Journal: Oncology Letters

Article Title: Methylglyoxal-derived glycated albumin enhances the stemness potential of invasive ductal carcinoma-derived breast cancer stem-like cell line KAIMRC1

doi: 10.3892/ol.2026.15541

Figure Lengend Snippet: Effects of GA on the expression of RAGE, vimentin and OCT3/4 in invasive ductal carcinoma-derived KAIMRC1 stem-like cells. Representative western blot analyses showing the protein expression levels of RAGE, vimentin and OCT3/4 in KAIMRC1 stem-like cells following 48 h of treatment with 100 µg/ml GA or unglycated BSA, compared with untreated control cells. GAPDH served as the loading control. Bar graphs present the relative protein expression levels normalized to GAPDH. Data are presented as the mean ± SD from three independent experiments. Statistical significance relative to the untreated control group is indicated as *P < 0.05 and **P < 0.01. RAGE, receptor for advanced glycation end products; GA, glycated albumin; BSA, bovine serum albumin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Mouse primary monoclonal antibodies directed against extracellular signal-regulated kinase (ERK)1 (clone G-8; cat. no. sc-271269), phosphorylated-ERK1/2 (p-ERK1/2; clone E-4; Tyr204 of ERK1; cat. no. sc-7383), OCT3/4 (clone A-9; cat. no. sc-365509) and RAGE (clone E-1; cat. no. sc-74473) were obtained from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Derivative Assay, Western Blot, Control

Journal: iScience

Article Title: Multifaceted role of POU5F1P1 in regulating its parental stem cell gene, POU5F1

doi: 10.1016/j.isci.2026.115137

Figure Lengend Snippet: 5′ UTR sequence of PG1 interacts with the 5′ UTR of OCT4A, leading to translational repression (A) Fluorescence intensity of FLAG immunostaining in NIH3T3 cells transfected with OCT4A-FLAG containing UTR, PG1 with UTR (PG1 full), or its 5′ UTR (PG1 5′ UTR) or 3′ UTR (PG1 3′ UTR) sequences. Transfection efficiency was normalized by dividing the fluorescence intensity of the co-transfected pRFP by n = 3. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. (B) Immunostaining with a specific OCT4 antibody (C-10; sc5279) was performed when full-length PG1 (PG1 full) and PG1 5′ UTR sequence plasmids were transfected into PA1 cells. To verify the transfected cells, pRFP was co-transfected, and the fluorescence signal intensity of OCT4A immunostaining was quantified in RFP-positive cells. Scale bars, 100 μm. n = 3. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. (C) Immunostaining with a specific OCT4A antibody (C-10; sc5279) when the RNA of the PG1 5′ UTR or 3′ UTR sequence was transfected into PA1 cells. n = 10 (calculated views). Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. Similar results were obtained in three independent experiments. (D) Quantity of OCT4A mRNA when the UTR sequence of PG1 was transfected into PA1 cells. Relative mRNA levels were measured using real-time PCR with specific primers ( D and S1E). Data are presented as relative expression levels normalized to HPRT1, with HPRT1 set to 1. n = 3. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001; ns = not significant. (E) Western blot analysis of OCT4A protein levels following the transfection of PG1 5′ UTR RNA into PA1 cells. PA1 cells were transfected with the PG1 5′ UTR sequence RNA at two concentrations (1: 5 nM and 2: 10 nM). Protein expression was detected using an OCT4A-specific antibody (C-10; sc5279). The graph on the right displays the quantification of Western blot band intensity from three independent experiments. Band intensities were normalized against the total protein amount visualized by Coomassie Brilliant Blue (CBB) staining of the gel, which served as a loading control. n = 3. Mean ± S.D. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. (F and G) ALP staining images (E) and nestin immunostaining (F) after the introduction of PG1 5′ UTR RNA into PA1 cells. Scale bars, 50 μm. (H) Fluorescence intensity of FLAG immunostaining following forced expression of full-length OCT4A-FLAG and full-length PG1, mutants with the 5′ UTR shortened by 60 nt were transfected into NIH3T3 cells. n = 3. Mean ± SD. (I) Relative positive rate of FLAG immunostaining following forced expression of full-length PG1 and full-length OCT4A-FLAG, 5′ or 3′ UTR deleted OCT4A-FLAG, transfected into NIH3T3 cells. n = 5. Mean ± SD. Unpaired two-tailed Student’s t test. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. (J) Fluorescence intensity of FLAG immunostaining following forced expression of full-length PG1 and full-length OCT4A, and OCT4A mutants with mutations in their 5′ UTR sequence, were transfected into NIH3T3 cells. Upper: Comparison of the sequence information of the 5′ UTRs of OCT4A and PG1, where the orientation of PG1 is complementary to that of OCT4A. The common nucleotide positions between OCT4A and PG1 are marked with an “∗.” The bottom row shows the sequences in which the common nucleotides in OCT4A were substituted with different bases. Left: Fluorescent images of FLAG immunostaining and co-transfection with RFP in both wild-type and mutant cells. Scale bars, 50 μm. Right: Relative positivity rate of FLAG-positive cells in mutant cells transfected with the vector or the full-length PG1 plasmid. The results for the wild type are shown in H n = 5. Mean ± SD. Unpaired two-tailed Student’s t test. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001; ns = not significant. (K) Fluorescence intensity of FLAG immunostaining following the forced expression of OCT4A wild-type and PG1 5′ UTR or its variants with different 5′ UTR lengths in the complementary region to OCT4A, transfected into NIH3T3 cells. n = 5. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. (L) Fluorescence intensity per cell stained with the antibody C-10 against OCT4A. PA1 cells were transfected with three different amounts of synthesized RNA, each containing an equal number of RNA molecules for each type of synthesized RNA (Control RNA, PG1 5′ UTR wild, and PG1-5′ UTR -119 nt). Normalization was performed by dividing the number of DAPI-stained nuclei to account for differences in the number of cells per well. n = 3. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. Plasmid vectors were introduced as mock controls in (A, H–K), and control RNA was used in (C–G, and L).

