Journal: bioRxiv

Article Title: XIST Drives X-Chromosome Inactivation and Safeguards Female Extraembryonic Cells in Humans

doi: 10.1101/2025.11.19.689206

Figure Lengend Snippet: A. Schematic illustration showing wild type (WT) and XIST inducible knock down (iKD) H9 naive hESCs under trophoblast differentiation medium with or without Doxycycline (Dox) and assaying cell identity and X-chromosome inactivation using microscopy, scRNA-seq, bulk RNA-seq and flow cytometry. B. Representative bright-field microscopy images showing the morphology of H9 XIST iKD clone-1 hESCs at Day 15 of culture in trophoblast differentiation medium with or without Dox. Note: Feeders only are seen in the +Dox conditions. Scale bar, 200 µm. The experiment was performed in 3 independent XIST iKD clones of H9 hESCs. Out of which one is shown here and the other two are shown in figure S5E (total n=3 independent clones). C. Representative IF of GATA3 (Green) of cells mentioned in 4B. DAPI is shown in blue. Scale bar, 20 µm. The experiment was performed in 3 independent XIST iKD clones of H9 hESCs. Out of which one is shown here and the other two are shown in figure S5F (total n=3 independent clones). D. The proportion of GATA3 positive nuclei in both WT and XIST iKD H9 hESCs at Day 15 of trophoblast differentiation with or without Dox. The number of nuclei counted is indicated above each bar. Each dot represents one of three independent XIST iKD clones (n=3) or WT clones (n=3). Statistical significance was assessed using a t-test. A p-value <0.05 was considered statistically significant. The number of nuclei counted is indicated in supplemental table S1. E. Flow cytometry quantification of the proportion of ENPEP positive cells at day 15 of trophoblast differentiation of XIST iKD hESCs with or without Dox. Statistical significance was tested using a Chi² test, resulting in p<0,0001. Each dot represents one of three independent XIST iKD clones (n=3). F. UMAP of scRNA-seq time course during differentiation of H9 WT and XIST iKD hESCs with or without Dox under trophoblast culture conditions, 13 clusters are shown using different colors. The scRNA-seq data was generated using one of the above 3 clones both for the WT and the XIST iKD hESCs with or without Dox (n=1). G. As in 4F but colored by cell types. Lineage information where Naive = Naive epiblast, Differ Naive = Differentiated naive epiblast, Epi interim 1 = Epiblast intermediate type 1, Epi interim 2 = Epiblast intermediate type 2, Epi interim 3 = Epiblast intermediate type 3, Epi interim 4 = Epiblast intermediate type 4, TSC = Trohpblast stem cells, and EXMC = Extraembryonic mesoderm cells. H. UMAPs from showing the expression of KLF4 and DNMT3L (naive hESC markers), GATA3 and GATA2 (TSC markers), LUM and NID2 (EXMC markers) in time course scRNA-seq data. I. Dot plot showing the expression of marker genes for core pluripotency, naive pluripotency, trophoblast and embryo extraembryonic mesoderm (EXM) in time course scRNA-seq data from this study. J. The proportion of each cell type at different time points during differentiation of naive hESCs into TSCs and EXMCs. Colored by cell types. K. Differential gene expression as detected by scRNA-seq between Day 8 XIST iKD (Top) and WT cells (Bottom) with Dox vs without Dox. Dashed lines indicate -log10 adjusted p-Value <0.05 and log2 fold change < -0.5 or > 0.5. L. GO enrichment analysis of differentially expressed genes as a result of XIST depletion comparing Day 8 XIST iKD cells with and without Dox.

Article Snippet: In summary, starting from day 1 or day 2 after seeding primed hESCs in E8 onto Geltrex, cells were lipofected daily with KLF4 mRNA (Miltenyi, 130-101-115) for 9 days .

