Journal: bioRxiv

Article Title: Basement membrane mechanics drives patterned response to developmental signalling

doi: 10.64898/2026.02.13.705301

Figure Lengend Snippet: a. Normalised counts obtained from RNA-sequencing data of hPSCs on stiff and soft substrates for BMPR1A and BMPR1B . Differential expression analysis was performed using DESeq2 with the Wald test. Data was generated from N = 3 independent experiments representing biological replicates. b. Pipeline used for quantification of the apical/basolateral ratio of PH Akt-GFP (labelling PIP3) signal intensity. See Materials and Methods .

Article Snippet: The following primary antibodies were used: Brachyury (1:500; Cell Signalling Technology, #81694S), integrin β5 (1:200; Cell Signalling Technology, #3629S), pFAK (1:200; Thermo Scientific, #44-624G), active integrin β1 (1:100; Abcam, #ab30394), ZO-1 (1:250; Thermo Scientific, #33-9100), BMPR1A (1:50; Santa Cruz Biotechnology, #sc-518037), Myosin Regulatory Light Chain 2 (1:100; Cell Signalling Technology, #3672S), Claudin-6 (1:100; Santa Cruz Biotechnology, #sc-17669), laminin (1:200; Thermo Scientific, #PA1-16730).

Techniques: RNA Sequencing, Quantitative Proteomics, Generated

Journal: bioRxiv

Article Title: Basement membrane mechanics drives patterned response to developmental signalling

doi: 10.64898/2026.02.13.705301

Figure Lengend Snippet: a. Top: Schematic showing the part of the hPSC colonies that was imaged using confocal microscopy (red box). Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates, stained for BMPR1A and DAPI. Images were reconstructed in x-z plane (along the apicobasal axis); 50 imaging planes were projected in y-axis. Scale bar = 15 μm. b. Top: Schematic showing the part of the hPSC colonies that was imaged using structured illumination microscopy (SIM). Bottom: Representative immunofluorescence images (maximum projection) of hPSCs on stiff and soft substrates, stained for BMPR1A and with CellMask HCS dye staining the cytoplasm. Images were reconstructed in x-z (i.e., along the apicobasal axis), and the top 10 μm of the apicobasal axis were imaged; 40 imaging planes were projected in y-axis. Scale bar = 10 μm. c. Plot of the fraction of BMPR1A-positive pixels against the distance from cell surface from SIM images. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates. d. Schematic of apical and basolateral membrane domains in epithelial hPSCs, defined by the localisation of phospholipids PIP2 and PIP3. Schematic also represents the hypothesis of polarity disruption as a consequence of substrate stiffness change. e. Representative images of hPSCs on stiff and soft substrates in control conditions and upon FAK inhibitor (FAKi) or PI3K inhibitor (PI3Ki) treatment, transfected with the plasmid encoding the GFP-tagged PH domain of Akt. Images were reconstructed in the x-z plane, i.e., along the apicobasal axis. Scale bar = 20 μm. f. Quantification of the apical/basolateral ratio of PH Akt-GFP (labelling PIP3) signal intensity in individual hPSCs on stiff and soft substrates in control condition as well as on stiff substrates upon FAK inhibitor (FAKi) or PI3K inhibitor (PI3Ki) treatment. See for quantification pipeline. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. **** indicates p-value < 0.0001. g. Top: Schematic showing the part of the hPSC colonies that was imaged using confocal microscopy (red box). Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates, stained for the active form of integrin β1 and DAPI. Images were reconstructed in x-z plane (along the apicobasal axis); 50 imaging planes were projected in y-axis. Scale bar = 15 μm. h. Top: Schematic showing the part of the hPSC colonies that was imaged using structured illumination microscopy (SIM) (red box). Bottom: Representative immunofluorescence images (maximum projection) of hPSCs on stiff and soft substrates, stained for the active form of integrin β1 and with HCS dye staining the cytoplasm. Images were reconstructed in x-z (i.e., along the apicobasal axis), and the top 10 μm of the apicobasal axis were imaged; 20 imaging planes were projected in the y-axis. Scale bar = 10 μm. i. Plot of the fraction of active integrin β1-positive pixels against the distance from the cell surface from SIM images. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates.

Article Snippet: The following primary antibodies were used: Brachyury (1:500; Cell Signalling Technology, #81694S), integrin β5 (1:200; Cell Signalling Technology, #3629S), pFAK (1:200; Thermo Scientific, #44-624G), active integrin β1 (1:100; Abcam, #ab30394), ZO-1 (1:250; Thermo Scientific, #33-9100), BMPR1A (1:50; Santa Cruz Biotechnology, #sc-518037), Myosin Regulatory Light Chain 2 (1:100; Cell Signalling Technology, #3672S), Claudin-6 (1:100; Santa Cruz Biotechnology, #sc-17669), laminin (1:200; Thermo Scientific, #PA1-16730).