Article Snippet: Mouse monoclonal anti-OCT3/4; C-10 , Santa Cruz , Cat #sc-5279; RRID: AB_628051.

Techniques: Sequencing, Fluorescence, Immunostaining, Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining, Control, Two Tailed Test, Comparison, Cotransfection, Mutagenesis, Plasmid Preparation, Synthesized

Journal: Nature Physics

Article Title: Boundary-guided cell alignment drives mouse epiblast maturation

doi: 10.1038/s41567-026-03176-9

Figure Lengend Snippet: a , Schematic (top) illustrating morphological changes at the cellular and tissue levels during the E4.5–E5.25 window, from blastocyst to cup-shaped egg-cylinder structure with elongated, radially oriented EPI cells (cyan). The EPI is surrounded by pTE/ExE (grey) and PrE/VE (red); other parts of the embryo are shown in light grey. Immunofluorescence images (first row) and the corresponding membrane segmentation (second row) of representative embryos from E4.5 to E5.25, stained for Oct3/4 (EPI; cyan) and cell membrane (orange), generated by combining E-cadherin and phalloidin signals. The third row shows the 3D visualization of segmented cell volumes viewed from a specific angle, with coordinate axes (arrows) indicating the x , y and z dimensions. The bottom row shows the cell long axes extracted by computing principal inertia vectors in three dimensions from the segmented volumes. n = 37 (E4.5), 26 (E4.75), 22 (E5.0) and 12 (E5.25) embryos segmented and analysed from at least three independent embryo recovery experiments. b , Heat map showing the normalized distribution of angles between the long axes of neighbouring cells, binned in 10° intervals (0°–90°). Data from E4.5 to E5.25 embryos are grouped by the EPI cell number in intervals of 15. Colour intensity represents normalized frequency within each group. Sample sizes by the EPI cell number: [15–29], n = 732 cells from 36 embryos; [30–44], 1,226 from 36; [45–59], 890 from 18; [60–74], 1,077 from 17; [75–89], 626 from 8; [90–104], 373 from 4. c , d , Angle measurement between the cell long axis and the normal vector to the tissue boundary, represented as violin plots with individual data points overlaid. Each plot shows the distribution of angles for EPI cells in contact with the ExE-boundary ( c ) and VE-boundary ( d ). Dot colours indicate the embryo stage. Data are grouped by the EPI cell number, with median values shown by red bars. Sample sizes for c : [15–29], n = 593 cells from 36 embryos; [30–44], 750 from 35; [45–59], 220 from 15; [60–74], 196 from 12; [75–89], 104 from 8; [90–104], 61 from 5. For d : [15–29], n = 618 cells from 36 embryos; [30–44], 951 from 35; [45–59], 682 from 15; [60–74], 649 from 12; [75–89], 540 from 8; [90–104], 446 from 5. Mann–Whitney U -test (two sided) without correction for multiple comparisons; each group compared with the reference group [15–29 cells]. *** P < 0.001, NS, not significant. Exact P values for d : [45–59], P = 2.35 × 10 −7 ; [60–74], P = 3.97 × 10 −16 ; [75–89], P = 3.02 × 10 −13 ; [90–104], P = 3.54 × 10 −64 . e , Time-lapse images of representative Sox2-Cre;mTmG embryos developed in 3D-geec culture system. Green, mG (EPI); magenta, mT (other tissues). Time shown as hours:minutes (hh:mm), with t = 00:00 marking the start of imaging. n = 5 embryos. f , Angle measurement between the cell long axis and the normal vector to the VE-boundary for tracked EPI cells. Each line represents an individual tracked EPI cell, with line colours indicating cell lineages. n = 4 initial cells tracked through 2 rounds of cell divisions to 16 cells, from the embryo shown in e . Scale bars, 20 µm (Extended Data Fig. and Supplementary Video ).

