Journal: bioRxiv

Article Title: Nascent extracellular matrix converts biomaterial cues into cell fate decisions

doi: 10.64898/2026.01.10.698826

Figure Lengend Snippet: a. Timeline and schematic showing the blocking of cell adhesion to nECM using an integrin β1 (ITGB1) function perturbing antibody for 1 hour pre-embedding and for 7 consecutive days . b. Representative fluorescent images of the incorporation of EdU in chondrocytes cultured without (Ctrl) and with ITGB1 inhibition (ITGB1i) in high and low modification hydrogels at day 7 (scale bar = 100 μm). c. Quantification of the incorporation of EdU in chondrocytes cultured without (Ctrl) and with ITGB1 inhibition (ITGB1i) in high and low modification hydrogels at day 7 (low-Ctrl: n = 23 ROIs, N = 3; low-ITGB1i: n = 30 ROIs, N = 3; high-Ctrl: n = 23 ROIs, N = 3; low-ITGB1i: n = 27 ROIs, N = 3). d. Representative fluorescent images of Sox9 immunofluorescence (dashed line outlines nuclei) of chondrocytes cultured without (Ctrl) and with ITGB1 inhibition (ITGB1i) in low and high modification hydrogels at day 7 (scale bar = 10 μm). e. Quantification of Sox9 nucleus-to-cytoplasm (NC) ratio of chondrocytes cultured without (Ctrl) and with ITGB1 inhibition (ITGB1i) in low and high modification hydrogels at day 7 (low-Ctrl: n = 138 cells, N = 4; low-ITGB1i: n = 133 cells, N = 4; high-Ctrl: n = 124 cells, N = 4; high-ITGBi: n = 138 cells, N = 4). a-e. N = number of independent experiments ****p < 0.0001, *p < 0.05, error bar = standard deviation, ns: no significant difference by two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: For perturbation studies, media was supplemented with either an anti-CD44 antibody (DSHB, H4C4, 2.5 μg/mL, day 0-3) or anti-integrin β1 antibody (anti-ITGB1, DSHB, AIIB2, 2.5 μg/mL, day 0-7).

Techniques: Blocking Assay, Cell Culture, Inhibition, Modification, Immunofluorescence, Standard Deviation

Journal: bioRxiv

Article Title: Extracellular matrix rigidity controls breast cancer metastasis via TYK2-mediated mechanotransduction

doi: 10.1101/2025.10.30.681251

Figure Lengend Snippet: a) Bright field images of the human MCF10A acini that were grown on 3D-PA gels and treated with β1 integrin AIIB2 blocking antibody or vehicle control at indicated concentrations for 5 days. Scale bar represents 100μm. b) Invasive structures of the β1-integrin blocking antibody AIIB2 or vehicle control treated human MCF10A acini grown on 3D-PA gels for 5 days were quantified and represented as fraction of total structures. Dots represent individual fields. ****p < 0.0001; ns, not significant. c) Bright field images of the TYK2 overexpressed human MCF10A acini that were grown on 3D-PA gels and treated with the β1 integrin blocking antibody AIIB2, FAK inhibitor VS-4718 (0.2μM) or vehicle control (DMSO) at indicated concentrations for 5 days. Scale bar represents 100μm. d) Invasive structures of the β1-integrin blocking antibody AIIB2, FAK inhibitor VS-4718 (0.2μM) or vehicle control (DMSO) the TYK2 overexpressed human MCF10A acini grown on 3D-PA gels for 5 days were quantified and represented as fraction of total structures. Dots represent individual fields. *p < 0.05; * * p < 0.01; ****p < 0.0001; ns, not significant. e) Immunoblot analysis of pFAK, total FAK and TYK2 expression in lysates from the TYK2 overexpressed human MCF10A treated with the β1-integrin blocking antibody AIIB2, FAK inhibitor VS-4718 (0.2μM) or vehicle control (DMSO) that were grown on 3D-PA gels. GAPDH is used as a loading control. f) MCF10A acini overexpressing FLAG-TYK2 WT and treated with the β1-integrin blocking antibody AIIB2, FAK inhibitor VS-4718 (0.2μM) or vehicle control (DMSO) that were grown on 3D-PA gels. Lysates were subjected to anti-FLAG immunoprecipitation were analysed with immunoblotting. g) MCF10A acini overexpressing FLAG-TYK2 WT and treated with the β1-integrin blocking antibody AIIB2 or vehicle control that were grown on 3D-PA gels. and then immunostained for TYK2 (red) and DAPI (blue). Scale bar represents 25μm. h) Plots of DAPI and TYK2 intensity profiles plotted as a function of distance across tissue sections versus their pixel intensities in human MCF10A acini overexpressing FLAG-tagged TYK2 and treated with the β1-integrin blocking antibody AIIB2 or vehicle control that were grown on 3D-PA gels.

