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 representation of DDX3X protein showing pathogenic mutations identified in DDX3X syndrome patients (bold- multiple occurrences, blue- polymicrogyria, brown- male patients). B . Schematic representation of DDX3X functional domains indicating critical DDX3X syndrome-associated mutations selected based on their predicted structural impact. Blue, RNA binding sites; Red, ATP binding and hydrolysis regions; Magenta, regions of interaction between ATP binding and RNA binding residues. C . Ribbon structural representation of DDX3X (dark grey) bound to dsRNA (red) (PDB ID: 6O5F), displaying surface accessibility and RNA interface proximity of DDX3X syndrome mutations. Mutated residues are color-coded in the represented structure. D . Heat map depicting the AlphaMissense prediction for the likely pathogenicity of selected DDX3X syndrome missense mutations, with dark red being the likely pathogenic and white being the likely benign mutation. E . Table depicting selected mutations in DDX3X that have been associated with different cancer types. F . Immunoblotting analysis of DDX3X showing the impact of DDX3X syndrome mutations on protein expression in Neuro2a cells. G . Fluorescence images of N2a cells expressing DDX3X-mCherry with DDX3X syndrome mutations, captured using Incucyte imager.

Article Snippet: Full length DDX3X-mCherry construct (pReceiver-M56-DDX3X-mCherry ) was procured from GeneCopoeia.

Techniques: Functional Assay, RNA Binding Assay, Binding Assay, Mutagenesis, Western Blot, Expressing, Fluorescence

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 . Immunoblot analysis showing levels of DDX3X protein in HeLa WT & Ddx3x -/- cells. B . Representative confocal microscopy images of HeLa cells expressing WT or mutant DDX3X-mCherry constructs and subjected to SA (200µM) stress and immunostained for stress granule marker G3BP1 (green) and DAPI (blue). Scale bar=10µm. C-E . Quantification of numbers of DDX3X (red), G3BP1 (green), and DDX3X-G3BP1 (yellow) granules per cell quantified from ≥50 cells across three independent experiments exhibiting DDX3X colocalising with stress granules in HeLa cells. ****p < 0.0001, ***p = 0.0008 (DDX3X granules/cell), **p = 0.0022 (WT vs I190S), **p = 0.0028 (WT vs T323I), **p = 0.0031 (WT vs L556S) (G3BP1 granules/cells), ****p < 0.0001, **p = 0.0034 (G3BP1-DDX3X colocalised granules/cells) (One-way ANOVA test). Data shown are mean ± SEM.

Article Snippet: Full length DDX3X-mCherry construct (pReceiver-M56-DDX3X-mCherry ) was procured from GeneCopoeia.

Techniques: Western Blot, Confocal Microscopy, Expressing, Mutagenesis, Construct, Marker

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. Representative confocal images of N2a cells expressing WT or mutant DDX3X-mCherry subjected to SA (100µM) stress for 2 hours and immunostained for stress granule marker G3BP1 (green) and DAPI (blue). Scale bar=10µm. B-D . Quantification of numbers of DDX3X (red), G3BP1 (green) and DDX3X-G3BP1 (yellow) granules per cell from ≥ 30cells across three independent experiments exhibiting DDX3X colocalising with stress granules in N2a cells. ****p < 0.0001, ***p = 0.0003, *p = 0.0181 (DDX3X granules/cell), ****p < 0.0001, ***p = 0.0001 (WT VS T198P), ***p = 0.0003 (WT vs L559H),**p = 0.0016 (G3BP1 granules/cells), ****p < 0.0001, ***p = 0.0001, **p = 0.0013 (WT vs I190S), **p = 0.0024 (WT vs L559H) (G3BP1-DDX3X colocalised granules/cells) (One-way ANOVA test). Data shown are mean ± SEM.

Article Snippet: Full length DDX3X-mCherry construct (pReceiver-M56-DDX3X-mCherry ) was procured from GeneCopoeia.

Techniques: Expressing, Mutagenesis, Marker

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 showing the protocol used for monitoring persistent granules in cells. Cells were treated with sodium arsenite (SA), followed by removing SA and supplementing fresh media (wash-off, recovery period) to monitor persistent granules. B . Representative confocal microscopy images of HeLa cells expressing WT or mutant DDX3X-mCherry showing stress granules (SGs) upon SA treatment and after wash-off (recovery), immunostained for G3BP1 (green) and DAPI (blue). Scale bar = 10µm. C-D . Quantification of the number of DDX3X (red) and DDX3X-G3BP1 (yellow) granules per cell from ≥ 30 cells across three independent experiments to monitor the formation of persistent DDX3X-SGs due to DDX3X syndrome mutations in HeLa cells. ****p < 0.0001, ***p = 0.0002, **p = 0.0073 (WT vs WT-WO), **p = 0.0046 (R475G vs R475G-WO), ns = not significant (DDX3X granules/cell), ****p < 0.0001, ***p = 0.0001, **p = 0.0096 (G3BP1-DDX3X granules/cell) (one-way ANOVA test). Data shown are mean ± SEM. E . Representative confocal microscopy images of N2a cells expressing WT or mutant DDX3X-mCherry showing SGs upon SA treatment and after wash-off (recovery), immunostained for G3BP1 (green) and DAPI (blue). Scale bar = 10µm. F-G . Quantification of the number of DDX3X (red) and DDX3X-G3BP1 (yellow) granules per cell from ≥ 50 cells across three independent experiments to monitor the formation of persistent DDX3X-SGs due to DDX3X syndrome mutations in N2a cells., ***p = 0.0006, **p = 0.0033 (WT vs WT-WO), **p = 0.0020 (F182V vs F182V-WO), ns = non-significant (DDX3X granules/cell), ****p < 0.0001, ***p = 0.0003 (G3BP1-DDX3X granules/cell) (One-way ANOVA test). Data shown are mean ± SEM.