Article Snippet: Primary antibodies against Oct3/4 (Santa Cruz Biotechnology, sc-5279 AF647), E-cadherin (BD Biosciences, 560064), active integrin β1 (9EG7, BD Biosciences, 553715) and phosphorylated ERK1/2 (p44/42 MAPK; Cell Signaling, 4370) were diluted at 1:100.

Techniques: Immunofluorescence, Membrane, Staining, Generated, Plasmid Preparation, MANN-WHITNEY, Imaging

Journal: Nature Physics

Article Title: Boundary-guided cell alignment drives mouse epiblast maturation

doi: 10.1038/s41567-026-03176-9

Figure Lengend Snippet: a , Schematic illustrating two different surfaces that favour distinct cell alignments: tangential alignment at the ExE-boundary (grey) and perpendicular alignment at the VE-boundary (green). b , Schematic of the in silico geometry based on the EPI tissue. Axially symmetric EPI tissue shape was represented by two spherical caps S μ \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(\mu =\alpha ,\beta)$$\end{document} ( μ = α , β ) with radius R μ and centre C μ . These geometrical parameters were obtained by fitting the ExE-boundary (dark grey) and VE-boundary (dark green) data from Fig. . c , Degree of global order P of the order parameter field minimizing the effective free energy as a function of the correlation and anchoring lengths ( ξ and λ ) relative to the characteristic system size \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${R}_{0}=(3{V}_{0}/{4\uppi )}^{1/3}$$\end{document} R 0 = ( V 0 / 4 π ) 1 / , where V 0 is the EPI tissue volume. Stars show the position in the parameter space of the three developmental stages studied, obtained by parameter fitting ( d – f ). The dotted line marks the transition between defect-free and defect-containing regimes. d , Order parameter fields p for the best set of parameters for each embryo stage based on EPI cell numbers. The colour map shows their magnitude \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p=\left|{\bf{p}}\right|$$\end{document} p = p . The black arrowhead marks the position of the topological defect. e , Square difference between the magnitude of the experimentally determined field, p exp , and that of the fitted order parameter field, p , at each point. Data are from the embryos analysed in Fig. . f , Degree of global order P grows with the stage of the embryos grouped by the EPI cell numbers both in the model (square) and in the experiments (circle). g , Absolute degree of alignment between the direction of the experimentally determined field, p exp , and that of the fitted order parameter field, p , at each point . Note that the directional information was not used during the fitting procedure. h , i , Material parameters obtained by fitting the theoretical field to the experimentally determined cell orientation field, plotted against the stage of embryos grouped by the EPI cell numbers. Both the correlation length ξ ( h ) and the anchoring length λ ( i ) relative to the characteristic system size R 0 increase with EPI cell number. j , Immunofluorescence images of representative embryos, stained for Oct3/4 (EPI; cyan) and cell membrane (orange). The white arrowheads indicate the rosette structures or a nascent lumen. Tissue length L is measured as the distance between the EPI–ExE interface and the distal tip. n = 7 embryos. k , Order parameter field calculated with the geometry and material parameters of the embryo stage at which the EPI cell number ranges from 90 to 114, with a uniform boundary that has a perpendicular alignment preference. A white arrowhead marks the position of the topological defect at position z along the system height L . l , Scatter plots showing topological defect (model; square) and rosette or lumen (in utero and ExE-devoid embryos; circle) position along the distal–proximal axis, relative to the tissue centre ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c}_{L}=L/2$$\end{document} c L = L / ) and scaled by tissue length L . ExE-devoid embryo data adapted from ref. . n = 7 (in utero) and 11 (ExE-devoid) embryos. Black bars indicate mean ± s.d. Student’s t -test (two sided), *** P = 1.11 × 10 −5 . Scale bars, 20 µm (Extended Data Figs. and ).