Techniques: Knockdown, Microscopy, RNA Sequencing, Flow Cytometry, Clone Assay, Generated, Expressing, Marker, Gene Expression

Journal: Nagoya Journal of Medical Science

Article Title: Acupuncture alleviates the progression of knee osteoarthritis by inhibiting X-inactive specific transcript-mediated activation of the mechanosensitive ion channel Piezo1 signaling

doi: 10.18999/nagjms.87.4.644

Figure Lengend Snippet: XIST knockdown inhibits apoptosis and proliferation of CHON-001 cells (A) Aggrecan , (B) MMP13 and (C) lncRNA XIST mRNA levels. (D) Transfection efficiency, (E) cell proliferation. In relation to IL-1β+si-NC, * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001 exist. XIST: X-inactive specific transcript MMP: matrix metalloproteinase IL: interleukin NC: normal control mRNA: messenger RNA lncRNA: long non-coding RNA si-XIST: siRNA targeting XIST si-NC: siRNA negative control OD: optical density

Article Snippet: Antibodies against insulin-like growth factor 2 messenger RNA (mRNA) binding proteins 2 (IGF2BP2) (Proteintech, cat no. 11601-1-AP) were coupled to protein A/G magnetic beads to capture IGF2BP2-bound complexes.

Techniques: Knockdown, Transfection, Control, Negative Control

Journal: Nagoya Journal of Medical Science

Article Title: Acupuncture alleviates the progression of knee osteoarthritis by inhibiting X-inactive specific transcript-mediated activation of the mechanosensitive ion channel Piezo1 signaling

doi: 10.18999/nagjms.87.4.644

Figure Lengend Snippet: Downregulation of XIST inhibits ECM-degeneration and levels of inflammatory factors in IL-1β-induced chondrocytes (A)–(F) After IL-1β stimulation and lncRNA XIST knockdown, the mRNA levels of MMP13, MMP3, ADAMTS-4, Aggrecan, COL2A1 and SOX9; (G) IL-6 and TNF-α levels was tested during IL-1β treatment and knockdown of lncRNA XIST. * P < 0.05, ** P < 0.01, *** P < 0.001. IL: interleukin XIST: X-inactive specific transcript ECM: extracellular matrix MMP: matrix metalloproteinase ADAMTS: a disintegrin and metalloproteinase with thrombospondin COL2A1: collagen type II alpha 1 SOX9: sex determining region Y box protein 9 TNF: tumor necrosis factor mRNA: messenger RNA lncRNA: long non-coding RNA si-XIST: siRNA targeting XIST si-NC: siRNA negative control

Article Snippet: Antibodies against insulin-like growth factor 2 messenger RNA (mRNA) binding proteins 2 (IGF2BP2) (Proteintech, cat no. 11601-1-AP) were coupled to protein A/G magnetic beads to capture IGF2BP2-bound complexes.

Techniques: Knockdown, Negative Control

Journal: Nagoya Journal of Medical Science

Article Title: Acupuncture alleviates the progression of knee osteoarthritis by inhibiting X-inactive specific transcript-mediated activation of the mechanosensitive ion channel Piezo1 signaling

doi: 10.18999/nagjms.87.4.644

Figure Lengend Snippet: Overexpression of Piezo1 reverses effects of XIST silencing on IL-1β-induced chondrocytes (A) Effectiveness of Piezo1 overexpression. After IL-1β stimulation, IL-1β stimulation + si-XIST, and IL-1β stimulation + si-XIST + Piezo1 expression, (B) the mRNA levels of Aggrecan, MMP13, MMP3, ADAMTS-4, COL2A1 and SOX9. (C) TNF-α and IL-6 levels was tested utilizing ELISA assay. * P < 0.05, ** P < 0.01, *** P < 0.001. XIST: X-inactive specific transcript IL: interleukin ADAMTS: a disintegrin and metalloproteinase with thrombospondin MMP: matrix metalloproteinase COL2A1: collagen type II alpha 1 SOX9: recombinant sex determining region Y box protein 9 TNF: tumor necrosis factor mRNA: messenger RNA si-XIST: siRNA targeting XIST si-NC: siRNA negative control ELISA: enzyme-linked immunosorbent assay

Article Snippet: Antibodies against insulin-like growth factor 2 messenger RNA (mRNA) binding proteins 2 (IGF2BP2) (Proteintech, cat no. 11601-1-AP) were coupled to protein A/G magnetic beads to capture IGF2BP2-bound complexes.