Techniques: Confocal Microscopy, Immunofluorescence, Staining, Imaging, Microscopy, Standard Deviation, Membrane, Disruption, Control, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Basement membrane mechanics drives patterned response to developmental signalling

doi: 10.64898/2026.02.13.705301

Figure Lengend Snippet: a. Representative immunofluorescence images of the colony apical surface of hPSCs on stiff and soft substrates, without permeabilization, stained for pan-laminin and DAPI. Scale bar = 50 μm. b. Quantification of apical laminin signal intensity across images. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. *** indicates p-value < 0.001. c. Top: Schematic of the hypothesized apical cell surface in hPSCs on stiff and soft substrates. Green arrows point to microvilli, red rectangle represents the plane imaged with SEM. Bottom: SEM images of the apical surface of hPSCs on stiff and soft substrates. Green arrows point to individual microvilli. Scale bar = 10 μm; zoomed in images scale bar = 2 μm. Representative images came from N = 3 independent experiments representing biological replicates. d. Top: Schematic representing the experimental protocol involving soluble laminin addition into the cell medium of hPSCs on stiff substrates before the BMP4 treatment. Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff substrates stained for pSMAD1/5 and DAPI after 1 h of BMP4 treatment, with or without addition of soluble laminin into the cell medium. Scale bar = 50 μm. e. Quantification of pSMAD1/5 signal from immunofluorescence images of hPSCs on stiff substrates, with or without the addition of soluble laminin into the cell medium and after 1 h of BMP4 treatment. The signal intensity in each nucleus is plotted as a function of the distance of that nucleus from the colony edge, normalized by the 98 th percentile value of each image. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates. f. Top: Schematic showing the part of the hPSC colonies that was imaged using confocal microscopy (red box). Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff substrates, with or without the addition of soluble laminin, stained for the active form of integrin β1 and DAPI. Images were reconstructed in the x-z plane (along the apicobasal axis); 50 imaging planes were projected in y-axis. Scale bar = 20 μm. g. Top: Schematic showing the part of the hPSC colonies that was imaged using structured illumination microscopy (SIM). Bottom Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates, with or without the addition of soluble laminin, and stained for BMPR1A and with HCS dye staining the cytoplasm. Images were reconstructed x-z (i.e., along the apicobasal axis), and the top 10 μm of the apicobasal axis was imaged; 40 imaging planes were projected in y-axis. Scale bar = 10 μm. h. Plot of the fraction of BMPR1A-positive pixels against the distance from the cell surface. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates. i. Representative immunofluorescence images (maximum projection) of colony top (top 5 confocal planes) of hPSCs on stiff substrates, with or without soluble laminin addition into the medium, stained for Claudin-6. Scale bar = 50 μm; zoomed in images scale bar = 20 μm. j. Quantification of junctional Claudin-6 signal intensity across maximum projection images. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. **** indicates p-value < 0.0001. k. Representative immunofluorescence images (maximum projection) of colony bottom (bottom 5 confocal planes) of hPSCs on stiff and soft substrates, in control condition or upon addition of soluble laminin into the cell medium, stained for pFAK and F-Actin. Scale bar = 50 μm. l. Quantification of normalised signal intensity in pFAK focal points in hPSCs on stiff substrates in control condition and upon addition of soluble laminin into the cell medium. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. **** indicates p-value < 0.0001.

Article Snippet: The following primary antibodies were used: Brachyury (1:500; Cell Signalling Technology, #81694S), integrin β5 (1:200; Cell Signalling Technology, #3629S), pFAK (1:200; Thermo Scientific, #44-624G), active integrin β1 (1:100; Abcam, #ab30394), ZO-1 (1:250; Thermo Scientific, #33-9100), BMPR1A (1:50; Santa Cruz Biotechnology, #sc-518037), Myosin Regulatory Light Chain 2 (1:100; Cell Signalling Technology, #3672S), Claudin-6 (1:100; Santa Cruz Biotechnology, #sc-17669), laminin (1:200; Thermo Scientific, #PA1-16730).

Techniques: Immunofluorescence, Staining, Standard Deviation, Confocal Microscopy, Imaging, Microscopy, Control

Journal: bioRxiv

Article Title: Basement membrane mechanics drives patterned response to developmental signalling

doi: 10.64898/2026.02.13.705301

Figure Lengend Snippet: a. Normalised counts obtained from RNA-sequencing data of hPSCs on stiff and soft substrates for BMPR1A and BMPR1B . Differential expression analysis was performed using DESeq2 with the Wald test. Data was generated from N = 3 independent experiments representing biological replicates. b. Pipeline used for quantification of the apical/basolateral ratio of PH Akt-GFP (labelling PIP3) signal intensity. See Materials and Methods .

