Journal: iScience

Article Title: Probiotic DNA epigenetically modulates KDM4A degradation to mitigate airway allergy

doi: 10.1016/j.isci.2025.114065

Figure Lengend Snippet: LR-DNA modulates regulatory T cell (Tr1) populations in the airways of airway allergy mice Single-cell suspensions were prepared from lung tissues and analyzed by flow cytometry (10 5 cells per sample). (A) Representative flow cytometry plots illustrating the gating strategy for CD3 + CD4 + T cells. (B) Representative flow cytometry plots illustrating the gating strategy for LAG3 + CD49b + Tr1 cells, identified from the CD3 + CD4 + T cell population. (C) Representative flow cytometry plots illustrating the gating strategy for c-Maf + IL-10 + cells, identified within the Tr1 (LAG3 + CD49b + ) population. (D–F) Bar graphs showing the frequency of total Tr1 cells (LAG3 + CD49b + ) (D), the frequency of CD3 + CD4 + c-Maf + IL-10 + Tr1 cells (E), and the absolute number of Tr1 cells (F) in the lungs. Data in bar graphs are presented as mean ± standard deviation (SD), with each individual dot representing one biological replicate. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s post-hoc test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. AA, airway allergy; NC, naive control.

Article Snippet: CD49b , Santa Cruz Biotech , sc-74466, AF594.

Techniques: Flow Cytometry, Standard Deviation, Control

Journal: bioRxiv

Article Title: Collagen I promotes cancer cell survival via amino acid import and mTORC1 activation

doi: 10.1101/2025.11.26.690708

Figure Lengend Snippet: (A) MDA-MB-231 and PANC1 cells were seeded on 2 mg/ml collagen I and treated with 5 µM, 10 µM BTT-3033 (BTT) or DMSO (Ctrl) for 4 days under Glc starvation. (B) MDA-MB-231 and PANC1 cells were seeded on 2 mg/ml collagen I and transfected with an siRNA targeting β1 integrin (β1 si), an siRNA targeting α2 integrin (α2 si) or a non-targeting siRNA control (Nt si) for 4 days under Glc starvation. Cells were fixed and stained with Hoechst 33342. Images were collected by ImageXpress micro and analysed by MetaXpress software. (C) MDA-MB-231-GFP spheroids were generated by the hanging drop method, embedded in 3 mg/ml collagen I and Geltrex (50:50) mixture and starved in Glc-free media for 2 days, in the presence of 10 µM BTT-3033 (BTT) or DMSO (Ctrl). Live images were collected by a Nikon A1 Confocal microscope. (D) MDA-MB-231-GFP cells were transfected with an siRNA targeting α2 integrin (α2 si) or a non-targeting siRNA control (Nt si) for 24 hours. Spheroids were generated by the hanging drop method, embedded in 3 mg/ml collagen I and Geltrex (50:50) mixture in Glc starvation media for 3 days. Live images were collected by a Nikon A1 Confocal microscope. (E) PANC1 spheroids were generated by the hanging drop method, embedded in 3 mg/ml collagen I and Geltrex (50:50) mixture in Glc starvation media for 6 days in the presence of 10 µM BTT-3033 (BTT) or DMSO (Ctrl). Spheroids were imaged live every 2 days with an Olympus E45O microscope. (F) PANC1 cells were transfected with an siRNA targeting α2 integrin (α2 si) or a non-targeting siRNA control (Nt si) for 24 hours. Spheroids were generated by the hanging drop method, embedded in 3 mg/ml collagen I and Geltrex (50:50) mixture in Glc starvation media for 6 days. Live images were collected by an Olympus E45O microscope. (G, H) E0771 mouse tumour organoids were grown in a 3 mg/ml collagen I and Geltrex (50:50) mixture in Glc starvation media for 3 days in the presence of 2.5 µM, 5 µM BTT-3033 (BTT) or DMSO (Ctrl). Spheroids were imaged live every day with an Olympus E45O microscope. Bar, 250 µm. Data are presented as mean ± SEM, N=3 independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; (A, B and H) Kruskal-Wallis, Dunn’s multiple comparisons test; (C-G) Two-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236 and LC3A/B were from Cell Signalling; α2 integrin (CD49b)-FITC conjugated and β1 integrin (CD29)-Alexa Fluor 488 conjugated from BioLegend; α2 integrin for Western blotting from BD-bioscience; SLC3A2 (CD98) and SLC7A5 (LAT1) from Proteintech and GAPDH from SANTA CRUZ Biotechnology.

