real time cell death analysis  (Sartorius AG)

Journal: bioRxiv

Article Title: DDX3X syndrome mutations lock DDX3X-RNA conformational states to drive persistent pathological condensation and neuronal death

doi: 10.64898/2026.01.29.702492

Figure Lengend Snippet: A . Real-time time-lapse confocal images of HeLa cells expressing WT or mutant DDX3X-mCherry and subjected to SA (200µM) stress for 2 hours. A single SG (indicated by white dashed circle, as the region of interest) was photobleached and fluorescence recovery was recorded over 180s using confocal microscopy. B . The relative fluorescence intensity of mCherry (tagged to DDX3X) in SGs in HeLa cells before and after photobleaching as a function of time. Curves are representative of four independent experiments. C. Real-time time-lapse confocal images of N2a cells expressing WT or mutant DDX3X-mCherry and subjected to SA (100µM) stress for 2 hours. A single SG (indicated by a dashed circle, as the ROI) was photobleached, and fluorescence recovery was recorded over 150s using confocal microscopy. D. The relative fluorescence intensity of mCherry (tagged to DDX3X) in SGs in N2a cells before and after photobleaching as a function of time. Curves are representative of five independent experiments.

Article Snippet: Real-time cell death analysis were performed using a two-colour IncuCyte S3 Live-Cell analysis instrument (Sartorius).

Techniques: Expressing, Mutagenesis, Fluorescence, Confocal Microscopy

Journal: bioRxiv

Article Title: DDX3X syndrome mutations lock DDX3X-RNA conformational states to drive persistent pathological condensation and neuronal death

doi: 10.64898/2026.01.29.702492

Figure Lengend Snippet: A. Schematic of the assay protocol to assess the effect of persistent SGs on global translation and cell death. B. Representative polysome profiles (A260 absorbance) of DDX3X syndrome mutants expressing N2a cell extracts treated with SA (100uM) followed by wash-off and recovery in SA-free media, indicating sedimentation (through 10%-50% sucrose gradient) of 40S & 60S ribosomal subunits, 80S monosomes and polysomes (n=3). C. Quantification of DDX3X-dependent mRNAs in the polysome fractions plotted as relative log2-fold change ratio of translated/untranslated fraction using gene-specific primers normalised to GAPDH (internal control). ****p < 0.0001, ***p = 0.0007 (WT vs L559H, RPL36A gene), ***p = 0.0002 (WT vs L559H, RPL13 gene), **p = 0.0034 (WT vs I190S, EIF3I gene), **p = 0.0024 (WT vs L556S, STAT1 gene), **p = 0.0035 (WT vs I190S, TOPBP1 gene), **p = 0.0016 (WT vs L556S, TOPBP1), **p = 0.00365 (WT vs F182V, GNB2 gene), *p = 0.0347 (One-way ANOVA). Data shown are mean ± SEM. D. Real-time cell death analysis by Sytox green staining of N2a cells expressing WT or mutant DDX3X-mCherry constructs treated with SA **p = 0.0079, *p = 0.0432 (WT vs I190S), *p = 0.0306 (WT vs L559H) (two-way ANOVA test). Data shown are mean ± SEM. E. Real-time cell death analysis by Sytox green staining of N2a cells expressing WT or mutant DDX3X constructs treated with Aβ peptide with or without SA treatment and wash off. ****p < 0.0001, ***p = 0.0004, **p = 0.0068 (two-way ANOVA test). Data shown are mean ± SEM. F. Cell death measurement by Sytox green staining of N2a cells expressing WT or mutant DDX3X treated with SA, followed by TNF and zVAD treatment. ****p < 0.0001, **p = 0.0020 (WT vs T198P), **p = 0.0038 (WT vs R480G), **p = 0.0053 (WT vs L556S), *p = 0.0182 (two-way ANOVA test). Data shown are mean ± SEM.

Article Snippet: Real-time cell death analysis were performed using a two-colour IncuCyte S3 Live-Cell analysis instrument (Sartorius).

Techniques: Expressing, Sedimentation, Control, Staining, Mutagenesis, Construct