Article Snippet: BMPR1A was stained with the anti-BMPR1A antibody (Santa Cruz Biotechnology, #sc-518037) which was given to the live cells for 45 minutes at the concentration of 1:20 in mTeSR Plus before the cells were fixed.

Techniques: RNA Sequencing, Quantitative Proteomics, Generated

Journal: bioRxiv

Article Title: Basement membrane mechanics drives patterned response to developmental signalling

doi: 10.64898/2026.02.13.705301

Figure Lengend Snippet: a. Top: Schematic showing the part of the hPSC colonies that was imaged using confocal microscopy (red box). Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates, stained for BMPR1A and DAPI. Images were reconstructed in x-z plane (along the apicobasal axis); 50 imaging planes were projected in y-axis. Scale bar = 15 μm. b. Top: Schematic showing the part of the hPSC colonies that was imaged using structured illumination microscopy (SIM). Bottom: Representative immunofluorescence images (maximum projection) of hPSCs on stiff and soft substrates, stained for BMPR1A and with CellMask HCS dye staining the cytoplasm. Images were reconstructed in x-z (i.e., along the apicobasal axis), and the top 10 μm of the apicobasal axis were imaged; 40 imaging planes were projected in y-axis. Scale bar = 10 μm. c. Plot of the fraction of BMPR1A-positive pixels against the distance from cell surface from SIM images. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates. d. Schematic of apical and basolateral membrane domains in epithelial hPSCs, defined by the localisation of phospholipids PIP2 and PIP3. Schematic also represents the hypothesis of polarity disruption as a consequence of substrate stiffness change. e. Representative images of hPSCs on stiff and soft substrates in control conditions and upon FAK inhibitor (FAKi) or PI3K inhibitor (PI3Ki) treatment, transfected with the plasmid encoding the GFP-tagged PH domain of Akt. Images were reconstructed in the x-z plane, i.e., along the apicobasal axis. Scale bar = 20 μm. f. Quantification of the apical/basolateral ratio of PH Akt-GFP (labelling PIP3) signal intensity in individual hPSCs on stiff and soft substrates in control condition as well as on stiff substrates upon FAK inhibitor (FAKi) or PI3K inhibitor (PI3Ki) treatment. See for quantification pipeline. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. **** indicates p-value < 0.0001. g. Top: Schematic showing the part of the hPSC colonies that was imaged using confocal microscopy (red box). Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates, stained for the active form of integrin β1 and DAPI. Images were reconstructed in x-z plane (along the apicobasal axis); 50 imaging planes were projected in y-axis. Scale bar = 15 μm. h. Top: Schematic showing the part of the hPSC colonies that was imaged using structured illumination microscopy (SIM) (red box). Bottom: Representative immunofluorescence images (maximum projection) of hPSCs on stiff and soft substrates, stained for the active form of integrin β1 and with HCS dye staining the cytoplasm. Images were reconstructed in x-z (i.e., along the apicobasal axis), and the top 10 μm of the apicobasal axis were imaged; 20 imaging planes were projected in the y-axis. Scale bar = 10 μm. i. Plot of the fraction of active integrin β1-positive pixels against the distance from the cell surface from SIM images. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates.

Article Snippet: BMPR1A was stained with the anti-BMPR1A antibody (Santa Cruz Biotechnology, #sc-518037) which was given to the live cells for 45 minutes at the concentration of 1:20 in mTeSR Plus before the cells were fixed.

Techniques: Confocal Microscopy, Immunofluorescence, Staining, Imaging, Microscopy, Standard Deviation, Membrane, Disruption, Control, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Basement membrane mechanics drives patterned response to developmental signalling