Techniques: Transfection, Control, Staining, Software, Generated, Microscopy

Journal: bioRxiv

Article Title: Collagen I promotes cancer cell survival via amino acid import and mTORC1 activation

doi: 10.1101/2025.11.26.690708

Figure Lengend Snippet: (A) MDA-MB-231 and (B) PANC1 cells were transfected with a non-targeting control siRNA (Nt si), an siRNA targeting α2 integrin (α2 si) or an siRNA targeting β1 integrin (β1 si) for 4 days. Cells were fixed and stained with Hoechst 33342 (blue) and α2 integrin or β1 integrin (grey). Images were collected by ImageXpress micro. Bar, 50 µm. Integrin signal intensity was quantified by MetaXpress software. Data are presented as mean ± SEM, N=3 independent experiments. p< 0.0001 Mann-Whitney test.

Article Snippet: Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236 and LC3A/B were from Cell Signalling; α2 integrin (CD49b)-FITC conjugated and β1 integrin (CD29)-Alexa Fluor 488 conjugated from BioLegend; α2 integrin for Western blotting from BD-bioscience; SLC3A2 (CD98) and SLC7A5 (LAT1) from Proteintech and GAPDH from SANTA CRUZ Biotechnology.

Techniques: Transfection, Control, Staining, Software, MANN-WHITNEY

Journal: bioRxiv

Article Title: Collagen I promotes cancer cell survival via amino acid import and mTORC1 activation

doi: 10.1101/2025.11.26.690708

Figure Lengend Snippet: (A) MCF10CA1 cells were seeded on 2 mg/ml collagen I and grown under glucose starvation in the presence of 15 µM BTT-3033 (BTT) or DMSO (Ctrl) control for 4 days. (B) MCF10CA1 cells were transfected with a non-targeting control siRNA (Nt si) or an siRNA targeting α2 integrin (α2 si) and grown on 2 mg/ml collagen I under glucose starvation for 4 days. Cells were fixed and stained with Hoechst 33342. Innages were collected by ImageXpress micro. Data are presented as mean ± SEM, N=3 independent experiments. p < 0.0001 Mann-Whitney test. (C) MCF10CA1 cells were transfected as in B, lysed and the levels of α2 integrin and GAPDH were measured by Western blotting.

Article Snippet: Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236 and LC3A/B were from Cell Signalling; α2 integrin (CD49b)-FITC conjugated and β1 integrin (CD29)-Alexa Fluor 488 conjugated from BioLegend; α2 integrin for Western blotting from BD-bioscience; SLC3A2 (CD98) and SLC7A5 (LAT1) from Proteintech and GAPDH from SANTA CRUZ Biotechnology.

Techniques: Control, Transfection, Staining, MANN-WHITNEY, Western Blot

Journal: bioRxiv

Article Title: Collagen I promotes cancer cell survival via amino acid import and mTORC1 activation

doi: 10.1101/2025.11.26.690708

Figure Lengend Snippet: MDA-MB-231 (A-E) and PANC1 (F-O) cells were transfected with an siRNA targeting SLC3A2 (3A2 si), an siRNA targeting α2 integrin (α2 si) or a non-targeting siRNA control (Nt si) and grown on 2 mg/ml collagen I in Glc starvation media for one (A-E), two (F-J) and three days (K-O). Protein level of phosphorylated S6 (pS6, B,G,L), LC3II (C,H,M), SLC3A2 (D,I,N) and α2 integrin (E,J,O) were measured via Western blotting. Data are presented as mean ± SEM, N≥4 independent experiments, *p < 0.05, **p < 0.01 Kruskal-Wallis, Dunn’s multiple comparisons test.

Article Snippet: Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236 and LC3A/B were from Cell Signalling; α2 integrin (CD49b)-FITC conjugated and β1 integrin (CD29)-Alexa Fluor 488 conjugated from BioLegend; α2 integrin for Western blotting from BD-bioscience; SLC3A2 (CD98) and SLC7A5 (LAT1) from Proteintech and GAPDH from SANTA CRUZ Biotechnology.

Techniques: Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: Collagen I promotes cancer cell survival via amino acid import and mTORC1 activation

doi: 10.1101/2025.11.26.690708

Figure Lengend Snippet: Under Glc starvation, collagen I promoted α2β1 integrin-dependent mTORCI activation in breast and pancreatic cancer cells. Collagen I also increased the membrane levels of the LAT1-4F2hc amino acid transporter heavy chain, SLC3A2 (3A2), and light chain, SLC7A5 (7A5), stimulating amino acid import. This resulted in mTORCI activation and autophagy inhibition, leading to increased survival, proliferation and invasion, in 2D and 3C contexts.

Article Snippet: Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236 and LC3A/B were from Cell Signalling; α2 integrin (CD49b)-FITC conjugated and β1 integrin (CD29)-Alexa Fluor 488 conjugated from BioLegend; α2 integrin for Western blotting from BD-bioscience; SLC3A2 (CD98) and SLC7A5 (LAT1) from Proteintech and GAPDH from SANTA CRUZ Biotechnology.

Techniques: Activation Assay, Membrane, Inhibition