal: Cells

Article Title: Dynamics of T-Cell Intracellular Antigen 1-Dependent Stress Granules in Proteostasis and Welander Distal Myopathy under Oxidative Stress

doi: 10.3390/cells11050884

Figure Lengend Snippet: TIA1a WT/WDM expression does not alter phosphorylation/dephosphorylation dynamics in an oxidative stress model. ( A , B ) Western blot of total cell extracts obtained from FT293 GFP-TIA1a WT ( A ) and FT293 GFP-TIA1a WDM ( B ) lines subjected to different treatments indicated in the legend at the top. Analyses were performed with anti-TIA1, anti-eIF2α-phosphorylated (P), anti-eIF2α (Total), anti-HuR, and anti-tubulin-α (TUBA) antibodies from three independent experiments. Molecular weight markers (kDa) and identification of each band are shown. The + and − signs indicate whether the sample contains the reagent (Tet, NaAsO 2 , CHX) or not. The +/− sign indicates that NaAsO 2 is added and removed after 1 h. Abbreviations: Tet, tetracy-cline; NaAsO 2 , sodium arsenite; CHX, cycloheximide.

Article Snippet: Oxidative stress was induced as above, and cycloheximide (CHX; 5 μg/mL; Merck, Darmstadt, Germany) was also used, as a control.

Techniques: Expressing, Phospho-proteomics, De-Phosphorylation Assay, Western Blot, Molecular Weight

Journal: Journal of Cancer Prevention

Article Title: Overcoming the Intrinsic Gefitinib-resistance via Downregulation of AXL in Non-small Cell Lung Cancer

doi: 10.15430/JCP.2019.24.4.217

Figure Lengend Snippet: Enhancement of AXL degradation by yuanhuadine (YD) in H1299 cells. (A, C) Cells were treated with 25 μg/mL cycloheximide (CHX) and 10 nM YD for the indicated times. Cell lysates were analyzed by Western blot with an antibody against AXL. β-actin was used as a loading control. (B, D) AXL expression levels were quantifies by densitometry using ImageJ.

Article Snippet: Cycloheximide (CHX) was purchased from A.G. Scientific (San Diego, CA, USA).

Techniques: Western Blot, Control, Expressing

Journal: Molecular Oncology

Article Title: Endothelin‐converting enzyme‐1c promotes stem cell traits and aggressiveness in colorectal cancer cells

doi: 10.1002/1878-0261.12609

Figure Lengend Snippet: Mutation of Lys‐6 to Arg enhances stability of ECE1c. (A) DLD‐1 clone cells expressing Flag‐tagged ECE1c WT or ECE1c K6R proteins were incubated with 20 μg·mL −1 cycloheximide (CHX) in the absence (for 36 h) or presence (for 12 h) of 25 μ m silmitasertib. ECE1c proteins were detected by western blot with an anti‐Flag antibody, using β‐actin as loading control. Representative blots are shown (upper). Relative levels (%) of Flag‐ECE1c proteins from three independent experiments were calculated (lower). (B) DLD‐1 clones described in A were grown in medium without FBS for 48 h. Culture supernatants were used to measure ET‐1 levels using ELISA according to manufacturer’s instructions (Thermo Fisher). Data represent average ± SEM ( n = 3). ANOVA and Tukey tests were used. * P ≤ 0.05, ** P ≤ 0.01.

Article Snippet: Cells (5 × 10 5 ) were seeded into P60 plates and cultured for 36 h in complete medium under normal conditions, with 20 μg·mL −1 cycloheximide (CHX) in the absence or presence of 25 μ m silmitasertib (ApexBio Technology LLC, Houston, TX, USA).

Techniques: Mutagenesis, Expressing, Incubation, Western Blot, Clone Assay, Enzyme-linked Immunosorbent Assay