Journal: eLife

Article Title: A single cysteine residue in vimentin regulates long non-coding RNA XIST to suppress epithelial–mesenchymal transition and stemness in breast cancer

doi: 10.7554/eLife.104191

Figure Lengend Snippet: ( A ) Volcano plot showing differentially expressed genes (DEGs) between WT and C328S cells. Gene Ontology (GO) showing overview of cellular functions, ( B ) downregulated, and ( C ) upregulated by DEGs. ( D ) RNA-Seq analysis showing log2-fold expression and validation by RT-qPCR for DEGs of interest. ( E ) XIST , the most upregulated gene, has been implicated in a large number of solid tumours . ( F ) Linear regression analysis of log2-fold changes from RT-qPCR and RNA-Seq of DEGs. ( G ) Immunostaining of WT and C328S cells with V9, rabbit anti-K8, and rabbit anti-K18 using AF-488 (green) goat anti-mouse and AF-594 (red) goat anti-rabbit were used. Nuclei are in blue, overlapping images are shown as Merge. Leica DM4000B Epi-fluorescence microscope was used for imaging (scale bar = 20 µm). ( H ) VIM, K18, K19, K8, CDH2/N-cadherin, and TWIST1 expression by western blotting in WT and C328S cells. Relevant bands were cropped from the original blots shown in and . ( I ) Quantification of the protein expression in panel (H) using ImageJ. ( J ) Relative log2-fold changes in the expression of EMT transcription factors, and ( K ) breast cancer stem cell markers in WT and C328S cells by RNA-Seq analysis. ( L ) Flow cytometry overlay dot plot of CD56-RY586 vs CD201-APC after gating on single and live cells for immunophenotype. ( M ) Comparison of mean fluorescence intensity (MFI) of CD56 in WT and C328S cells. ( N ) Comparison of MFI of CD201 in WT and C328S cells. ( O ) Transplantation of WT and C328 cells in nude mice without oestrogen. ( P ) Average tumour burden after 2 weeks in nude mice injected with WT and C328S cells. ( Q ) H&E-stained tumour sections scale bar = 50 µm. ( R ) Representative image from immunohistochemical staining of CD56 and CD201 in tumour sections compared with IgG control, scale bar = 50 µm. ( S ) Quantification of CD56 and ( T ): CD201 staining in tumour sections and control using ImageJ. Statistical analyses: n = 3, Error bars = ± SEM, Student’s t -test to calculate p values using Microsoft Excel and are given as asterisks (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001). Figure 3—source data 1. Full-size western blots indicating the relevant bands cropped for . Figure 3—source data 2. Original files for western blots analysis displayed in .

Article Snippet: Sections were then incubated with anti-CD56 or anti-CD201 antibodies listed in or the equivalent concentration of normal rabbit or normal goat IgG as control, at 4°C for 16 h. Slides were washed in TBS, and then incubated sequentially with biotinylated anti-rabbit/mouse (for CD56) or biotinylated anti-goat (for CD201) secondary antibodies and streptavidin-peroxidase reagent (Vectastain Elite ABC-HRP Kit; Vector Laboratories, cat# PK-6100) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Expressing, Biomarker Discovery, Quantitative RT-PCR, Immunostaining, Fluorescence, Microscopy, Imaging, Western Blot, Flow Cytometry, Comparison, Transplantation Assay, Injection, Staining, Immunohistochemical staining, Control