Article Snippet: Antibodies used in this study included TYK2 specific for human, Sigma-Aldrich, HPA005157; TYK2, GeneTex, GTX61449; pTyr1054/1055 TYK2, Cell Signaling Technology, #68790; Phospho-Tyrosine, Cell Signaling Technology, # 9411; TWIST, Santa Cruz Biotechnology, sc-81417; G3BP2, Sigma-Aldrich, HPA018425; E-cadherin, BD Biosciences,610181;Vimentin, Cell Signaling Technology, #5741; Fibronectin, Sigma-Aldrich, F3548; STAT3, Cell Signaling Technology, #9139; pTyr705 STAT3, Cell Signaling Technology, #9145; STAT1, Cell Signaling Technology, #9172; pTyr701 STAT1, Cell Signaling Technology, #9167; STAT5, Cell Signaling Technology, #57580; pTyr694 STAT5, Cell Signaling Technology, #4322; EPHA2, Cell Signaling Technology, #6997; pS897 EPHA2, Cell Signaling Technology, #6347; LYN, Cell Signaling Technology, #2796; pY416 SFK, Cell Signaling Technology, #2101; IFNAR1, Abcam, ab45172; FLAG, Cell Signaling Technology, #14793; Rabbit IgG, Cell Signaling Technology, #2729; FAK, Cell Signaling Technology, #13009; pTyr397 FAK, Invitrogen, # 44-625G; AIIB2 β1-integrin-blocking antibody, Developmental Studies Hybridoma Bank, AB_528306; Laminin V, kind gift from M. Aumailley, University of Cologne, Germany; KRT5, Invitrogen, # PA5-32465; Ki-67, Invitrogen, # MA5-14520; GAPDH, GeneTex, GTX100118.

Techniques: Blocking Assay, Control, Western Blot, Expressing, Immunoprecipitation

Journal: Molecular Therapy. Nucleic Acids

Article Title: ACE-2 blockade and TMPRSS2 inhibition mitigate SARS-CoV-2 severity following cigarette smoke exposure in airway epithelial cells in vitro

doi: 10.1016/j.omtn.2025.102580

Figure Lengend Snippet: ACE-2 and TMPRSS2 expression are elevated in cigarette-smoke-exposed ferret lung (A) Dot plot shown from RNA-seq analysis of SARS-CoV-2 receptor and associated gene expression in lung tissue of ferrets exposed to cigarette smoke for 6 months. (B) ACE-2 and (C) TMPRSS2 mRNA expression in lung tissue from smoke-exposed vs. air control ferrets as assessed by real-time qPCR. (D) Representative western blot and (E) quantification of ACE-2 expression in smoke-exposed ferret lung. (F) Quantification of TMPRSS2 expression in smoke-exposed ferret lung. (G) Representative immunofluorescence images of smoke-exposed and air-control ferret lung sections stained with ACE-2 antibody (red) and DAPI (blue). Scale bars, 50 μM. Areas of magnification (magnification 40X) are outlined by the red dashed line. (H) Representative immunofluorescence images of smoke-exposed and air-control ferret lung sections stained with TMPRSS-2 antibody (Green), Muc5AC (red), and DAPI (blue). Scale bars, 50 μM. Areas of magnification (magnification 40X) are outlined by the red dashed line. Real-time qPCR analysis of (I) ACE2 and (J) TMPRSS2 mRNA expression in terminally differentiated ferret tracheal epithelial cells (FTECs) exposed to cigarette smoke extract (CSE) or vehicle control. ∗ p < 0.05, ∗∗ p < 0.005,∗∗∗ p < 0.0005.

Article Snippet: TMPRSS2 was detected by western blotting using monoclonal anti-TMPRSS2 antibody (clone P5H9-A3; Millipore Sigma).

Techniques: Expressing, RNA Sequencing, Gene Expression, Control, Western Blot, Immunofluorescence, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: ACE-2 blockade and TMPRSS2 inhibition mitigate SARS-CoV-2 severity following cigarette smoke exposure in airway epithelial cells in vitro