Article Snippet: Primary antibodies against Oct3/4 (Santa Cruz Biotechnology, sc-5279 AF647), E-cadherin (BD Biosciences, 560064), active integrin β1 (9EG7, BD Biosciences, 553715) and phosphorylated ERK1/2 (p44/42 MAPK; Cell Signaling, 4370) were diluted at 1:100.

Techniques: In Silico, Immunofluorescence, Staining, Membrane, In Utero

Journal: Nature Physics

Article Title: Boundary-guided cell alignment drives mouse epiblast maturation

doi: 10.1038/s41567-026-03176-9

Figure Lengend Snippet: ( A ) Single-cell transcriptome data analysis using a published dataset ( 38 Bondarenko et al. 2023). Gene Ontology terms for ECM molecules were used to identify expression levels in single cells isolated from E4.5 and E5.25 embryos. EPI, PrE, VE, TE, and ExE were annotated by Leiden clustering and known markers. ( B ) Time-lapse images of representative Col4a2-eGFP embryos developed in 3D-geec culture system. Time is shown as hours:minutes (hh:mm), with t = 00:00 marking the start of imaging. Signals accumulated at the VE-boundary (yellow arrowhead) and diminished at the ExE-boundary (open arrowhead). n = 3 embryos. ( C ) Immunofluorescence images of representative E4.75 embryos stained for laminin α1, laminin α5, laminin β1, and laminin γ1 (magenta), together with cell membrane (orange, derived from E-cadherin and phalloidin), and Oct3/4 (EPI, cyan). n = 3 embryos for each laminin subunit from 2 independent litters. Scale bars, 20 µm. See also Fig. .

Article Snippet: Primary antibodies against Oct3/4 (Santa Cruz Biotechnology, sc-5279 AF647), E-cadherin (BD Biosciences, 560064), active integrin β1 (9EG7, BD Biosciences, 553715) and phosphorylated ERK1/2 (p44/42 MAPK; Cell Signaling, 4370) were diluted at 1:100.

Techniques: Single Cell, Expressing, Isolation, Imaging, Immunofluorescence, Staining, Membrane, Derivative Assay

Journal: Nature Physics

Article Title: Boundary-guided cell alignment drives mouse epiblast maturation

doi: 10.1038/s41567-026-03176-9

Figure Lengend Snippet: a , Immunofluorescence images of representative embryos from E4.5 to E5.0, stained for laminin (green), Oct3/4 (EPI; cyan) and cell membrane (orange). n = 29 (E4.5), 9 (E4.75) and 7 (E5.0) embryos analysed from at least three independent embryo recovery experiments. b , Quantification of laminin distribution at the tissue boundary, shown as the ratio of intensity at the VE-boundary to that at the ExE-boundary, based on the embryos in a . Each dot represents an individual embryo, plotted against the EPI cell number. Data are presented as mean ± s.d. from three z sections (centre ± 6.4 µm) to account for intensity variation (technical replicates). n = 29 (E4.5), 9 (E4.75) and 7 (E5.0) embryos. c , Scatter plot of EPI cell orientation with respect to surface normal versus normalized laminin intensity at the local tissue boundary, based on the images in a . Cells on the middle plane of the 3D EPI volume were used for the analysis. Each dot represents an individual EPI cell in contact with the VE-boundary (green) or ExE-boundary (red). The black lines show linear regression with a 95% confidence interval (shaded band). E4.5: slope = –0.25, R 2 = 4.88 × 10 −5 , P = 0.982; E5.0: slope = –20.6, R 2 = 0.033, P = 0.336. d , Immunofluorescence images of representative embryos from E4.5 to E4.75, stained for active integrin β1 (green), Oct3/4 (EPI; cyan) and cell membrane. n = 24 (E4.5) and 8 (E4.75) embryos analysed from at least three independent embryo recovery experiments. e , Quantification of active integrin β1 distribution at the tissue boundary, shown as the ratio of intensity at the VE-boundary to that at the ExE-boundary, in embryos shown in d . Each dot represents an individual embryo, plotted against the EPI cell number. Data are presented as mean ± s.d. from three z sections (centre ± 6.4 µm) to account for intensity variation (technical replicates). n = 24 (E4.5) and 8 (E4.75) embryos. f , Scatter plot of EPI cell orientation versus normalized active integrin β1 intensity at the local tissue boundary, based on the images in d . Cells on the middle plane of the 3D EPI volume were used for the analysis. Each dot represents an individual EPI cell in contact with the VE-boundary (green) or ExE-boundary (red). The black lines show linear regression with a 95% confidence interval (shaded band). E4.5: slope = 17.7, R 2 = 0.092, P = 0.171; E4.75: slope = –74.6, R 2 = 0.260, P = 0.013. g , Order parameter fields with weak anchoring to the surface (λ/ R 0 < 1.0) for the average boundary geometry at the EPI cell number stage, [65–89 cells]. Scale bars, 20 µm (Extended Data Fig. ).