Article Snippet: Full length DDX3X-mCherry construct (pReceiver-M56-DDX3X-mCherry ) was procured from GeneCopoeia.

Techniques: Confocal Microscopy, Expressing, Mutagenesis

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: Full length DDX3X-mCherry construct (pReceiver-M56-DDX3X-mCherry ) was procured from GeneCopoeia.

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: Full length DDX3X-mCherry construct (pReceiver-M56-DDX3X-mCherry ) was procured from GeneCopoeia.

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

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. Cell death measurement by Sytox green staining of N2a cells ectopically expressing WT or mutant DDX3X-mCherry. ns = non-significant (Two-way ANOVA test). Data shown are mean ± SEM. B. Cell death measurement by Sytox green staining of N2a cells ectopically expressing WT or mutant DDX3X-mCherry treated with TNF and zVAD. Ns = non-significant, (Two-way ANOVA test). Data shown are mean ± SEM.

Article Snippet: Full length DDX3X-mCherry construct (pReceiver-M56-DDX3X-mCherry ) was procured from GeneCopoeia.

Techniques: Staining, Expressing, Mutagenesis

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 showing the protocol used for the cross-seeding kinetics of Aβ 1-42 aggregation by DDX3X seeds. B . Boltzmann fitting of the cross-seeding experiment depicting the co-aggregation kinetics of Aβ 1-42 when cross-seeded with sonicated seeds of DDX3X variants. C . Representative confocal images of N2a cells expressing WT or mutant DDX3X-mCherry, subjected to SA stress (100µM), wash-off, and recovery, followed by ThT staining and immunostained with DAPI (blue). ThT fluorescence is represented in green. Scale bar= 10µm. D-F . Quantification of numbers of DDX3X (red), ThT stained (green) and ThT-DDX3X (yellow) granules per cell from ≥ 20 cells across three independent experiments exhibiting DDX3X colocalization with ThT granules in N2a cells. ****p < 0.0001 (DDX3X granules/cell), ****p < 0.0001, ***p = 0.0005, **p = 0.0022 (WT vs I190S), **p = 0.0080 (WT vs T198P) (ThT granules/cells) ****p < 0.0001, ***p = 0.0003, **p = 0.0021 (WT vs I190S), **p = 0.0057 (WT vs T198P) (ThT-DDX3X colocalised granules/cells) (One-way ANOVA test). Data shown are mean ± SEM.

Article Snippet: Full length DDX3X-mCherry construct (pReceiver-M56-DDX3X-mCherry ) was procured from GeneCopoeia.

Techniques: Sonication, Expressing, Mutagenesis, Staining, Fluorescence

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A , Structure of Lenti-KRT5mCherry (L-KRT5mCherry). B , Co-localization of KRT5 immunostaining (KRT5, green) and mCherry (L-KRT5) expression (magenta). Orange, overlay. Counterstaining with DAPI (blue). Scale bar, 60 µm. C , Quantification of cells expressing both KRT5 and L-KRT5 (KRT5+ L-KRT5+), or only KRT5 (KRT5+) or L-KRT5+. D , Experimental design of isolation of cells expressing both L-KRT5mCherry and L-hUbC-GFP or L-hUbC-GFP alone. E, PCR detection of L-hUbC-GFP (GFP) and L-KRT5mCherry (mCherry) DNA in both KRT5+ (K5+) and KRT5- (K5-) cells. All error bars denote s.d.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Immunostaining, Expressing, Isolation

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A and B , Organoids derived from SKOV3 cells expressing both lentiviruses (A, GFP and mCherry, orange) or GFP alone (B, green). Scale bar, 100 µm. C , Quantification of KRT5+ (blue symbols, pink bars) and KRT5- (pink symbols, yellow bars) cancer organoids in 6 consecutive passages. All error bars denote s.d. D, Volume of tumors formed by serially diluted (1 x 10 5 , 1 x 10 4 , 1 x 10 3 ) of KRT5+ and KRT5- cells after their s.c. transplantation into different flanks of NSG mice. KRT5-group did not form tumors. E and F , mCherry (E) and KRT5 (F) expression in KRT5+ cell derived xenografts. Elite ABC method. Hematoxylin counterstaining. Scale bar, 60 µm. All error bars denote s.d. G. Live microscopy of cells were isolated by FACS based on their expression of GFP (green) and mCherry (magenta) after coinfection with Lenti-UbC-GFP and Lenti-KRT5mCherry. Orange, Overlay. Individual frames of live microscopy. Scale bar, 60 µm.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Derivative Assay, Expressing, Transplantation Assay, Microscopy, Isolation