Journal: iScience

Article Title: Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors

doi: 10.1016/j.isci.2022.104907

Figure Lengend Snippet: Biosensors fused to HER2-Nb precisely report [K + ] ex changes upon immobilization on HER2 expressing HEK293 cells (A) Coomassie-stained SDS PAGE of 1 μg protein (left panel) and immunoblot analysis using anti-VHH antibody (right panel) of purified HER2-Nb-GEPII 1.0 and HER2-Nb-pH-Lemon proteins are shown. (B and C) Representative confocal images of living HEK293 cells transiently overexpressing HER2 (upper row) or untransfected HEK293 cells (lower row) following incubation with HER2-Nb-GEPII 1.0 (B) or HER2-Nb-pH-Lemon (C) are shown. Scale bar 10 μm, N= 4. (D) Response of HER2-Nb-GEPII 1.0 immobilized on HEK293 cell transiently overexpressing HER2 in response to buffers with different K + . Shown is a representative measurement of four cells (mean ± SD in red, traces from individual cells in black). (E) Respective single wavelength traces (FRET in red, mseCFP in pink) of the ratio curve as shown in (D) in response to K + alterations.

Article Snippet: For immobilization of SPOT-Nb-sensors on HEK293 cells, the cells were transfected with a plasmid encoding a GPI-anchored SPOTtag (GPI-SPOT) using Lipofectamine 2000 reagent (Thermo Fisher) according to the manufacturer's instructions 2 days before experiments, followed by the removal of the transfection reagent after 6 h. For transient HER2 overexpression in HEK293 cells, the cells were transfected with a plasmid encoding human HER2 wildtype ( )(Addgene plasmid #16257) the day before measurement with removal of the transfection reagent after 6 hours.

Techniques: Expressing, Staining, SDS Page, Western Blot, Purification, Incubation

Journal: iScience

Article Title: Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors

doi: 10.1016/j.isci.2022.104907

Figure Lengend Snippet: HER2-Nb-biosensors specifically label endogenous HER2 on HER2 positive breast cancer cells (A) Schematic illustration of a HER2 positive SkBr3 cell endogenously expressing HER2 on the cell surface. Biosensors fused to HER2 can be bound to HER2 for immobilization on the plasma membrane. Figure created using BioRender. (B) Representative images of SkBr3 cells following incubation with HER2-Nb-GEPII 1.0 and HER2-Nb-pH-Lemon. Scale bar 10 μm, n= 4 experiments representing biological replicates. (C) Representative confocal images of HER2 negative MCF7 breast cancer cells following incubation with HER2-Nb-GEPII 1.0 and HER2-Nb-pH-Lemon are shown. Scale bar 10 μm, N= 4. (D) Determination of cell viability of SkBr3 cells using MTT in response to vehicle (ctrl), unfused HER2-Nb and HER2-Nb-GEPII 1.0 after 3, 6, 24, and 48 h after immobilization. Not significant as determined using one-way ANOVA, Dunn’s Multiple Comparison Test.

Article Snippet: For immobilization of SPOT-Nb-sensors on HEK293 cells, the cells were transfected with a plasmid encoding a GPI-anchored SPOTtag (GPI-SPOT) using Lipofectamine 2000 reagent (Thermo Fisher) according to the manufacturer's instructions 2 days before experiments, followed by the removal of the transfection reagent after 6 h. For transient HER2 overexpression in HEK293 cells, the cells were transfected with a plasmid encoding human HER2 wildtype ( )(Addgene plasmid #16257) the day before measurement with removal of the transfection reagent after 6 hours.

Techniques: Expressing, Clinical Proteomics, Membrane, Incubation, Comparison

Journal: iScience

Article Title: Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors

doi: 10.1016/j.isci.2022.104907

Figure Lengend Snippet:

Article Snippet: For immobilization of SPOT-Nb-sensors on HEK293 cells, the cells were transfected with a plasmid encoding a GPI-anchored SPOTtag (GPI-SPOT) using Lipofectamine 2000 reagent (Thermo Fisher) according to the manufacturer's instructions 2 days before experiments, followed by the removal of the transfection reagent after 6 h. For transient HER2 overexpression in HEK293 cells, the cells were transfected with a plasmid encoding human HER2 wildtype ( )(Addgene plasmid #16257) the day before measurement with removal of the transfection reagent after 6 hours.

Techniques: Virus, Recombinant, Staining, Plasmid Preparation, Protease Inhibitor, Software

Journal: bioRxiv

Article Title: Transitions in density, pressure, and effective temperature drive collective cell migration into confining environments

doi: 10.1101/2023.04.10.536258

Figure Lengend Snippet: Cell density distribution after collective migration into (a) wide and (b) narrow channels for MCF10A wildtype, 10A+RhoA, and 10A+ErbB2 cell variants, with (i-iii) nuclear intensity and (iv-vi) cell density heatmaps. Temporal variation of difference in cell density between inside and outside the microchannels, Δρ , for (c) WT, (d) 10A+RhoA and (e) 10A+ErbB2 cells. (b) Cell density difference averaged from t = 18-24 hr. * p < 0.05, by two-way ANOVA. N≥4 ROIs.

