Journal: bioRxiv
Article Title: Vimentin intermediate filaments organize organellar architecture in response to ER stress
doi: 10.1101/2022.03.24.485587
Figure Lengend Snippet: A) Validation of Vimentin knockout in U2OS cells. Western blot analysis of two Vimentin knockout U2OS cells created using CRISPR/Cas9. Membranes were stained for endogenous Vimentin, while Vinculin was stained as a loading control. MW markers indicated. B) Distribution of RNF26 as a function of Vimentin. Representative confocal microscopy images of overexpressed GFP-RNF26 distribution in WT and VIM KO#1 U2OS, immunostained for VAP-A to visualize the ER.. Boxed zoom-ins designate region spanning from the perinuclear area (PN) towards the plasma membrane (periphery, PP). Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10μm. C) Colocalization (Manders’) of VAP-A over RNF26 in WT vs VIM KO#1 cells, indicating the fraction of the ER that is covered by RNF26. Error bars indicate mean +/− SD (t-test, **** p<0,001, n = 22 cells (WT) vs 12 cells (KO). D) Colocalization (Manders’) of RNF26 over VAP-A in WT vs VIM KO#1 cells, indicating presence of RNF26 in the ER after VIM KO. Error bars indicate mean +/− SD (t-test, N.S. = non-significant). E, F) Late (E) and early (F) endosomal distribution as a function of Vimentin (and RNF26 (E)). Shown are Z-slices of representative parental U2OS cell, siRNF26#1 (E), VIM KO#1 cells, and VIM KO#2 cells that were transfected with Vimentin. Cells were fixed and immunostained for LAMP1 (E) (and Vimentin (rescue, E) or EEA1 (F) prior to imaging with a confocal microscope. Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10μm. G) Strategy for quantification of perinuclear vs peripheral fluorescent signals used in this paper. Briefly, cell nucleus and PM were manually annotated after which the cytoplasm was classified into 4 bins ranging from nucleus to PM. Then, using a custom Fiji plugin, fluorescent signals were merged with the binned image and mean fluorescence per bin and bin size were calculated. Average intensity per pixel in each bin was calculated, weighed for bin size and compared among bin 1 (PN) and bin 3 (PP) to obtain a ratio of perinuclear vs peripheral signal. H) Endosomal distribution analysis of late (LAMP1) and early (EEA1) endosomal distributions using Fiji plugin as described in (G). 51 (WT), 55 (VIM KO#1), 65 (VIM KO#2), 19 (VIM KO#1 + rescue), 42 (siC) and 48 (siRNF26) cells were analyzed (Mann-Whitney U test, (* p<0,05; ** p<0,01; **** p<0,0001). Error bars indicate median and 95% confidence interval
Article Snippet: ( Confocal microscopy) mouse anti-EEA1 (1:200, mAb 610457, BD transduction laboratories), mouse anti-CD63 NKI-C3 (1:500, , rabbit anti-LAMP1 (1:200, Sino Biological), rabbit anti-VAP-A (1:100, 15272-1-AP, Proteintech), goat anti-CNX, mouse anti-CLIMP63 (followed by anti-Rabbit/Mouse/goat Alexa-dye coupled antibodies (1:400, Invitrogen).
Techniques: Knock-Out, Western Blot, CRISPR, Staining, Confocal Microscopy, Transfection, Imaging, Microscopy, Fluorescence, MANN-WHITNEY
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