Journal: bioRxiv

Article Title: Identification of cellular double-stranded RNAs in mammalian embryonic stem cells

doi: 10.64898/2026.04.13.718158

Figure Lengend Snippet: ( A ) Expression of the pluripotency markers Pou5f1 and Nanog and differentiation markers Acta2 and Tagln in ESCs and differentiated cells was measure by RT-qPCR, comparing mock and dsRNA-stimulated cells. Actb was used as a normaliser. Data represent the average of three biological replicates ± SD relative to untreated ESC. One-way ANOVA was used to calculate significant differences between dsRNA- treated samples within each cell line, followed by a Tukey HSD. *p-val ≤ 0.05, **p-val ≤ 0.01, ***p-val ≤ 0.001. ( B ) Expression of Ifnb1 and interferon stimulated genes Oas1 and Stat1 in ESCs and differentiated cells upon dsRNA or mock stimulation by RT-qPCR analysis. Actb was used as a normaliser. Data represent the average of three biological replicates ± SD relative to untreated ESC. One-way ANOVA was used to calculate significant differences between dsRNA-treated cell lines, followed by a Tukey HSD. *p-val ≤ 0.05, **p-val ≤ 0.01, ***p-val ≤ 0.001. ( C ) Western blot analysis of phosphorylated IRF3 levels in ESCs and differentiated cells. GAPDH serves as a loading control ( D ) Western blot analysis of phosphorylated PKR in ESCs and differentiated cells. Tubulin serves as a loading control ( E ) rRNA electrophoresis from ESCs and differentiated cells, mock and dsRNA-stimulated. (+) represents lower dsRNA and (++) higher dsRNA stimulation.

Article Snippet: Cells were transfected with the dsRNA analogue poly(I:C) (Invivogen, tlrl-pic) using Lipofectamine 2000 (ThermoFisher, 11668027).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Electrophoresis

Journal: bioRxiv

Article Title: Identification of cellular double-stranded RNAs in mammalian embryonic stem cells

doi: 10.64898/2026.04.13.718158

Figure Lengend Snippet: ( A ) Flow cytometry analysis against dsRNA using the dsRNA-specific antibody J2 in ESC vs differentiated (diff) cells in the presence or absence of RNase III. RNase III treatment tests specificity of J2 antibody signal. Data represent the average of three biological replicates ± SD. One-way ANOVA was used to calculate significant differences amongst comparisons, followed by a Tukey HSD. *p-val ≤ 0.05, **p-val ≤ 0.01, ***p-val ≤ 0.001. ( B ) Representative examples of normalised Flow cytometry intensity in ESCs and differentiated cells after mock and RNaseIII treatment. For all replicates and gating strategy see Supplementary figure 2a ( C ) Immunofluorescence for dsRNA. J2 antibody was used to detect dsRNA, and DAPI to stain the nucleus. Scalebar is 10 µm ( D ) Setup of dsRNA-immunoprecipitation using the J2 antibody. DsRNAs bound to J2 were either mock treated, treated with RNaseI or RNaseIII prior to purification and followed high-throughput RNA sequencing. ( E ) Bioanalyzer electropherogram of purified RNAs J2-IP using the different treatments shown in ( D ). ( F ) Detailed view of Bioanalyzer electropherogram showing accumulation of shorter fragments upon treatment with RNase I and III.

Article Snippet: Cells were transfected with the dsRNA analogue poly(I:C) (Invivogen, tlrl-pic) using Lipofectamine 2000 (ThermoFisher, 11668027).

Techniques: Flow Cytometry, Immunofluorescence, Staining, Immunoprecipitation, Purification, High Throughput Screening Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Identification of cellular double-stranded RNAs in mammalian embryonic stem cells

doi: 10.64898/2026.04.13.718158

Figure Lengend Snippet: ( A ) Normalised counts of the four major classes of transposable elements compared to input controls. One-way ANOVA was used to calculate significant differences between input and immunoprecipitated samples, followed by a Tukey HSD. *p-val ≤ 0.05, **p-val ≤ 0.01, ***p-val ≤ 0.001. ( B ) Violin plots showing differential enrichment (log₂ fold change) of TE subfamilies within selected families for dsRNA IP (mock) versus input. Points represent individual subfamilies, coloured by differential enrichment (red: upregulated, blue: downregulated, grey: not significant; log₂FC > 1, adjusted p < 0.05). ( C ) Violin plots showing differential enrichment (log₂ fold change) of TE subfamilies within selected families after RNase I treatment versus dsRNA (mock). Points represent individual subfamilies, coloured by differential enrichment (red: upregulated, blue: downregulated, grey: not significant; log₂FC > 1, adjusted p < 0.05). ( D ) Levels of A-to-I editing in retrotransposons for input and dsRNA-IPs. One-way ANOVA was used to calculate significant differences between input and immunoprecipitated samples, followed by a Tukey HSD. *p-val ≤ 0.05, **p-val ≤ 0.01, ***p-val ≤ 0.001. ( E ) Levels of A-to-I editing by TE class. Statistical analysis as in ( D ). ( F ) Percentage of sense reads mapping to TEs with a corresponding antisense match, for input and dsRNA IPs. One-way ANOVA was used to calculate significant differences between input and immunoprecipitated samples, followed by a Tukey HSD. *p-val ≤ 0.05, **p-val ≤ 0.01, ***p- val ≤ 0.001. ( G ) Sense/antisense overlap by TE class. Statistical analysis as in ( F ). ( H ) Examples of specific TE locus. The combined normalised reads from the biological replicates mapped are shown, for inputs and dsRNA IPs. Above black line represents sense (or ‘plus’), below represents antisense reads (or ‘minus’). Chromosomal location of each locus is represented at the top

Article Snippet: Cells were transfected with the dsRNA analogue poly(I:C) (Invivogen, tlrl-pic) using Lipofectamine 2000 (ThermoFisher, 11668027).

Techniques: Immunoprecipitation

Journal: bioRxiv

Article Title: Evolved differences in microglial cell biology between surface and cave populations of Astyanax mexicanus

doi: 10.64898/2026.04.11.717796

Figure Lengend Snippet: (A) Schematic of the microinjection setup. (B–D) Zymosan A injection into the brain parenchyma followed by neutral red staining in (B) Zebrafish, (C) A. mexicanus surface morphs, and (D) A. mexicanus cave morphs. (E) Quantification of microglial density in the midbrain following Zym A injection. Each data point represents a single larva. Graph shows mean ± SD. Statistical analysis was performed using an unpaired t -test. (F-H) Poly(I:C) injections in zebrafish, surface fish, and cavefish larvae followed by neutral red labeling.

Article Snippet: One nanoliter of poly(I:C) (Tocris Bioscience, 4287; 1 mg/ml in 1X PBS), zymosan (Sigma-Aldrich, Z4250; 10 mg/ml in 1X PBS, boiled to solubilize), Magic Red Cathepsin (Immunochemistry Technologies, 937; vial 6133 suspended in 50 μL of DMSO and diluted 1:1 in 1X PBS before use), E. coli Texas Red (Thermo Fisher Scientific, E2863; 10 mg/ml in PBS), and Amyloid-β (1-42) HiLyte-Fluor 488 (Anaspec, AS-60479; 1 mg/ml in 1X PBS with 1% NH 4O H) were injected into one hemisphere of the midbrain.

Techniques: Microinjection, Injection, Staining, Labeling