dmem/ham’s f12 (fad) medium with low ca 2  (Biochrom)

Journal: Journal of Cell Science

Article Title: Cell volume regulates terminal differentiation of cultured human epidermal keratinocytes

doi: 10.1242/jcs.264242

Figure Lengend Snippet: Bulk RNA sequencing of keratinocytes seeded on S1 or S2 substrates. (A) Schematic overview. Cells were cultured for 1 h, 4 h or 12 h in complete FAD medium prior to RNA extraction. RNA was extracted from cells in three independent experiments (replicate experiments are denoted e.g. 1 h S1_1, 1 h S1_2 and 1 h S1_3). Created in BioRender by Zijl, S., 2025. https://BioRender.com/udy2d42 . This figure was sublicensed under CC-BY 4.0 terms. (B) Principal component analysis (PCA) plots comparing cells on S1 (red dots) with cells on S2 (blue dots) at different time points. Each data point in the graph represents a different sample. PC, principal component. (C) Volcano plot showing global transcriptional differences between cells on S1 and S2 at 12 h. Each data point represents one gene. The log 2 fold change (LFC) of each gene is shown on the x -axis and the −log 10 of its Benjamini–Hochberg adjusted P -value ( P adj) is shown on the y -axis. Genes with a P adj<0.05 and LFC>1 (red dots) are upregulated on S2. Genes with P adj<0.05 and LFC<−1 (green dots) are downregulated on S2. (D,E) The most significantly differentially expressed genes at 12 h. (D) Heatmaps of the 30 most significantly differentially expressed genes (DEGs) on S1 and S2 at 12 h. Genes were sorted by their P adj (left-hand column) or expression based on log 2 transformed expression values and LFC in expression (right-hand column). Left-hand column, the most significantly DEGs (lowest P adj) are shown first. Right-hand column, highly expressed genes upregulated on S2 (compared to S1) are shown first, followed by highly expressed genes downregulated on S2. Each square represents the (log 2 transformed) expression level for a different gene (rows) or sample (columns). (E) Heatmaps of the top 10 upregulated genes (highest LFC) (left-hand column) and downregulated genes (highest negative LFC) (right-hand column) on S2 at 12 h (compared to S1). Genes were selected based on a P adj<0.05 and ranked according to their LFC. Each square represents the log2 transformed expression level for a different gene (rows) or sample (columns). (F) Heatmaps of selected AP1 transcription factors showing changes in expression at 4 h or 12 h relative to 1 h. Each square represents the log 2 transformed expression level for a different gene (rows) or sample (columns).

Article Snippet: pLenti-LifeAct-EGFP-INV-mCherry-infected keratinocytes were disaggregated and seeded on polystyrene (PS) topographies for 45–60 min. Non-adherent cells were removed, and adherent cells were incubated at 37°C for 4–6 h before being transferred to Phenol Red-free FAD medium supplemented with 1 mM sodium pyruvate, 36.5 mM sodium bicarbonate (Gibco) and 100 mM HEPES.

Techniques: RNA Sequencing, Cell Culture, RNA Extraction, Expressing, Transformation Assay

Journal: Journal of Cell Science

Article Title: Cell volume regulates terminal differentiation of cultured human epidermal keratinocytes

doi: 10.1242/jcs.264242

Figure Lengend Snippet: Effects of PEG300 and DI on cells on micropatterned islands and S1 and S2 substrates. (A) Representative images of anti-involucrin (IVL, red) and anti-keratin14 (K14, magenta) staining of cells grown on 20 μm and 50 μm diameter micropatterned islands. Cells were counterstained with DAPI (blue). Cells were grown in the presence of 5% PBS, 5% PEG300, 30% PBS or 30% DI for 24 h. Scale bars: 50 μm. (B,C) Quantification of (B) the percentage of differentiated cells (% IVL+ cells) and (C) the fold change in the percentage of differentiated cells (% IVL+ cells, normalized to 20 μm islands treated with 5% PBS). Results are from three independent experiments performed with three technical replicates per condition. (D) Quantification of the percentage of differentiated cells (% IVL+ cells) on flat, S1 and S2 substrates. Cells were cultured in the presence of 5% PEG300 for 24 h and fixed or allowed to recover in complete FAD medium for 24 h (washout). (E) Cells grown on S2 for 24 h in the presence of 30% PBS or 30% DI. More than 1000 cells were analysed per experiment. * P <0.05; *** P <0.001 [two-way ANOVA lpus Šídák's) test (B,C); one-way ANOVA plus Tukey's test (D,E)]. Error bar show s.d.

Article Snippet: pLenti-LifeAct-EGFP-INV-mCherry-infected keratinocytes were disaggregated and seeded on polystyrene (PS) topographies for 45–60 min. Non-adherent cells were removed, and adherent cells were incubated at 37°C for 4–6 h before being transferred to Phenol Red-free FAD medium supplemented with 1 mM sodium pyruvate, 36.5 mM sodium bicarbonate (Gibco) and 100 mM HEPES.

Techniques: Staining, Cell Culture

Journal: bioRxiv

Article Title: Functional analyses and integrated mechanisms of cellular destruction by L-amino acid oxidase

doi: 10.1101/2024.09.16.613219

Figure Lengend Snippet: A , Monomeric structure of B. atrox LAAO showing the predicted catalytic and substrate binding residues as sticks: Y372, R322, R90, H223, N172. B , Surface of the substrate binding pocket of LAAO ( A. H. pallas venom (PDB 1TDN) showing positively charged amino acid residues (blue), polar residues (cyan), and hydrophobic residues (green). L-leucine substrate is shown as magenta stick. C , L-amino acid oxidation reaction catalysed by LAAO WT with co-factor FAD (E-FAD) generating H 2 O 2 and ammonia as by products. D - E, Activity of purified LAAO WT or LAAO mutants (52.72 nM) compared to wild type (D, arbitrarily set at 100%) or their initial velocity V (E; µM per second, Y axis). The initial velocity V is defined as the change in the concentration of H 2 O 2 produced by LAAO at each concentration of substrate L-leucine (mM, X axis). In the bottom panel, catalytic activities of selected mutants are shown magnified (100X). F , Keratinocyte cell viability was assessed by incubation 14 nM of LAAO WT or mutants. G-H , Cellular reactive oxygen species (ROS) generated by keratinocytes untreated or treated with 280 nM LAAO WT or LAAO R90A and live stained with CellRox Green (green) and fixed before confocal imaging. Representative images (G) and quantification (H) are shown. Bars indicate mean and error bars show standard deviation. The assays were done as multiple technical replicates in three independent biological replicates (thereafter N=3), represented in blue, green, or pink colours. Black asterisks represent comparison within a single treatment group; red asterisk compares across different treatments. **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Number of all images used for the analysis are indicated in File S1. Scale bar: 20 µm.

Article Snippet: Normal human epidermal keratinocytes at 4000 cells/well (passages 3-6) were co-cultured with 3T3-J2 fibroblasts in a 96 well plate (Corning, 3585; cell viability assay) or a CellCarrier-96 ultra microplates (PerkinElmer, 6055300; confocal imaging) in FAD standard medium (FAD medium (Gibco, custom made) as described previously ).

Techniques: Binding Assay, Activity Assay, Purification, Concentration Assay, Produced, Incubation, Generated, Staining, Imaging, Standard Deviation, Comparison