Article Snippet: Human mammary gland wildtype (WT) MCF10A (10A) epithelial cells with green fluorescence protein-labelled nuclei, MCF10A cell line with constitutively active RhoA (10A+RhoA; courtesy of Gregory Longmore, Washington University in St. Louis), and MCF10A cell line with overexpressing ErbB2 (10A+ErbB2; courtesy of Dihua Yu, The University of Texas MD Anderson Cancer Center) mutant cell lines were cultured using Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Invitrogen, Waltham, MA), supplemented with 5% horse serum (Hyclone heat-inactivated, Cytiva, Marlborough, MA), 20 ng/mL epidermal growth factor (EGF; Miltenyi Biotec, Gaithersburg, MD), 0.5 mg/mL hydrocortisone (H0888, MilliporeSigma), 100 ng/mL cholera toxin (C8052, Sigma-Aldrich), 10 μg/mL insulin (I6634, Sigma-Aldrich) and 100 μg/mL proprietary antimicrobial (Normocin; InvivoGen, San Diego, CA).

Techniques: Migration

Journal: bioRxiv

Article Title: Transitions in density, pressure, and effective temperature drive collective cell migration into confining environments

doi: 10.1101/2023.04.10.536258

Figure Lengend Snippet: Migration tracks of cells within (a) MCF10A WT, (b) 10A+RhoA and (c) 10A+ErbB2 monolayers entering wide and narrow channels, with tracks color-coded according to the track mean speed. Root-mean-squared (RMS) velocities of (d) WT, (e) 10A+RhoA, and (f) 10A+ErbB2 cells outside (blue) and inside (red) channels. (g) Average difference in RMS velocity of cells outside and inside the channels. ** p < 0.05, **** p < 0.001

Article Snippet: Human mammary gland wildtype (WT) MCF10A (10A) epithelial cells with green fluorescence protein-labelled nuclei, MCF10A cell line with constitutively active RhoA (10A+RhoA; courtesy of Gregory Longmore, Washington University in St. Louis), and MCF10A cell line with overexpressing ErbB2 (10A+ErbB2; courtesy of Dihua Yu, The University of Texas MD Anderson Cancer Center) mutant cell lines were cultured using Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Invitrogen, Waltham, MA), supplemented with 5% horse serum (Hyclone heat-inactivated, Cytiva, Marlborough, MA), 20 ng/mL epidermal growth factor (EGF; Miltenyi Biotec, Gaithersburg, MD), 0.5 mg/mL hydrocortisone (H0888, MilliporeSigma), 100 ng/mL cholera toxin (C8052, Sigma-Aldrich), 10 μg/mL insulin (I6634, Sigma-Aldrich) and 100 μg/mL proprietary antimicrobial (Normocin; InvivoGen, San Diego, CA).

Techniques: Migration

Journal: bioRxiv

Article Title: Transitions in density, pressure, and effective temperature drive collective cell migration into confining environments

doi: 10.1101/2023.04.10.536258

Figure Lengend Snippet: Persistence and order parameter within the monolayers across cell lines. (a) The snapshots of cell tracks within the monolayers color-coded according to track-wise persistence. (b) Curves of persistence and order parameter within the monolayers across cell lines. (i) WT_wide: n = 17; (ii) 10A+RhoA_wide: n = 4; 10A+ErbB2_wide: n = 7; (iv) WT_narrow: n = 8; (v) 10A+RhoA_narrow: n = 6; (vi) 10A+ErbB2_narrow: n=18.

Article Snippet: Human mammary gland wildtype (WT) MCF10A (10A) epithelial cells with green fluorescence protein-labelled nuclei, MCF10A cell line with constitutively active RhoA (10A+RhoA; courtesy of Gregory Longmore, Washington University in St. Louis), and MCF10A cell line with overexpressing ErbB2 (10A+ErbB2; courtesy of Dihua Yu, The University of Texas MD Anderson Cancer Center) mutant cell lines were cultured using Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Invitrogen, Waltham, MA), supplemented with 5% horse serum (Hyclone heat-inactivated, Cytiva, Marlborough, MA), 20 ng/mL epidermal growth factor (EGF; Miltenyi Biotec, Gaithersburg, MD), 0.5 mg/mL hydrocortisone (H0888, MilliporeSigma), 100 ng/mL cholera toxin (C8052, Sigma-Aldrich), 10 μg/mL insulin (I6634, Sigma-Aldrich) and 100 μg/mL proprietary antimicrobial (Normocin; InvivoGen, San Diego, CA).

Techniques:

Journal: bioRxiv

Article Title: Transitions in density, pressure, and effective temperature drive collective cell migration into confining environments

doi: 10.1101/2023.04.10.536258

Figure Lengend Snippet: Temporal variation of effective pressure difference ΔP eff = ρ out υ out 2 − ρ in υ in 2 for (a) MCF10A WT, (b) 10A+RhoA and (c) 10A+ErbB2 monolayers entering wide (green) and narrow (purple) channels in 48 hours.