Techniques: Over Expression, Expressing, Enzyme-linked Immunosorbent Assay, Recombinant, Negative Control

Journal: Nagoya Journal of Medical Science

Article Title: Acupuncture alleviates the progression of knee osteoarthritis by inhibiting X-inactive specific transcript-mediated activation of the mechanosensitive ion channel Piezo1 signaling

doi: 10.18999/nagjms.87.4.644

Figure Lengend Snippet: LncRNA XIST promotes Piezo1 expression through IGF2BP2 pathway (A–B) Prediction and determination of subcellular localization of lncRNA XIST using online database and subcellular fractionation test respectively; (C) si-XIST significantly reduced the Piezo1 mRNA, and overexpression of IGF2BP2 reversed this effect; (D) the interplay between lncRNA XIST and Piezo1 was examined by RIP assay; (E) stability of Piezo1 mRNA was decreased and IGF2BP2 overexpression reverses it. * P < 0.05, *** P < 0.001. XIST: X-inactive specific transcript IGF2BP2: insulin-like growth factor 2 mRNA binding proteins 2 RIP: binding protein immunoprecipitation assay mRNA: messenger RNA lncRNA: long non-coding RNA si-XIST: siRNA targeting XIST si-NC: siRNA negative control GAPDH: glyceraldehyde-3-phosphate dehydrogenase

Article Snippet: Antibodies against insulin-like growth factor 2 messenger RNA (mRNA) binding proteins 2 (IGF2BP2) (Proteintech, cat no. 11601-1-AP) were coupled to protein A/G magnetic beads to capture IGF2BP2-bound complexes.

Techniques: Expressing, Fractionation, Over Expression, Binding Assay, Immunoprecipitation, Negative Control

Journal: Nagoya Journal of Medical Science

Article Title: Acupuncture alleviates the progression of knee osteoarthritis by inhibiting X-inactive specific transcript-mediated activation of the mechanosensitive ion channel Piezo1 signaling

doi: 10.18999/nagjms.87.4.644

Figure Lengend Snippet: Schematic representation illustrating how acupuncture attenuates the progression of knee osteoarthritis by inhibiting XIST-mediated activation of the mechanosensitive ion channel Piezo1 signaling XIST: X-inactive specific transcript IGF2BP2: insulin-like growth factor 2 mRNA binding proteins 2 lncRNA: long non-coding RNA MMP: matrix metalloproteinase COL2A1: recombinant collagen type II alpha 1 SOX9: sex determining region Y box protein 9 IL: interleukin TNF: tumor necrosis factor ADAMTS: a disintegrin and metalloproteinase with thrombospondin

Article Snippet: Antibodies against insulin-like growth factor 2 messenger RNA (mRNA) binding proteins 2 (IGF2BP2) (Proteintech, cat no. 11601-1-AP) were coupled to protein A/G magnetic beads to capture IGF2BP2-bound complexes.

Techniques: Activation Assay, Binding Assay, Recombinant

Journal: Nagoya Journal of Medical Science

Article Title: Acupuncture alleviates the progression of knee osteoarthritis by inhibiting X-inactive specific transcript-mediated activation of the mechanosensitive ion channel Piezo1 signaling

doi: 10.18999/nagjms.87.4.644

Figure Lengend Snippet: Activation of Piezo1 reverses effects of AP treatment among MIA-induced rats After AP stimulation, and AP stimulation + Piezo1, (A–B) western blot assay accustomed to observing the protein levels of MMP13, MMP3, ADAMTS-4, Aggrecan, COL2A1, SOX9. (C) TNF-α and IL-6 levels. (D) The XIST and Piezo1 mRNA levels. * P < 0.05, ** P < 0.01, *** P < 0.001. AP: acupuncture MIA: monosodium iodoacetate MMP: matrix metalloproteinase COL2A1: recombinant collagen type II alpha 1 SOX9: sex determining region Y box protein 9 XIST: X-inactive specific transcript IL: interleukin TNF: tumor necrosis factor mRNA: messenger RNA GAPDH: glyceraldehyde-3-phosphate dehydrogenase ADAMTS: a disintegrin and metalloproteinase with thrombospondin