doi: 10.1016/j.omtn.2025.102580

Figure Lengend Snippet: ACE-2 and TMPRSS2 expression are elevated in CSE-exposed human airway cells Real-time qPCR measurement of (A) ACE-2 and (B) TMPRSS2 mRNA expression in Calu-3 cells exposed to cigarette smoke extract (CSE) or vehicle control for 48 h Real-time qPCR measurement of (C) ACE-2 and (D) TMPRSS2 mRNA expression in primary human bronchial epithelial (HBE) cells derived from healthy control donors and treated with CSE or vehicle control for 48 h. N = 3 monolayers/condition across 3–4 different donors. Western blot image HBE cells treated with cigarette smoke extract for 24 h (E) and quantitation of western blot for ACE2 (F) and TMPRSS2 (G). Real-time qPCR measurement of (H) ACE-2 and (I) TMPRSS2 mRNA expression in bronchial epithelial cells derived from patients with COPD (COPD HBE) or healthy non-smoker donors. N = 3 monolayers/condition across 3–4 different donors. Western blot analysis of lung tissue from COPD and healthy non-smoker donors (J), with corresponding quantification of TMPRSS2 protein expression (K). (G) Representative western blot for ACE2 (L), with quantification of ACE2 protein expression comparing non-smokers vs. COPD (M) and non-smokers vs. smokers (N). N = 3–4 monolayers/conditions derived from 3 to 4 different donors. ∗ p < 0.05, ∗∗ p < 0.005.

Article Snippet: TMPRSS2 was detected by western blotting using monoclonal anti-TMPRSS2 antibody (clone P5H9-A3; Millipore Sigma).

Techniques: Expressing, Control, Derivative Assay, Western Blot, Quantitation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: ACE-2 blockade and TMPRSS2 inhibition mitigate SARS-CoV-2 severity following cigarette smoke exposure in airway epithelial cells in vitro

doi: 10.1016/j.omtn.2025.102580

Figure Lengend Snippet: SARS-CoV-2 genomic replication is increased in CSE-exposed normal HBE cells and HBE cells derived from COPD patients (A) Transepithelial electrical resistance (TEER) measurements in vehicle-treated, cigarette smoke extract (CSE)-treated, and COPD human bronchial epithelial (HBE) monolayer cultures infected with SARS-CoV-2 or mock infection. The data were obtained by averaging three independent Transwell reads, each of which represented the mean of three separate readings. ( N = 3–5) HBE. Each line corresponds to a distinct donor (3–5 donors in total), and each donor is represented by 3–5 replicates. (B) Quantification of viral load by real-time qPCR of SARS-CoV-2 mRNA at 72 h post-infection with SARS-CoV-2 in CSE- or vehicle-exposed HBE cells. N = 3 different donors, with each line representing a separate donor. (C) Quantification of viral load by real-time qPCR of SARS-CoV-2 mRNA at 72 h post-infection with SARS-CoV-2 in healthy non-smoker or COPD HBE cells. N = monolayers/condition derived from 3 to 5 different donors. (D and E) Representative images using RNAscope in situ hybridization for comparable detection of genomic RNA with only the SARS-CoV-2-specific S probe. Images show CSE- or vehicle-exposed HBE cells, and healthy non-smoker or COPD HBE cells, at 72 h post-inoculation with (D) mock infection or (E) SARS-CoV-2. SARS-CoV-2 (red), TMPRSS2 (green), ACE-2 (white), nuclei (blue). ∗∗ p < 0.01.

Article Snippet: TMPRSS2 was detected by western blotting using monoclonal anti-TMPRSS2 antibody (clone P5H9-A3; Millipore Sigma).

Techniques: Derivative Assay, Infection, RNAscope, In Situ Hybridization

Journal: Molecular Therapy. Nucleic Acids

Article Title: ACE-2 blockade and TMPRSS2 inhibition mitigate SARS-CoV-2 severity following cigarette smoke exposure in airway epithelial cells in vitro

doi: 10.1016/j.omtn.2025.102580

Figure Lengend Snippet: Simultaneous ACE2 blockade and TMPRSS2 inhibition reduces SARS-CoV-2 infection after CSE exposure in FTECs (A) Scheme depicting the approach to ACE2 antisense oligonucleotide (ASO) treatment, cigarette smoke extract (CSE) exposure, and SARS-CoV-2 infection in ferret tracheal epithelial cells (FTECs). Seven days of treatment with ACE2 ASO (20 μM) or control ASO was followed by a 48-h incubation period in CSE or vehicle control prior to inoculation with SARS-CoV-2 or no virus for 72 h of infection. Assessment of mRNA expression of (B) ACE-2 and (C) TMPRSS2 following the scheme depicted in (A). (D) Scheme depicting the approach to ACE2 ASO and camostat mesylate treatment, CSE exposure, and SARS-CoV-2 infection in FTECs. Camostat mesylate (100 mM) was added for 2 h on day 9, prior to inoculation with SARS-CoV-2. Assessment of (E) viral load and mRNA expression of (F) ACE-2 and (G) SARS-Cov2 infection for a shorter duration following the scheme depicted in (D). Viral load following shorter duration of infection (H) and (I). Real-time qPCR was used for mRNA quantification in all studies. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: TMPRSS2 was detected by western blotting using monoclonal anti-TMPRSS2 antibody (clone P5H9-A3; Millipore Sigma).

Techniques: Inhibition, Infection, Control, Incubation, Virus, Expressing