Journal: Science Advances

Article Title: Targeting oncoproteins with a positive selection assay for protein degraders

doi: 10.1126/sciadv.abd6263

Figure Lengend Snippet: ( A ) Immunoblot and ( B ) RT-qPCR analysis of the NCI-H1876 SCLC cell line that endogenously expresses ASCL1 infected to express the indicated sgRNAs. n = 3 biological replicates. ( C and E ) Immunoblot and ( D and F ) RT-qPCR analysis of NCI-H1876 human SCLC cells (C and D) and 97-2 mouse SCLC cells (E and F) after treatment with the CDK2 PROTAC degraders (TMX-2138 and TMX-2172) or the indicated negative controls, all used at 500 nM for either 36 hours (C and D) or 8 hours (E and F). Neg Deg, negative control degrader ZXH-7035. n = 3 biological replicates. ( G ) Immunoblot analysis and ( H ) quantification of ASCL1 protein levels in 97-2 cells first treated with the CDK2 PROTAC degrader or negative control (500 nM) for 4 hours and then treated with cycloheximide (CHX) (150 μg/ml) for the indicated times. S.E., short exposure; L.E., long exposure. n = 4 biological replicates. In all experiments, error bars represent SD except in (H), where error bars represent SEM. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following compounds were purchased: POM (Selleck, #S1567), LEN (Selleck, #S1029), MG132 ( N -carbobenzyloxy- l -leucyl- l -leucyl- l -leucinal; Thermo Fisher Scientific, #47479020MG), MLN4924 (Active Biochem, #A-1139), MLN7243 (Thermo Fisher Scientific, #NC1129906), Spautin-1 (BioTechne; #5197/10), cycloheximide (VWR, #97064-724), BVdU (Chem-Impex International Inc., catalog no. 27735), actinomycin D (Thermo Fisher Scientific, #11805017), and dinaciclib (Selleck, #S2768).

Techniques: Western Blot, Quantitative RT-PCR, Infection, Negative Control

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: bioRxiv

Article Title: Functional and structural deficiencies of Gemin5 variants associated with neurological disease

doi: 10.1101/2022.01.25.477707

Figure Lengend Snippet: (A) HEK293 cells expressing the wild type version of G5 845-1508 , side by side to the variants R1016C, D1019E or L1367P during 12 h were treated (+) or not (-) with cycloheximide (CHX) for additional 16 h. Samples were taken at 0, 12 and 16 h post CHX treatment. The intensity of each protein at the indicated time was determined by WB. A long exposure is shown for L1367P protein. (B) Steady-state analysis of Xpress-G5 845-1508 mRNA levels present in transfected cells at the time of harvesting determined by RTqPCR. (C) Values represent the protein intensity (mean ± SEM) of three independent experiments relatively to time 0. Asterisks denote P -values (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: For cycloheximide (CHX) chase experiments, CHX (100 μg/ml) (Merck) was added to stop translation at 12 h or 24 h post-transfection (hpt) for Xpress-G5 845-1508 or Xpress-Gemin5 proteins, respectively.

Techniques: Expressing, Transfection

Journal: Epigenetics

Article Title: An HP1 isoform-specific feedback mechanism regulates Suv39h1 activity under stress conditions

doi: 10.1080/15592294.2016.1278096

Figure Lengend Snippet: (A) Cycloheximide (CHX) chase experiment to determine endogenous Suv39h1 protein stability in absence or presence of empty vector (−) or FLAG-tagged HP1α, β or γ after 0, 2, 5, 10, and 24 h of CHX treatment. (B) Ubiquitination of Suv39h1 in presence or absence of Suv39h1 isoforms. Myc-Suv39h1 was purified from 293F cells expressing Ubiquitin-HA and the indicated HP1 constructs. After immunoprecipitation, equal levels of immunopurified Suv39h1 were loaded in a gel and the levels of ubiquitination (HA-tag incorporation) were tested. The inputs of the initial immunoprecipitation experiment are shown in Fig. S2D. (C) Levels of Suv39h1 in presence or absence of SirT1 or HP1α. The relative levels were normalized compared to Suv39h1 levels in WT cells upon overexpression of empty vector (−). **P < 0.005; ****P < 0.0001. (D) Effect of HP1α, β, and γ in MDM2-dependent degradation of Suv39h1. Western blot of the levels of Suv39h1 in H1299 cells transfected with MDM2−/+ HP1 indicated isoforms.