Article Snippet: Primary antibodies against Oct3/4 (Santa Cruz Biotechnology, sc-5279 AF647), E-cadherin (BD Biosciences, 560064), active integrin β1 (9EG7, BD Biosciences, 553715) and phosphorylated ERK1/2 (p44/42 MAPK; Cell Signaling, 4370) were diluted at 1:100.

Techniques: Immunofluorescence, Staining, Membrane

Journal: Nature Physics

Article Title: Boundary-guided cell alignment drives mouse epiblast maturation

doi: 10.1038/s41567-026-03176-9

Figure Lengend Snippet: a , Immunofluorescence images of representative Lamc1 +/+ , Lamc1 +/− and Lamc1 −/− embryos from E4.5 to E5.0, stained for Oct3/4 (EPI; cyan) and cell membrane (orange). Sample sizes: Lamc1 +/+ , n = 9 (E4.5), 13 (E4.75), 11 (E5.0); Lamc1 +/− , n = 12, 28, 20; Lamc1 −/− , n = 4, 11, 9 embryos from 5, 7 and 5 independent Lamc1 +/− × Lamc1 +/− litters. b , Cell orientation towards the VE-boundary, shown as violin plots with individual data points. Dot colours indicate the embryo stage. Data are grouped by the EPI cell number, with median values shown by red bars. Sample sizes: Lamc1 +/+ , n = 120 cells from 6 embryos (E4.5), 306 from 8 (E4.75) and 440 from 10 (E5.0); Lamc1 +/− , n = 141 from 7 and 503 from 14, 529 from 10; Lamc1 − / − , n = 66 from 3, 233 from 7 and 250 from 8. Subset of the samples in a with sufficient 3D segmentation quality. Mann–Whitney U -test (two sided) without correction for multiple comparisons, each group compared with the reference group [15–29 cells]. * P < 0.05, *** P < 0.001. Exact P values: Lamc1 +/+ [45–59], P = 0.027; [60–74], P = 0.026; [75–89], P = 4.58 × 10 −7 ; Lamc1 +/− [60–74], P = 2.41 × 10 −5 ; Lamc1 − / − [45–59], P = 0.018; [60–74], P = 0.013. c , Cross-embryo average of nematic cell alignment maps. Colour indicates nematic alignment magnitude, and lines represent mean orientation. The grey and green thick lines indicate the ExE-boundary and VE-boundary, respectively. Black dots with error bars show the mean ± s.d. position of the interface between ExE-boundary and VE-boundary. n = 14 ( Lamc1 +/+ ), 14 ( Lamc1 +/− ) and 9 ( Lamc1 −/− ) embryos. d , Scatter plot of the mean nematic magnitude versus the EPI cell number analysed in c . Each dot represents an individual embryo. The solid lines show linear regression: Lamc1 +/+ , slope = 0.0020, R 2 = 0.0690, P = 0.364; Lamc1 +/− , slope = 0.0011, R 2 = 0.0078, P = 0.764; Lamc1 −/− , slope = –0.0032, R 2 = 0.233, P = 0.188. The dashed line shows wild type from Fig. . No significant differences among genotypes (Kruskal–Wallis test, P = 0.219). e , Immunofluorescence images of representative Itgb1 +/+ , Itgb1 +/− and Itgb1 −/− embryos from E4.5 to E5.0, stained for Oct3/4 (EPI; cyan) and cell membrane (orange). Sample sizes: Itgb1 +/+ , n = 5 (E4.5), 13 (E4.75), 9 (E5.0); Itgb1 +/− , n = 13, 28, 26; Itgb1 −/− , n = 8, 14, 11 embryos from 5, 8 and 7 independent Itgb1 +/− × Itgb1 +/− litters. f , Cell orientation towards the VE-boundary, shown as violin plots with individual data points. Dot colours indicate the embryo stage. Data are grouped by the EPI cell number, with median values shown by red bars. Sample sizes: Itgb1 +/+ , n = 94 cells from 3 embryos (E4.5), 338 from 8 (E4.75), 213 from 4 (E5.0); Itgb1 +/− , n = 156 from 5, 477 from 11, 495 from 10; Itgb1 −/− , n = 148 from 7, 310 from 11, 254 from 9. Subset of the samples in e , with sufficient 3D segmentation quality. Mann–Whitney U -test (two sided) without correction for multiple comparisons, each group compared with the reference group [15–29 cells]. * P < 0.05, *** P < 0.001. Exact P values: Itgb1 +/+ [60–74], P = 0.024; [75–89], P = 1.30 × 10 −4 ; Itgb1 +/− [30–44], P = 0.013; [45–59], P = 0.020; [60–74], P = 1.72 × 10 −5 ; [75–89], P = 1.59 × 10 −9 . g , Cross-embryo average of nematic cell alignment maps. Colour indicates the nematic alignment magnitude, and lines represent the mean orientation. The grey and green thick lines indicate the ExE-boundary and VE-boundary, respectively. Black dots with error bars show the mean ± s.d. position of the interface between the ExE-boundary and VE-boundary. n = 12 ( Itgb1 +/+ ), 22 ( Itgb1 +/− ) and 12 ( Itgb1 −/− ) embryos. h , Scatter plot of the mean nematic magnitude versus the EPI cell number analysed in g . Each dot represents an individual embryo. The solid lines show linear regression: Itgb1 +/+ , slope = 0.0037, R 2 = 0.152, P = 0.211; Itgb1 +/− , slope = 0.0045, R 2 = 0.244, P = 0.0195; Itgb1 −/− , slope = 0.0035, R 2 = 0.343, P = 0.0455. The dashed line shows the wild type from Fig. . No significant differences among genotypes (Kruskal–Wallis test, P = 0.324). Scale bars, 20 µm (Extended Data Figs. and ).

