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: 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

Journal: NPJ Biofilms and Microbiomes

Article Title: Assessing the inflammatory response to in vitro polymicrobial wound biofilms in a skin epidermis model

doi: 10.1038/s41522-022-00286-z

Figure Lengend Snippet: 24-hour biofilms were treated with 3% H 2 O 2 , 10% w/v PVP-I, and 0.05% CHX for 24 hours at 37 o C under anaerobic conditions. Following treatment, viability of the biofilms were assessed using live/dead qPCR analyses for total bacterial counts (16 S; a ), total fungal counts (18 S; b ) and combined counts ( c ). % viability was calculated using the above counts for total vs. viable cells ( d ). Statistical significance was determined using a two-tailed Student’s t-test to compare the means of treated biofilms vs untreated biofilms (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: These treatments, hydrogen peroxide (H 2 O 2 ), povidone iodine (PVP-I) and chlorhexidine (CHX) (all Merck Life Sciences, Gillingham, UK), were prepared fresh in sterile ddH 2 O prior to use.

Techniques: Two Tailed Test

Journal: NPJ Biofilms and Microbiomes

Article Title: Assessing the inflammatory response to in vitro polymicrobial wound biofilms in a skin epidermis model

doi: 10.1038/s41522-022-00286-z

Figure Lengend Snippet: Skin epidermis was exposed to treated and untreated biofilms for 24 h at 37°C, 5% CO 2 . Appropriate negative and positive controls were used. Following stimulation, RNA was extracted, cDNA synthesized, and gene expression was profiled using a custom array containing primers for different pro-inflammatory genes and upregulated proteins from the Olink technology (Fig. ). Panel a depicts the Log 2 fold change in RHE tissue stimulated with PMA (POS CON), untreated biofilms (UT), CHX-treated, PVP-I-treated, or H 2 O 2 -treated biofilms, all relative to unstimulated negative controls. Data presented as a n = 6, encompassing three technical replicates from two independent experiments. Each column represents these 6 replicates. Panel b shows a principal component analysis plot showing distinct clustering of the transcriptional profiles in the tissue following stimulation.

Article Snippet: These treatments, hydrogen peroxide (H 2 O 2 ), povidone iodine (PVP-I) and chlorhexidine (CHX) (all Merck Life Sciences, Gillingham, UK), were prepared fresh in sterile ddH 2 O prior to use.

Techniques: Synthesized, Expressing

Journal: NPJ Biofilms and Microbiomes

Article Title: Assessing the inflammatory response to in vitro polymicrobial wound biofilms in a skin epidermis model

doi: 10.1038/s41522-022-00286-z

Figure Lengend Snippet: Volcano plots show the log fold change in protein concentration after correction of the p values using Benjamini–Hochberg false discovery rate (FDR) of 5%. Proteins that are significantly downregulated are shown in blue, and those upregulated are red. The dotted line depicts significance value of p < 0.01 (-log 2.0). PMA-stimulated positive controls (PC) were first compared with unstimulated control tissue (NC) ( a ), then tissue stimulated with untreated biofilms with negative controls ( b ). Each treatment modality was then compared with untreated biofilms (CHX vs UT, panel c ; PVP-I vs UT, panel d and H 2 O 2 vs UT, panel e ).

Article Snippet: These treatments, hydrogen peroxide (H 2 O 2 ), povidone iodine (PVP-I) and chlorhexidine (CHX) (all Merck Life Sciences, Gillingham, UK), were prepared fresh in sterile ddH 2 O prior to use.

Techniques: Protein Concentration

Journal: NPJ Biofilms and Microbiomes

Article Title: Assessing the inflammatory response to in vitro polymicrobial wound biofilms in a skin epidermis model

doi: 10.1038/s41522-022-00286-z

Figure Lengend Snippet: 11-species biofilm was formed on and throughout the porous cellulose matrix substrate using the serum-containing hydrogel system. These biofilms were also treated with CHX, H 2 O 2 or PVP-I following growth on the hydrogel ( a ). For the co-culture set-up, the matured biofilms were removed from the hydrogel and transferred to the reconstructed human epidermis ( b ). Experimental outputs for the study are shown in panels c , d , for the biofilm and RHE tissue experiments, respectively. In brief, the main outputs for the biofilm part of the study involved scanning electron microscopic imaging, compositional analysis and live/dead qPCR. For the tissue part of the study, this included gene expression and proteomic analyses including presentation of data as heatmaps, volcano plots and/or principal component analyses.

Article Snippet: These treatments, hydrogen peroxide (H 2 O 2 ), povidone iodine (PVP-I) and chlorhexidine (CHX) (all Merck Life Sciences, Gillingham, UK), were prepared fresh in sterile ddH 2 O prior to use.

Techniques: Co-Culture Assay, Imaging, Expressing

Journal: Cell

Article Title: Parental genome unification is highly error-prone in mammalian embryos

doi: 10.1016/j.cell.2021.04.013

Figure Lengend Snippet:

Article Snippet: Cyclohexamide (Merck #239763) was diluted in embryo tested water (Sigma-Aldrich #TMS-006-C) to make a 10 mg/ml stock and was added to activated eggs at a final concentation of 10 μg/ml.

Techniques: Recombinant, RNA HS Assay, Clone Assay, Plasmid Preparation, Software, Microscopy