Article Snippet: Human mammary gland wildtype (WT) MCF10A (10A) epithelial cells with green fluorescence protein-labelled nuclei, MCF10A cell line with constitutively active RhoA (10A+RhoA; courtesy of Gregory Longmore, Washington University in St. Louis), and MCF10A cell line with overexpressing ErbB2 (10A+ErbB2; courtesy of Dihua Yu, The University of Texas MD Anderson Cancer Center) mutant cell lines were cultured using Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Invitrogen, Waltham, MA), supplemented with 5% horse serum (Hyclone heat-inactivated, Cytiva, Marlborough, MA), 20 ng/mL epidermal growth factor (EGF; Miltenyi Biotec, Gaithersburg, MD), 0.5 mg/mL hydrocortisone (H0888, MilliporeSigma), 100 ng/mL cholera toxin (C8052, Sigma-Aldrich), 10 μg/mL insulin (I6634, Sigma-Aldrich) and 100 μg/mL proprietary antimicrobial (Normocin; InvivoGen, San Diego, CA).

Techniques:

Journal: bioRxiv

Article Title: Transitions in density, pressure, and effective temperature drive collective cell migration into confining environments

doi: 10.1101/2023.04.10.536258

Figure Lengend Snippet: For MCF10A WT, 10A+RhoA and 10A+ErbB2 monolayers entering the wide and narrow channels, (a) immunostaining of cell-cell junctions (P120), (b) aspect ratio (AR), (c) shape index (SI), and (d) combined representation AR and SI according to the indicated colormap. Comparison of averaged (e) cell area, (f) aspect ratio, and (g) shape index across cell type and confinement variations, where the markers and bars represent median and interquartile range, respectively. Kruskal-Wallis test is used here for pairwise comparison between groups, *p<0.05 compared to wide channels. #p<0.05 compared to WT. N≥1700 cells from at least 4 ROIs.

Article Snippet: Human mammary gland wildtype (WT) MCF10A (10A) epithelial cells with green fluorescence protein-labelled nuclei, MCF10A cell line with constitutively active RhoA (10A+RhoA; courtesy of Gregory Longmore, Washington University in St. Louis), and MCF10A cell line with overexpressing ErbB2 (10A+ErbB2; courtesy of Dihua Yu, The University of Texas MD Anderson Cancer Center) mutant cell lines were cultured using Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Invitrogen, Waltham, MA), supplemented with 5% horse serum (Hyclone heat-inactivated, Cytiva, Marlborough, MA), 20 ng/mL epidermal growth factor (EGF; Miltenyi Biotec, Gaithersburg, MD), 0.5 mg/mL hydrocortisone (H0888, MilliporeSigma), 100 ng/mL cholera toxin (C8052, Sigma-Aldrich), 10 μg/mL insulin (I6634, Sigma-Aldrich) and 100 μg/mL proprietary antimicrobial (Normocin; InvivoGen, San Diego, CA).

Techniques: Immunostaining, Comparison

Journal: bioRxiv

Article Title: Transitions in density, pressure, and effective temperature drive collective cell migration into confining environments

doi: 10.1101/2023.04.10.536258

Figure Lengend Snippet: (a) Nuclear intensity within 10A+ErbB2+Metformin monolayers entering (i) wide and (ii) narrow channels. (b) Temporal variation of cell density difference Δρ between outside and inside channels, (c) averaged Δρ , (d) temporal persistence, and (e) averaged difference in RMA velocity Δv RMS between outside and inside channels for 10A+ErbB2+Metformin cells. * p < 0.05. (f) P120 expression from immunostained images, (g) aspect ratio, (h) shape index and (i) combined colormap of +ErbB2+Metformin monolayers. Comparison of averaged (j) cell area, (k) aspect ratio and (l) shape index of 10A+ErbB2 cells with and without Metformin treatment, where markers and bars represent median and the interquartile range, respectively. Kruskal-Wallis test is used for pairwise comparison between groups. *p<0.05 compared to wide channels. #p<0.05 compared to ErbB2 untreated control. N≥3400 cells from at least 8 ROIs.

Article Snippet: Human mammary gland wildtype (WT) MCF10A (10A) epithelial cells with green fluorescence protein-labelled nuclei, MCF10A cell line with constitutively active RhoA (10A+RhoA; courtesy of Gregory Longmore, Washington University in St. Louis), and MCF10A cell line with overexpressing ErbB2 (10A+ErbB2; courtesy of Dihua Yu, The University of Texas MD Anderson Cancer Center) mutant cell lines were cultured using Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Invitrogen, Waltham, MA), supplemented with 5% horse serum (Hyclone heat-inactivated, Cytiva, Marlborough, MA), 20 ng/mL epidermal growth factor (EGF; Miltenyi Biotec, Gaithersburg, MD), 0.5 mg/mL hydrocortisone (H0888, MilliporeSigma), 100 ng/mL cholera toxin (C8052, Sigma-Aldrich), 10 μg/mL insulin (I6634, Sigma-Aldrich) and 100 μg/mL proprietary antimicrobial (Normocin; InvivoGen, San Diego, CA).

Techniques: Expressing, Comparison, Control