Extended Data Fig. 5: Assessment of cytoskeleton organization and lipid composition in TRE3G-NINJ1-IRES-mCherry stable HEK-293T cells.
From: NINJ1 regulates plasma membrane fragility under mechanical strain
a-b, Cells were treated Dox at various doses. Where applicable, mCherry fluorescence intensity were used to assess the relative expression levels of NINJ1. Quantification data are from cells from 3 separate fields for each group using one-way ANOVA followed by Bonferroni corrections. a, Representative images and quantitation of immunofluorescence staining of F-actin in cells express different levels of NINJ1. b, Representative images and quantitation of β-tubulin staining. Average intensity, tubule lengths and tubule count per cells were quantified. c-d, TRE3G-NINJ1-IRES-mCherry stable HEK-293T cells were transfected with Dox-inducible fluorescent probes of phosphatidic acid (PA) and phosphatidylserine (PS), then incubated with 300 ng ml−1 Dox to induce NINJ1 and lipid probe expression. Quantification data are from cells from 3 separate fields for each group and analysed by one-way ANOVA followed by Bonferroni corrections. c, Representative images and quantitation of average intensity of PA-GFP. d, Representative images and average intensity of PS-GFP. e, TRE3G-NINJ1-IRES-mCherry stable HEK-293T cells were incubated with Dox at various doses to induce NINJ1 expression, then stained for Annexin V and quantified for Annexin V intensity (n = 6 ~ 15 wells from 3 independent experiments, one-way ANOVA followed by Bonferroni corrections). f, Validation of Annexin V antibodies was carried out by staining cells treated with nigericin to induce cell death (n = 10 ~ 11 wells from 3 independent experiments, two-sided unpaired Student’s t-test, ** p <0.01 versus control). Unless otherwise noted, all data are presented as mean ± s.e.m. All scale bars are 25 µm.