Article Snippet: Primary antibodies against Oct3/4 (Santa Cruz Biotechnology, sc-5279 AF647), E-cadherin (BD Biosciences, 560064), active integrin β1 (9EG7, BD Biosciences, 553715) and phosphorylated ERK1/2 (p44/42 MAPK; Cell Signaling, 4370) were diluted at 1:100.

Techniques: Immunofluorescence, Staining, Membrane, MANN-WHITNEY

Journal: Nature Physics

Article Title: Boundary-guided cell alignment drives mouse epiblast maturation

doi: 10.1038/s41567-026-03176-9

Figure Lengend Snippet: ( A ) Cell orientation toward ExE-boundary, shown as violin plots with individual data points. Dot colours indicate embryo stage; red bars show medians. Sample sizes: Lamc1 +/+ , n = 114 cells from 6 embryos (E4.5), 167 from 8 (E4.75), 247 from 10 (E5.0); Lamc1 +/− , n = 132 from 7, 226 from 14, 179 from 10; Lamc1 − / − , n = 50 from 3, 91 from 7, 230 from 8. Same samples as Fig. . Mann-Whitney U test (two-sided) without correction for multiple comparisons, each group compared to the reference group [15-29 cells]. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Exact p-values: Lamc1 +/+ [75–89], p = 0.014; Lamc1 +/− [30–44], p = 0.0066; [45–59], p = 5.36 × 10 −4 ; [60–74], p = 0.0019; [75–89], p = 0.011; Lamc1 −/− [45–59], p = 0.043. ( B ) Immunofluorescence images of representative Lamc1 +/+ or +/− and Lamc1 −/− embryos from E4.5 to E5.0, stained for Oct3/4 (EPI, cyan) and active integrin β1 (white). Yellow arrowheads indicate signals at the VE-boundary. Same samples as Fig. . ( C ) Cell orientation toward ExE-boundary, as in ( A ). Sample sizes: Itgb1 +/+ , n = 46 cells from 3 embryos (E4.5), 132 from 8 (E4.75), 58 from 4 (E5.0); Itgb1 +/− , n = 98 from 5, 144 from 11, 212 from 10; Itgb1 −/− , n = 129 from 7, 258 from 11, 175 from 9. Same samples as Fig. . Mann-Whitney U test (two-sided) without correction for multiple comparisons, each group compared to the reference group [15-29 cells]. ns = not significant. ( D ) Immunofluorescence images of representative Itgb1 +/+ or +/− and Itgb1 −/− embryos at E4.75, stained for Oct3/4 (EPI, cyan), phospho-ERM (red), and phalloidin (yellow). Right panels show enlarged views. Same samples as Fig. . ( E ) Box plots of pERM intensity ratio from three concentric zones, normalised to the outer zone. Individual embryos shown as lines. Box plots show median (centre), 25th–75th percentiles (box), with whiskers extending to 1.5x interquartile range. n = 14 ( Itgb +/+ or +/− ), 11 ( Itgb −/− ) embryos at E4.75. One-sample Wilcoxon signed-rank test (two-sided) comparing each zone to the reference value of 1 (outer). **p < 0.01, ***p < 0.001. ns = not significant. Exact p-values: Itgb1 +/+ or +/− : Inner, p = 1.22 × 10 −4 . Itgb1 −/− : Middle, p = 0.0020; Inner, p = 9.77 × 10 −4 . Scale bars, 20 µm. See also Fig. .

