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: PLoS ONE

Article Title: Phenotypic Switching Induced by Damaged Matrix Is Associated with DNA Methyltransferase 3A (DNMT3A) Activity and Nuclear Localization in Smooth Muscle Cells (SMC)

doi: 10.1371/journal.pone.0069089

Figure Lengend Snippet: (A) Timecourse of intracellular DNMT3A expression/localization after plating cells on NC and DNC. DNC plated cells show stronger DNMT3A signals overall than NC plated cells. The 36 hour timepoint shows strong signal in the nucleus of DNC plated cells. At 48 hours there continues to be high expression in the DNC cells, though the nuclear stain was not as clear as the 36 hour timepoint. NC cells did not show nuclear staining. (B) DNMT3A nuclear localization is slightly affected by inhibitors of transcription (actinomycin D) and translation (cyclohexamide) on NC, but downregulation on DNC strongly depends on both functions. SMC were plated for 4 hours as in and treated with cyclohexamide or actinomycin for the next 44 hours.

Article Snippet: Dosages for each treatment were as follows: Nocodazole at 0.04 μg/mL (Sigma-Aldrich), 10 μg/mL cyclohexamide (LC Laboratories), PD98059 at 40 μM (Sigma), F11 antibody at 0.03 μM, (Sigma-Aldrich), FGF2 at 10 μg/mL (BD Transduction), EGF at 50 μg/mL (BD Transduction), DAC at 0.2, 1 or 3 μM as indicated (Sigma-Aldrich) and rapamycin at 5 ng/mL (LC Laboratories).

Techniques: Expressing, Staining

Journal: The Journal of investigative dermatology

Article Title: CCAR2 is required for proliferation and tumor maintenance in human Squamous Cell Carcinoma

doi: 10.1016/j.jid.2016.09.027

Figure Lengend Snippet: (a) Histology (H&E) and Immunofluorescence of CCAR2 (green) and Keratin 14 (K14, red) protein expression in normal human skin and Squamous Cell Carcinoma (SCC) frozen sections. DAPI (blue) stains the nucleus. Scale = 50μm. (b) Western blot analysis of CCAR2 and SIRT1 protein in keratinocytes (OKF6, P1-Ep and N) and SCC (SCC-13, SCC-25, JHU-029 and HO1N1) cell lines. GAPDH provides the loading control. (c) Quantitative RT-PCR of CCAR2 (left) and SIRT1 (right) mRNA expression in keratinocytes (Ker) and SCC cell lines. p > 0.05, not significant (N.S.). (d) Western blot of CCAR2 and SIRT1 protein expression in keratinocytes (N, P1-Ep) and SCC (HO1N1 and JHU-029) cell lines treated with Cyclohexamide (CHX, 100 μg/ml) for 0, 2, 4, 6 or 8 hours. GAPDH provides the loading control.

Article Snippet: For protein stability experiments, cells were treated with 100 μg/ml Cyclohexamide (VWR) for up to 8 hours.

Techniques: Immunofluorescence, Expressing, Western Blot, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Sonic hedgehog signaling instigates high-fat diet–induced insulin resistance by targeting PPARγ stability

doi: 10.1074/jbc.RA118.004411

Figure Lengend Snippet: Shh signaling decreased PPARγ stability via NEDD4-1–dependent ubiquitination. HEK 293 cells were transfected with PPARγ overexpression plasmid. A, cells were pretreated with cyclohexamide (CHX) (5 μg/ml) for 30 min before exposure to SAG (0.5 μmol/liter), followed by immunoblotting to detect PPARγ and β-actin (left). Quantifications of band intensity normalized to β-actin (right). B, cells were incubated with SAG (0.5 μmol/liter) for 24 h in the presence or absence of MG132 (10 μmol/liter) pretreatment. C, cells were infected with scramble shRNA lentivirus or different lentiviral shRNA constructs against NEDD4-1. PPARγ, NEDD4-1, and β-actin levels were analyzed by immunoblotting. D, immunoblotting (IB) of whole cell lysates (Input) and immunoprecipitates (IP) from PPARγ-overexpressing HEK 293 cells with or without SAG (0.5 μmol/liter) treatment for 24 h. MG132 (10 μmol/liter) was added to the medium 12 h before collecting. E, PPARγ-transfected HEK 293 cells were pretreated with MG132 (10 μmol/liter) and then incubated with or without SAG (0.5 μmol/liter) for 24 h. PPARγ was immunoprecipitated from cell lysates and immunoblotted with an anti-ubiquitin antibody (left). Band intensities were normalized to that of IgG (right). Immunoblots shown are representative of 3 independent experiments. *, p < 0.05.