doi: 10.64898/2026.02.13.705301

Figure Lengend Snippet: a. Representative immunofluorescence images of the colony apical surface of hPSCs on stiff and soft substrates, without permeabilization, stained for pan-laminin and DAPI. Scale bar = 50 μm. b. Quantification of apical laminin signal intensity across images. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. *** indicates p-value < 0.001. c. Top: Schematic of the hypothesized apical cell surface in hPSCs on stiff and soft substrates. Green arrows point to microvilli, red rectangle represents the plane imaged with SEM. Bottom: SEM images of the apical surface of hPSCs on stiff and soft substrates. Green arrows point to individual microvilli. Scale bar = 10 μm; zoomed in images scale bar = 2 μm. Representative images came from N = 3 independent experiments representing biological replicates. d. Top: Schematic representing the experimental protocol involving soluble laminin addition into the cell medium of hPSCs on stiff substrates before the BMP4 treatment. Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff substrates stained for pSMAD1/5 and DAPI after 1 h of BMP4 treatment, with or without addition of soluble laminin into the cell medium. Scale bar = 50 μm. e. Quantification of pSMAD1/5 signal from immunofluorescence images of hPSCs on stiff substrates, with or without the addition of soluble laminin into the cell medium and after 1 h of BMP4 treatment. The signal intensity in each nucleus is plotted as a function of the distance of that nucleus from the colony edge, normalized by the 98 th percentile value of each image. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates. f. Top: Schematic showing the part of the hPSC colonies that was imaged using confocal microscopy (red box). Bottom: Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff substrates, with or without the addition of soluble laminin, stained for the active form of integrin β1 and DAPI. Images were reconstructed in the x-z plane (along the apicobasal axis); 50 imaging planes were projected in y-axis. Scale bar = 20 μm. g. Top: Schematic showing the part of the hPSC colonies that was imaged using structured illumination microscopy (SIM). Bottom Representative immunofluorescence merged images (maximum projection) of hPSCs on stiff and soft substrates, with or without the addition of soluble laminin, and stained for BMPR1A and with HCS dye staining the cytoplasm. Images were reconstructed x-z (i.e., along the apicobasal axis), and the top 10 μm of the apicobasal axis was imaged; 40 imaging planes were projected in y-axis. Scale bar = 10 μm. h. Plot of the fraction of BMPR1A-positive pixels against the distance from the cell surface. The continuous line represents the mean signal intensity and edges of the shaded areas represent +/- standard deviation from N = 3 biological replicates. i. Representative immunofluorescence images (maximum projection) of colony top (top 5 confocal planes) of hPSCs on stiff substrates, with or without soluble laminin addition into the medium, stained for Claudin-6. Scale bar = 50 μm; zoomed in images scale bar = 20 μm. j. Quantification of junctional Claudin-6 signal intensity across maximum projection images. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. **** indicates p-value < 0.0001. k. Representative immunofluorescence images (maximum projection) of colony bottom (bottom 5 confocal planes) of hPSCs on stiff and soft substrates, in control condition or upon addition of soluble laminin into the cell medium, stained for pFAK and F-Actin. Scale bar = 50 μm. l. Quantification of normalised signal intensity in pFAK focal points in hPSCs on stiff substrates in control condition and upon addition of soluble laminin into the cell medium. Individual data came from N = 3 independent experiments representing biological replicates. P-value determined by Welch’s t-test. **** indicates p-value < 0.0001.

Article Snippet: BMPR1A was stained with the anti-BMPR1A antibody (Santa Cruz Biotechnology, #sc-518037) which was given to the live cells for 45 minutes at the concentration of 1:20 in mTeSR Plus before the cells were fixed.

Techniques: Immunofluorescence, Staining, Standard Deviation, Confocal Microscopy, Imaging, Microscopy, Control

Journal: JCI Insight

Article Title: Stem cell–associated osteogenic deficiency causes craniofacial deformities with progeroid accumulation of prelamin A

doi: 10.1172/jci.insight.196932

Figure Lengend Snippet: ( A ) Limiting dilution analysis of stem cell–mediated bone formation with renal capsule transplantation. Representative images of whole-mount von Kossa staining detecting ectopic bone formation in the mouse recipients transplanted by the indicated number of suture cells into the renal capsule. Arrowheads indicate the ectopic bones. ( B ) Representative images showing the analysis of Axin2-expressing cells using the Axin2 mGFP allele in the indicated 1-month-old (1M) suture. ( C ) Representative images examining the BMPR1A + and GLI1 + cell population within the indicated 1-month-old (1M) suture. ( D ) Graphs indicate the quantitation of the average percentage of BMPR1A + and GLI1 + cells in 3 independent experiments ( P < 0.005 or 0.05, n = 3, mean ± SEM, 2-tailed Student’s t test). SAG, sagittal; COR, coronal; AF, anterior frontal. Scale bars: 1 mm ( A ) and 50 μm ( B and C ).

Article Snippet: Mouse monoclonal antibodies against Bmpr1a (NBP2-37421, 1:75, Novus Biologicals), fascin1 (SC-21743, 1:100, Santa Cruz), and Alexa Fluor 568 phalloidin (A12380, 1:400, Invitrogen); rabbit polyclonal antibodies Osx (ab22552, 1:800, Abcam), OCN (23418-1-AP, 1:50, Proteintech), Gli1 (NBP1-78259, 1:100, Novus Biologicals), Myl2 (3671, 1:100, Cell Signaling Technology), and GM130 (2296, 1:200, Cell Signaling Technology); rabbit monoclonal antibodies lamin A/C (MA5-35284, 1:200, Invitrogen) and SUN2(EPR6557, 1:100, Abcam); anti-rabbit secondary antibody (BA-1000, 1:200, Vector laboratories); and anti-mouse IgG secondary antibodies (PK-2200, 1:250, Vector laboratories), were used in the immunostaining studies.

Techniques: Transplantation Assay, Staining, Expressing, Quantitation Assay