Article Snippet: Primary antibodies against Oct3/4 (Santa Cruz Biotechnology, sc-5279 AF647), E-cadherin (BD Biosciences, 560064), active integrin β1 (9EG7, BD Biosciences, 553715) and phosphorylated ERK1/2 (p44/42 MAPK; Cell Signaling, 4370) were diluted at 1:100.

Techniques: MANN-WHITNEY, Immunofluorescence, Staining

Journal: Nature Physics

Article Title: Boundary-guided cell alignment drives mouse epiblast maturation

doi: 10.1038/s41567-026-03176-9

Figure Lengend Snippet: ( A ) Box plots of EPI cell number, showing median (centre), 25th–75th percentiles (box), with whiskers extending to 1.5x interquartile range, with individual embryos shown as dots. Sample sizes: Lamc1 +/+ , n = 9 (E4.5), 13 (E4.75), 11 (E5.0); Lamc1 +/− , n = 12, 28, 20; Lamc1 −/− , n = 4, 11, 9 embryos from 5, 7, and 5 independent Lamc1 +/− × Lamc1 +/− litters. One-way ANOVA with Tukey’s post hoc test, ns = not significant. ( B ) Box plots of EPI cell number, as in ( A ). Sample sizes: Itgb1 +/+ , n = 5 (E4.5), 13 (E4.75), 9 (E5.0); Itgb1 +/− , n = 13, 28, 26; Itgb1 −/− , n = 8, 14, 11 embryos from 5, 8, and 7 independent Itgb1 +/− × Itgb1 +/− litters. One-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01, ns = not significant. At E5.0, Itgb1 +/+ vs Itgb1 −/− , p = 0.017; Itgb1 +/− vs Itgb1 −/− , p = 0.001. ( C ) Immunofluorescence images of representative Lamc1 +/+ or +/− and Lamc1 −/− embryos at E5.25, stained for Oct3/4 (EPI, cyan), phalloidin (yellow), and podocalyxin (magenta). n = 28 ( Lamc1 +/+ or +/− ), 6 ( Lamc1 −/− ) embryos from 4 independent Lamc1 +/− × Lamc1 +/− litters. ( D ) Immunofluorescence images of representative Itgb1 +/+ or +/− and Itgb1 −/− embryos at E5.25, stained for Oct3/4 (EPI, cyan), pan-laminin (green), and phalloidin (yellow). n = 13 ( Itgb1 +/+ or +/− ), 2 ( Itgb1 −/− ) embryos from 2 independent Itgb1 +/− × Itgb1 +/− litters. ( E ) Long axis length measurement, shown as violin plots with individual data points. Dot colours indicate embryo stage; red bars show medians. Sample sizes: Lamc1 +/+ , n = 163 cells from 6 embryos (E4.5), 397 from 8 (E4.75), 571 from 10 (E5.0); Lamc1 +/− , n = 199 from 7, 583 from 14, 637 from 10; Lamc1 − / − , n = 75 from 3, 277 from 7, 462 from 9. Mann-Whitney U test (two-sided) without correction for multiple comparisons, each group compared to the reference group [15-29 cells]. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Exact p-values: Lamc1 +/+ [30–44], p = 0.345; [45–59], p = 0.189; [60–74], p = 0.236; [75–89], p = 8.72 × 10 −9 . Lamc1 +/− [30–44], p = 0.051; [45–59], p = 1.00 × 10 −3 ; [60–74], p = 5.02 × 10 −6 ; [75–89], p = 7.42 × 10 −6 . Lamc1 −/− [30–44], p = 0.539; [45–59], p = 4.07 × 10 −4 ; [60–74], p = 0.059. ( F ) Long axis length measurement, as in ( E ). Sample sizes: Itgb1 +/+ , n = 110 cells from 3 embryos (E4.5), 399 from 8 (E4.75), 251 from 4 (E5.0); Itgb1 +/− , n = 200 from 5, 560 from 11, 618 from 10; Itgb1 −/− , n = 209 from 7, 470 from 11, 360 from 9. Mann-Whitney U test (two-sided) without correction for multiple comparisons, each group compared to the reference group [15-29 cells]. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Exact p-values: Itgb1 +/+ : [30–44], p = 2.36 × 10 −4 ; [45–59], p = 0.015; [60–74], p = 8.97 × 10 −3 ; [75–89], p = 2.34 × 10 −4 . Itgb1 +/− : [30–44], p = 0.845; [45–59], p = 0.335; [60–74], p = 0.518; [75–89], p = 0.022. Itgb1 −/− : [30–44], p = 5.65 × 10 −4 ; [45–59], p = 0.022; [60–74], p = 3.71 × 10 −4 . Scale bars, 20 µm. See also Fig. .