Article Snippet: For Suv39h1 stability assays, NIH3T3 cells expressing empty vector, FLAG-HP1α, β or γ were incubated with 200 μg/ml cyclohexamide (FLUKA) for 0, 2, 4, 6, 8, 24, 30 h and the levels of endogenous Suv39h1 were analyzed by western-blot (Millipore).

Techniques: Plasmid Preparation, Ubiquitin Proteomics, Purification, Expressing, Construct, Immunoprecipitation, Over Expression, Western Blot, Transfection

Journal: bioRxiv

Article Title: Mapping the adaptor protein complex interaction network in Arabidopsis identifies P34 as a common stability regulator

doi: 10.1101/2022.08.31.505729

Figure Lengend Snippet: a , FM4-64 uptake in the p34 iCRISPR -1 and p34 iCRISPR -2 mutants. Root epidermal cells of 5-day-old wild type (Col-0), p34 iCRISPR -1 , p34 iCRISPR -2 and ap2m-2 seedlings grown on DMSO or 10 μM β-estradiol (Est) were imaged after staining with FM4-64 (2 μM, 15 min). b, Relative intracellular to plasma membrane (PM) fluorescence intensity ratio of FM4-64 of images in (a). n =40 cells in 10 roots were analysed. c, d, Immunolocalization of PIN2 in epidermal (epi) and cortex (cox) cell in (c) and in lateral root cap (LRC) cells in (d) of 7-day-old Col-0, p34 iCRISPR -1 and p34 iCRISPR -2 roots treated with 10 μM β-estradiol. Arrow heads and arrows indicate the non-polar distribution of PIN2 in epidermal and lateral root cap cells and endosomal PIN2, respectively. The insets show the non-polar distribution of PIN2 in the epidermis. Scale bars, 10 µm. The ratio of PIN2 abnormal/normal localization in roots is indicated for three independent experiments. PIN2-GFP in the root cells of 5-day-old wild type and p34 iCRISPR -1 seedlings grown on DMSO and 10 μM β-estradiol. The seedlings were pretreated with cycloheximide (CHX) (50 μM, 1 h) and then treated with CHX (50 μM) and Brefeldin A (BFA) (50 μM) for 30 min. Seedlings were washed with media containing CHX (50 μM) and imaged 100 min after the wash. f , BFA body sizes before the washout. n, number of BFA bodies. At least seven roots were imaged and measured. g , Percentage of BFA body-containing cells in PIN2-GFP seedlings before and after BFA washout in (e). n , number of roots analysed. h , Fluorescence intensity images of SYP61-CFP in the root cells of 5-day-old Col-0 and p34 iCRISPR -1 grown on DMSO or 10 μM β-estradiol. i , Relative intracellular to PM fluorescence intensity ratios of SYP61-CFP in one representative experiment. ns, not significant. n, number of cells being analysed. At least five roots were imaged and measured. j , Fluorescence intensity images of VHAa1-mRFP in the root cells of 5-day-old Col-0 and p34 iCRISPR -1 grown on DMSO or 10 μM β-estradiol. k , Relative intracellular to PM fluorescence intensity ratios of VHAa1-mRFP in one representative experiment. n, n u mber of cells analysed. i, Localization of MTV1-GFP in the root cells of 5-day-old Col-0, p34 iCRISPR -1 and ap4b-1 grown on DMSO or 10 μM β-estradiol. m , Quantification of the presence of defined MTV1-GFP punctate signals by analysis of the skewness of the signal intensity from at least seven independent point populations (seven roots). Higher and lower skewness indicate and more diffuse signals, respectively. The imaging of all genotypes was repeated three times. One representative experiment is shown. Error bar represents standard deviation. *** P ≤ 0.001 [one-way ANOVA in (b, f, i, k, m)], ns, not significant, a.u., arbitrary units.

Article Snippet: β-estradiol (Sigma-Aldrich, 20 Mm stock in DMSO), BFA (Sigma-Aldrich, 50 mM stock in DMSO), CHX (Merck, 50 mM stock in DMSO), and FM4-64 (Invitrogen, 2 Mm stock in water), were used at the concentrations indicated.

Techniques: Staining, Fluorescence, Imaging, Standard Deviation