Article Snippet: Antibodies against insulin-like growth factor 2 messenger RNA (mRNA) binding proteins 2 (IGF2BP2) (Proteintech, cat no. 11601-1-AP) were coupled to protein A/G magnetic beads to capture IGF2BP2-bound complexes.

Techniques: Activation Assay, Western Blot, Recombinant

Journal: The EMBO Journal

Article Title: Germ granule localization of nematode Argonaute WAGO-4 ensures fidelity in small RNA loading

doi: 10.1038/s44318-025-00606-x

Figure Lengend Snippet: ( A ) Representative fluorescent micrographs of gonads (pachytene region) from live worms co-expressing PGL-3::mCherry and GFP::WAGO-4 (upper row) or RFP::ZNFX-1 and GFP::WAGO-4 (lower row) in WT and 3×FG mutant backgrounds at 20 °C. For each condition, a single confocal slice is shown for the indicated proteins and merged channels. Scale bar: 5 µm. ( B ) Quantification of GFP::WAGO-4 mean intensity signal inside granules vs. the cytoplasm. Ratios were calculated from images of live worms expressing GFP::WAGO-4 together with PGL-3::mCherry (P granules, purple hues) or RFP::ZNFX-1 (Z granules, blue hues) in WT and 3×FG backgrounds. Average ratios with SD are plotted, and numbers above bars indicate average values. Analysis was performed on the pachytene region, covering approximately 35 nuclei for P granules (35.3 ± 2.8) and 30 nuclei for Z granules (29.7 ± 2.7) from three live worms grown at 20 °C. To ensure precise granule volume definition and accurate intensity ratio measurement, the granule vs. cytoplasm signal ratios were calculated for each of 54 confocal planes (0.1 µm). Statistical analysis was performed using a two-tailed Mann–Whitney test, P values indicated in the graph. Representative fluorescence micrographs are shown in ( A ). ( C ) Schematic representation of the in vitro phase separation experiment. Two types of condensates were formed to approximate minimal P granules. Each condensate contained recombinant purified PGL-1, PGL-3, GLH-1, and GLH-4 proteins from an insect cell expression system, along with commercial poly(A)+ mRNA. The first type of condensate contained WT GLH-1 and GLH-4, while the second contained GLH-1 and GLH-4 with F → A mutations in FG dipeptides (identical to those introduced in live worms, shown in Fig. ). To each condensate type, the same amount of worm-purified mScarlet::WAGO-4 was added. For negative controls, the same amount of WAGO-4 storage buffer was added to the condensates. ( D ) Graph representing the ratio of mScarlet::WAGO-4 signal intensity between the condensed and dilute phases for WT and FG-mutant condensates. Results from a single experiment are shown; five to six ROIs per image were defined inside and outside condensates, with six images analyzed for each condition ( n = 32–36). Bars represent average values with SD and numbers above bars indicate average values. Statistical analysis was performed using a two-tailed Mann–Whitney test, P values indicated in the graph. A second independent experiment is shown in Fig. . mScarlet::WAGO-4 storage buffer was used as a negative control, and ratios were calculated in the same manner. ( E ) Schematic representation of WAGO-4 constructs expressed in worms. Fusions or point mutations were introduced at the native wago-4 locus using CRISPR-Cas. Introduced fusions or WAGO-4 predicted domains, identified based on sequence alignment with human Ago2, are depicted in different colors, as indicated. ( F ) Representative fluorescence micrographs of gonads (pachytene region) from live worms expressing different fluorescently labeled WAGO-4 proteins: GFP::WAGO-4, MTS::GFP::WAGO-4, PH::GFP::WAGO-4, and GFP::WAGO-4(Y611E). For each expressed protein, a single confocal slice is shown for worms grown at 20 °C. Scale bar: 5 µm. ( G ) Graph representing the number of progeny for worms of the indicated genotypes grown at 26 °C for one generation. Each dot represents the number of progeny from a single worm ( n = 17–19). Horizontal lines represent mean values, and error bars indicate SD. Statistical analysis was performed using the Kruskal–Wallis test, and the corresponding P values are reported in Dataset . Groups sharing at least one letter above the plot are statistically indistinguishable ( P < 0.05). ( H ) Quantification of unfertilized oocytes in the uteri of worms grown at 26 °C, categorized into three groups: (1) worms containing exclusively embryos, (2) worms containing at least one embryo and at least one unfertilized oocyte, and (3) worms containing exclusively unfertilized oocytes. Data represent the average proportion of worms in each category from four independent experiments ( n = 52.8 ± 6.1 worms per experiment). Dots represent values from individual experiments and error bars indicate SD. ( I ) Mortal germline assay at 25 °C comparing the 3×FG mutant, MTS::GFP::WAGO-4-expressing strain, and wago-4(ko) , with N2 as a control. For each strain, five replicates with five P 0 worms were used. .