Article Snippet: Primary antibodies against Oct3/4 (Santa Cruz Biotechnology, sc-5279 AF647), E-cadherin (BD Biosciences, 560064), active integrin β1 (9EG7, BD Biosciences, 553715) and phosphorylated ERK1/2 (p44/42 MAPK; Cell Signaling, 4370) were diluted at 1:100.

Techniques: Immunofluorescence, Staining, MANN-WHITNEY

Journal: Nature Physics

Article Title: Boundary-guided cell alignment drives mouse epiblast maturation

doi: 10.1038/s41567-026-03176-9

Figure Lengend Snippet: a , Immunofluorescence images of representative embryos from E4.75 to E5.5, stained for pERM (apical, red), phalloidin (white), Oct3/4 (EPI; cyan) and pERK (yellow). n = 21 (E4.75), 18 (E5.0) and 13 (E5.5) embryos analysed from 3 independent embryo recovery experiments. b , Quantification of mean pERK signal intensity within the EPI tissue. Each dot represents an individual embryo. n = 10 (E4.75), 12 (E5.0) and 10 (E5.5) embryos measured from the embryos shown in a . Box plots show the median (centre line), 25th–75th percentiles (box bounds) and whiskers extending to 1.5× the interquartile range. Points beyond the whiskers indicate outliers. One-way analysis of variance ( P = 4.56 × 10 −6 ) with Tukey’s post hoc test. *** P < 0.001. Exact P values: E4.75 versus E5.0, P = 0.64; E4.75 versus E5.5, P = 1.00 × 10 −5 ; E5.0 versus E5.5, P = 6.01 × 10 −5 . c , Immunofluorescence images of representative control and collagenase-treated embryos, stained for pERM (apical, red), phalloidin (white), Oct3/4 (EPI; cyan) and collagen IV (yellow). n = 8 (control) and 8 (collagenase-treated) embryos from 2 independent embryo culture experiments. d , Immunofluorescence images of representative embryos cultured for 16 h in control or collagenase-containing medium, stained for pERM (apical; red), phalloidin (white), Oct3/4 (EPI; cyan) and pERK (yellow). n = 14 (control) and 17 (collagenase-treated) embryos cultured from 3 independent experiments. e , Quantification of mean pERK signal intensity within the EPI tissue. Each dot represents an individual embryo. n = 7 (control) and 10 (collagenase-treated) embryos measured from the embryos shown in d . Box plots show the median (centre line), 25th–75th percentiles (box bounds) and whiskers extending to 1.5× the interquartile range. Points beyond the whiskers indicate outliers. Mann–Whitney U -test (two sided); * P = 0.014.

Article Snippet: Primary antibodies against Oct3/4 (Santa Cruz Biotechnology, sc-5279 AF647), E-cadherin (BD Biosciences, 560064), active integrin β1 (9EG7, BD Biosciences, 553715) and phosphorylated ERK1/2 (p44/42 MAPK; Cell Signaling, 4370) were diluted at 1:100.

Techniques: Immunofluorescence, Staining, Control, Embryo Culture, Cell Culture, MANN-WHITNEY