Article Snippet: Cyclohexamide was from Cayman Chemical (Ann Arbor MI).

Techniques: Ubiquitin Proteomics, Transfection, Over Expression, Plasmid Preparation, Western Blot, Incubation, Infection, shRNA, Construct, Immunoprecipitation

Journal: eLife

Article Title: Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome

doi: 10.7554/eLife.08467

Figure Lengend Snippet: 80 shRNAs targeting 20 different proteasome subunits and control hairpins were expressed in HepG2 ( A ), H838 ( B ), T47D ( C ), and H1792 ( D ) cells by viral transduction. Each subunit was targeted by 4 different shRNAs and the cell viability was measured as the relative cell number compared to the average of non-targeting shRNAs 5 days after the initial introduction of the shRNAs. ( E , F ) HepG2 cells with shRNAs targeting PSMC5, PSMD2, and green fluorescent protein (GFP) were grown out. Their relative growth was analyzed in the absence of bortezomib ( E ) and their protein content was analyzed 24 hr after the addition of either 8 or 12 nM of bortezomib ( F ). ( G ) The relative cell number of cells harboring a control shLacZ (black) or each of 5 individual shRNAs targeting shPSMC5 (Cayenne) was analyzed 4 days after addition of the indicated concentrations of bortezomib. ( H ) HepG2 cells stably expressing shRNAs targeting the PSMC5 subunit and a control shRNA (lacZ) were analyzed by Western blot for the indicated proteins 24 hr with or without bortezomib treatment. ( I – N ) The HepG2 cells with shRNAs targeting PSMC5, PSMD2, and GFP (described above) were further exposed to a short panel of stress inducers including bortezomib ( I ), tunicamycin ( J ), rohinitib-RHT ( K ), Hsp90 inhibition ( L ), withaferin A ( M ), and cyclohexamide ( N ) at indicated concentrations and the relative cell number (RFU) was examined after 4 days. The graphs represent the average of at least 4 different measurements and the SEM. DOI: http://dx.doi.org/10.7554/eLife.08467.006

Article Snippet: MG132 (EMD Millipore, Billerica, MA, United States), Bortezomib (LC Laboratories # B-1408, Woburn, MA, United States), Cyclohexamide (Enzo life sciences, Farmingdale, NY, United States), withaferin A, tunicamycin (Sigma St. Louis, MO, United States).

Techniques: Transduction, Stable Transfection, Expressing, shRNA, Western Blot, Inhibition

Journal: eLife

Article Title: Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome

doi: 10.7554/eLife.08467

Figure Lengend Snippet: Cells were then collected, washed, and plated in the absence of doxycycline 24 hr prior to exposure to increasing concentration of bortezomib ( A ), MG132 ( B ), cyclohexamide ( C ), withaferin A ( D ), tunicamycin ( E ), and rotenone ( F ). ( G – I ) PSMD2 KD was induced as described above and then cells were grown in the presence or absence of 15 nM bortezomib for the indicated time points (3, 6, 24 hr). ( G ) Immunoblot analysis of the total proteasome subunits following SDS-PAGE (upper panels) and the 20S proteasome complex levels after native gel electrophoresis. These results were quantified by Imagelab software and plotted ( H ). ( I ) Lysosomal degradation rate was measured in control and PSMD2 knock down (Dox) cells in the presence or absence of 10 nM Bortezomib treatment for 20 hr. DOI: http://dx.doi.org/10.7554/eLife.08467.010

Article Snippet: MG132 (EMD Millipore, Billerica, MA, United States), Bortezomib (LC Laboratories # B-1408, Woburn, MA, United States), Cyclohexamide (Enzo life sciences, Farmingdale, NY, United States), withaferin A, tunicamycin (Sigma St. Louis, MO, United States).

Techniques: Concentration Assay, Western Blot, SDS Page, Nucleic Acid Electrophoresis, Software