Article Snippet: poly-A+ mRNA , Takara , 636101.

Techniques: Expressing, Mutagenesis, Two Tailed Test, MANN-WHITNEY, Fluorescence, In Vitro, Recombinant, Purification, Negative Control, Construct, CRISPR, Sequencing, Labeling, Control

Journal: The EMBO Journal

Article Title: Germ granule localization of nematode Argonaute WAGO-4 ensures fidelity in small RNA loading

doi: 10.1038/s44318-025-00606-x

Figure Lengend Snippet: ( A ) Table showing the number of up- and downregulated genes in 3×FG, wago-4(Y611E) , and wago-4(ko) worms grown at 20 °C or 26 °C ( p < 0.05). ( B ) PCA plot based on the top 500 most variable genes. Three biological replicates were analyzed for each genotype and temperature condition, with each replicate individually plotted. The legend on the right indicates the symbol for each condition. ( C ) Table listing all histone genes downregulated in 3×FG vs. WT based on mRNA sequencing at 20 °C. Each column represents a histone gene, and rows indicate the histone type it encodes. Genes highlighted in bold also showed increase in WAGO-4-associated 22G-RNAs in 3×FG compared to WT (Fig. ). ( D ) Volcano plot showing fold enrichment of proteins from total worm lysates of worms grown at 26 °C (3×FG vs. WT), determined by mass spectrometry ( n = 3 biological replicates). Statistical significance was calculated using limma (Ritchie et al, ). Detected histone proteins or clusters and known germ granule proteins are highlighted.

Article Snippet: poly-A+ mRNA , Takara , 636101.

Techniques: Sequencing, Mass Spectrometry

Journal: The EMBO Journal

Article Title: Germ granule localization of nematode Argonaute WAGO-4 ensures fidelity in small RNA loading

doi: 10.1038/s44318-025-00606-x

Figure Lengend Snippet: ( A ) MA plot representing differential gene expression (3×FG vs.WT) based on mRNA sequencing from worms grown at 20 °C. Red dots indicate upregulated genes, blue dots indicate downregulated genes, and gray dots represent genes without significant changes in expression. The number of regulated genes is indicated at the bottom ( P < 0.05). ( B ) Enrichment analysis of genes classified as oogenic, spermatogenic, or germline-constitutive (Ortiz et al, ) compared to genes up- or downregulated in 3×FG vs. WT based on mRNA sequencing. Log₂ enrichment is indicated by color according to the scale shown, and significant enrichment or depletion (p < 0.05, Fisher’s exact test) is highlighted in bold and underlined. ( C ) Venn diagram showing the overlap of upregulated genes ( P < 0.05) from mRNA sequencing for three genotypes: 3×FG, wago-4(Y611E) , and wago-4(ko) . Numbers in brackets indicate the size of each gene group. ( D ) Venn diagram showing the overlap of downregulated genes ( P < 0.05) from mRNA sequencing for three genotypes: 3×FG, wago-4(Y611E) , and wago-4(ko) . Numbers in brackets indicate the size of each gene group. ( E ) Table presenting the percentage of up- or downregulated genes in 3×FG, wago-4(Y611E) , and wago-4(ko) that are identified as WAGO-4 targets according to Seroussi et al . Percentages are highlighted with yellow and green hues. The total number of up- and downregulated genes is indicated for each condition.

Article Snippet: poly-A+ mRNA , Takara , 636101.

Techniques: Gene Expression, Sequencing, Expressing

Journal: The EMBO Journal

Article Title: Germ granule localization of nematode Argonaute WAGO-4 ensures fidelity in small RNA loading

doi: 10.1038/s44318-025-00606-x

Figure Lengend Snippet: ( A ) Bar plots showing the 5’ nucleotide composition and length distribution of sRNAs in each Argonaute IP. Pie charts depict the proportion of sRNAs corresponding to each genetic element (biotype). Data represent the average of three biological replicates. IPs were performed using 3xFLAG::GFP::WAGO-4 in young adult worms. ( B ) Volcano plot showing Log 2 fold enrichment of 22G-RNAs targeting WAGO-4 targets in 3×FG vs. WT worms grown at 20 °C. Each dot represents the average Log 2 fold change ( n = 3 biological replicates). Differential expression and statistical analysis were performed using DESeq2 (Love et al, ). Targets with an average Log 2 fold change of <−1 or >1 and P < 0.01 were considered to have a significant decrease or increase in WAGO-4-associated 22G-RNAs, respectively (indicated by red dots). ( C ) Enrichment of WAGO-4 sRNA targets in four groups: WT-specific targets, 3×FG-specific targets, targets with a decrease in WAGO-4-associated sRNAs in 3×FG, and targets with an increase in WAGO-4-associated sRNAs in 3×FG. These groups were compared to genes up- or downregulated in 3×FG/WT based on mRNA sequencing. All data represent worms grown at 20 °C. Log₂ enrichment is indicated by color according to the scale shown, and significant enrichment or depletion ( P < 0.05, Fisher’s exact test) is highlighted in bold and underlined. ( D ) Venn diagram showing the overlap of WAGO-4 targets in WT worms (this study) with CSR-1 targets (Seroussi et al, ). Both datasets were derived from sRNA sequencing after Ago IP from adult worms grown at 20 °C. Numbers in brackets indicate the size of each group. ( E ) Metagene profiles for sRNAs complementary to protein-coding gene WAGO-4 targets (all identified targets in WT and 3×FG worms grown at 20 °C). Size-normalized targets were partitioned into 100 bins, and the mean coverage (RPM) for each bin was plotted. Solid lines represent IP values, and dashed lines represent input values. The number of identified targets for each genotype is indicated in brackets. ( F ) Metagene profiles for sRNAs complementary to protein-coding gene WAGO-4 and CSR-1 targets. CSR-1 targets were identified from CSR-1 IP in WT worms (Seroussi et al, ). Size-normalized targets were partitioned into 100 bins, and mean coverage (RPM) for each bin was plotted. Solid lines represent IP values, and dashed lines represent input values. The total number of genes analyzed was determined as WAGO-4 and CSR-1 shared targets based on published data (Seroussi et al, ), and the number of identified targets for each genotype is indicated in brackets.

Article Snippet: poly-A+ mRNA , Takara , 636101.

Techniques: Quantitative Proteomics, Sequencing, Derivative Assay